morphological transformation, dna damage, and chromosomal...

[CANCER RESEARCH 39, 2356-2365, July 1979]0008-5472/79/0039-0000$02.00

Morphological Transformation, DNA Damage, and Chromosomal AberrationsInduced by a Direct DNA Perturbation of Synchronized Syrian HamsterEmbryo Celli'

Takeki Tsutsui, J. Carl Barrett,2 and Paul 0. P. Ts'o3

Division of Biophysics, The Johns Hopkins university School of Hygiene and Public Health, Baltimore, Maryland 21205

number of photochemical lesions in BrdUrd-substituted DNAthan in nonsubstituted DNA. The major photoproduct is uracil,which is formed by the dissociation of bromouracil in the radicalformation, and these radicals may extract an electron from thefuranose backbone. This ultimately leads to a break in theDNA, particularly when it is subjected to alkaline conditions(22,35).

Several studies (3, 12, 25, 29, 30, 38, 39) have indicatedthat the DNA of mammalian chromosomes is replicated in agiven chronology and pattern during the S phase. Furthermore,it has been suggested that the time at which individual genesare replicated during the DNA synthetic period is specific andconstant from one cell generation to the next (2, 4, 19, 21 , 23,36). The period of S phase during which a given gene isreplicated can be determined by treatment of synchronizedcells with mutagens which are selective for replicating DNA, toproduce mutations in the gene (1, 13, 24, 31 , 34, 37). Onesuch specific mutagenic treatment for replicating DNA is theincorporation of BrdUrd, followed by illumination with near UV(1, 8, 14, 34, 37, 40-43).

To probe further the involvement of DNA perturbation in theinitiation of neoplastic transformation, we have conductedBrdUrd pulse experiments with synchronized Syrian hamsterembryo cells. Experiments with synchronized cells allow us todetermine whether DNA synthesis is required for the transformation of cells in culture induced by BrdUrd treatment plusnear-UV irradiation. Furthermore, when synchronized culturesare treated with 1-hr pulses of BrdUrd during different hr of Sphase, irradiation damage can be directed to the specificportion of DNA replicated during a pulse period. It is thuspossible to demonstrate whether damage to specific regions ofcellular DNA is involved in neoplastic transformation.

In this paper, we report the effects of BrdUrd treatment andnear-UV irradiation upon DNA replicated during different penods of S phase in synchronized Syrian hamster embryo cells,as well as the role of specific DNA damage in the induction ofmorphological transformation.

MATERIALS AND METHODS

Cells and Culture Conditions. Syrian hamster embryo cellcultures were established from 13-day gestation fetuses collected aseptically by cesarean section from inbred Syrian hamsters, strain LSH/ss LAK (Lakeview Hamster Colony, Newfield,N. J.). Pools of primary cultures from littermates were stored inliquid nitrogen (5). Secondary cultures were initiated from thefrozen stocks, checked for chromosome number, and pretested for their ability to undergo morphological transformationby treatment with BrdUrd plus near-UV irradiation without synchrony. Secondary cultures which exhibited both over 90% of

2356 CANCER RESEARCH VOL. 39

ABSTRACT

The cellular effects of a direct perturbation of DNA duringvarious portions of the DNA synthetic period (S phase) wereexamined. Early-passage Syrian hamster embryo cells weresynchronized by growth in medium containing 1% serum, followed by hydroxyurea treatment. This method resulted in about80% of the cells entering S phase synchronously and did notinduce any detectable chromosomal abnormalities. Cells atdifferent periods in the S phase were treated for 1 hr with 5-bromodeoxyuridine, followed by irradiation with near UV. Thistreatment induced chromosomal aberrations and DNA damage,as measured by changes in sedimentation profile in alkalinesucrose gradients. No specific period during S phase wassignificantly more sensitive to treatment with respect to cellsurvival, chromosomal aberrations, or DNA damage and itsrepair. The induction of morphological transformation was alsocell phase dependent, occurring only in cells synthesizing DNA.However,incontrastto theresultswiththeaboveparameters,the incidence of morphological transformation was dependenton the portion of the S phase during which treatment wasadministered. The highest frequency of morphological transformation was observed for treatment in early to middle Sphase, particularly in the second hr of S phase, whereas notransformation was observed in late S phase, G1-S boundary,and G2 phase. 5-Bromodeoxyuridine treatment or irradiationalone induced no changes. These results suggest that certainregion(s) in the DNA of Syrian hamster embryo cells, as designated by their specific temporal relationship in the S phase,may be the more sensitive targets to the perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for theinitiation of morphological transformation.

INTRODUCTION

We have demonstrated previously that a direct perturbationof DNA by combined BrdUrd4 and near-UV irradiation treatmentis sufficient to initiate neoplastic transformation of asynchronous Syrian hamster embryo cells in vitro (8, 42). BrdUrd isincorporated into only DNA of cells in place of its analog dThd,and irradiation with near UV produces a significantly higher

t This research is supported in part by National Cancer Institute Grants CA

16043 and CP 55713.2 Present address: National Institute of Environmental Health Sciences, P. 0.

Box 12233, Research Triangle Park, N. C. 27709.3 To whom requests for reprints should be addressed, at Division of Biophys

cs, Department of Biochemical and Biophysical Sciences, School of Hygieneand Public Health, The Johns Hopkins University, 615 North Wolfe Street,Baltimore, Md. 21205.

4 The abbreviations used are: BrdUrd, 5-bromodeoxyuridine; dThd, thymidine;

dCyd, deoxycytidine; HU, hydroxyurea; PBS, phosphate-buffered saline [0.14M NaCI:3 mM KCI:8 mM Na2HPO4:1 m@iKH2P04 (pH 7.4)].

Received June 5, 1978; accepted March 20, 1979.

Research. on February 4, 2020. © 1979 American Association for Cancercancerres.aacrjournals.org Downloaded from

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Effects of DNA Perturbation on Synchronized Hamster Cells

diploidy and a suitable response to the pretesting were restoredin liquid nitrogen. Ampuls from the same pool of the cryopreserved secondary cultures were used for all subsequent experiments.

The cell culture medium (complete medium) used was IBRmodified Dulbecco's Eagle's reinforced medium (Biolabs,Northbrook, III.) supplemented with 0.22 g NaHCO3per 100 mland 10% fetal bovine serum (Reheis Chemical Co., Kankakee,Ill.) without antimicrobial drugs. Cells were transferred bygentle trypsinization with 0.1 % trypsin solution (1:250; GrandIsland Biological Co., Grand Island, N. V.) for 5 mm at 37Â°.Allcells were tested periodically by Microbiological Associates(Walkersville, Md.) and found free of Mycoplasma contamination. dCyd at a concentration of 0.2 m@idecreased the cytotoxicity of BrdUrd (8, 28). For this reason, 0.2 mp@idCyd wasincluded in all experiments involving BrdUrd treatment.

Chemicals and Isotopes. BrdUrd, dCyd, and HU were purchased from Sigma Chemical Co. (St. Louis, Mo.). Chemicalswere dissolved in medium without serum, filter sterilized, anddiluted with complete medium to the desired concentrationimmediately before use. All BrdUrd-containing solutions, media, and treated cells were exposed only to red-filtered light.[3H]dThd (62 Ci/mmol) and [3H]BrdUrd (23.7 Ci/mmol) werepurchased from Schwarz/Mann (Orangeburg, N.Y.) and NewEngland Nuclear (Boston, Mass.), respectively.

Cell Synchronization. Cells (2 x 10@)on logarithmicgrowthwere plated in triplicate on 15-mm-diameter glass coverslips in16-mm tissue culture cluster dishes (Costar, Cambridge,Mass.) in complete medium. After an overnight incubation at37Â°,complete medium was replaced with medium containing1% serum, (or, in certain experiments, 2% serum), and thecultures incubated for -â€˜-36hr. At this time, the cultures weretreated with 0.32 mM HU in medium containing 10% serum foran additional 12 hr, the cultures were washed once with complete medium, and prewarmed medium was added. This wasthe 0-hr time point. At 0.5, 1.5, 2.5, . . . 16.5, and 17.5 hr afterrelease from HU block, [3H]dThd was added at a final concentration of 1 foCi/mI for a 30-mm pulse label. The uptake of[3HJdThdwas stopped by rinsing the coverslips twice with cold0.1 5 M NaCI and immersing twice in 5% cold trichloroaceticacid for 15 mm. After washing with deionized water and drying,the coverslips were transferred to scintillation vials and countedin a Beckman liquid scintillation spectrometer for determinationof tritium incorporation. After counting, the coverslips wereremoved from the vials, washed with acetone, dried, dipped inKodak NTB-2 liquid emulsion, and exposed for 2 days forautoradiography. Following development and fixation, theywere stained with hematoxylin.

General Experimental Scheme of BrdUrd Treatment andNear-UV Irradiation of Synchronized Cultures. As shown inChart 1, synchronized cells on 100-mm dishes (Falcon) weretreated with a 1-hr pulse of 1 or 10 fLMBrdUrd. Five 1-hr pulseswere administered during the entire S phase, and a single 1-hrpulse was administered both before and after the S phase.After BrdUrd treatment, each dish of cells was washed twicewith 5 ml of PBS, and 5 ml of PBS were added prior toirradiation. Dishes were uniformly irradiated at room temperature for specified times with near UV from a row of 6 Fl 5T8/BLBfluorescentbulbs(Sylvania,Inc.,Springfield,Va.)at adistance of 7.5 cm (-@-33J/sq m/sec) as determined by theBlak-Ray UV meter (Ultra-Violet Products, Inc., San Gabriel,

3@ 3

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I I I I I I I-1 0 1 2 3 4 5 6

Hours after hydrosyurea release

â€¢â€˜â€¢â€¢â€¢,hydroxyureoblock E@,BrdU treatment

3,IrradiattonChart 1. Experimental scheme of BrdUrd (BrdU) treatment and near-UV irra

diation. As indicated in Chart 2, the length of S phase is 5 hr after the removal ofHU.

Calif.). After irradiation, PBS was replaced with complete medium. Untreated cultures, cultures receiving irradiation alone,or cultures treated with BrdUrd alone were used as controls.

Cytotoxicity and MorphologicalTransformation. Cells(10@)were inoculated into each of five 100-mm dishes/group, synchronized, and treated with BrdUrd and irradiation as describedabove. After treatment, the cells were incubated for 7 days toallow colony formation. The colonies were fixed with methanoland stained with Giemsa, and both the number of colonies andthe number of morphologically transformed colonies werescored. The cloning efficiency of untreated cells was 1 to 2%.Colonies in which cells exhibited 3-dimensional growth withrandom orientation and extensive crisscrossing at the periphery of the colony (5, 10, 17) were scored as morphologicallytransformed. Figure 1 is a representative example of a colonytransformed by treatment with BrdUrd plus near-UV irradiation.The morphology of the normal colony or colonies transformedby benzo(a)pyrene was shown in our previous publication (5).

Chromosomal Aberrations. Cells were plated in 100-mmdishes at a density of 2 x 10@cells/dish and synchronized.After removal of HU, the cultures were treated with 1 and 10@iMBrdUrd for 1 hr during different periods of S phase andsubsequently irradiated for 5, 10, 20, and 30 mm, as shown inChart 1. Cultures were then incubated with complete mediumcontaining 10 @LMdThd. Colcemid (0.2 @@g/mI)was added atthe fifth hr after release from HU treatment, and the metaphaseblocked cells were harvested for chromosome preparation after4 hr. Over 100 metaphases were scored for chromosomalaberrations.

DNA Damage Assayed by Alkaline Sucrose Gradients.Cells were plated in 100-mm dishes at a density of 2 x 1O@cells/dish, synchronized, treated with medium containing both1 or 10 @LMBrdUrd and 2 zCi [3H]dThd per ml for 1 hr duringdifferent periods of the S phase, and incubated for 1 hr withmedium containing 10 @LMdThd to allow short pieces of newlysynthesized DNA to elongate. Cultures were then washed twicewith PBS and irradiated for 5 to 30 mm. Following irradiation,some cultures were reincubated with medium containing 10@LMdThd for 1 hr at 37Â° to examine repair of DNA breakage.

After harvesting by treatment with 0.5% trypsin for 3 mm at 4Â°,1o@cells in 0.02 ml of PBS were gently placed on a 0.25-mIlysing solution on top of a 5 to 20% alkaline sucrose gradientprepared in a 5-mI cellulose nitrate centrifuge tube. The lysingsolution consisted of 0.55 M NaCI, 0.45 M NaOH, and 0.01 Mdisodium EDTA. The gradient contained 0.55 M NaCI, 0.45 M

JULY1979 2357



T. Tsutsuietal.

NaOH, and 0.003 M disodium EDTA. After 1 hr lysis at 37Â°,tubes were centrifuged in a Beckman L2-65B ultracentrifugeat 106,000 x g (34,000 rpm; SW 50.1 rotor)for 90 mm at 12Â°as described previously (43, 44). The tubes were pierced atthe bottom and approximately thirty-five 9-drop fractions werecollected onto Whatman GF/C filters. The radioactivity on thefilters was counted by a liquid scintillation counter. It has beendemonstrated that the assay system results in sedimentationprofiles with DNA dissociated from lipoprotein (45) and that theaverage molecular weight of DNA from untreated cultures was2 to 3 x 108 (44 45).

BrdUrd Substitution for dThd. The extent of BrdUrdsubstitution for dThd in newly synthesized DNA during each 1-hrperiod of the S phase was assayed by CsCIgradient equilibriumcentrifugation according to the method of Chen et al. (15) andcalculated by using a formula described by Luk and Bick (27).Cells were plated at a density of 2 x l0@cells/iOO-mm dish,synchronized, and labeled with 2 @iCi[3H]BrdUrd per ml for 1hr of S phase at a final concentration of 1 or 10 ,.tMBrdUrd.Control cultures were labeled with 2 @.tCi[3H]dThd. Followingthe labeling period, cells were rinsed with PBS and harvestedby treatment with trypsin. Cells were collected by centrifugation; resuspended at a density of â€˜â€”106 cells/mI in a solutionwhich contained 0.01 5 M NaCI:l .5 mt@isodium citrate buffer(pH 7.3; NaCI:citrate), 20 m@iEDTA, 0.1 % Sarkosyl NL-30(Geigy Chemical Corp., Ardsley, N. Y.), and 100 @tgproteinaseK (EM Laboratories, Inc., Elmsford, N. Y.) per ml; and digestedfor 3 hr at 37Â°.After digestion, 0.2 ml of sample was added to4.0 ml of NaCI:citrate-buffered CsCI (Schwarz/Mann) at adensity of 1.745 g/mI, overlaid with 0.4 ml of mineral oil, andcentrifuged in the SW 50.1 rotor of a Beckman L2-65B ultracentrifuge at 33,000 rpm for 72 hr at 21Â°.Nine-drop fractionswere collected from the bottom of each gradient onto WhatmanGF/C glass fiber filters. The filters were dried and counted ina Beckman liquid scintillation counter. The buoyant density ofevery tenth fraction was determined from the refractive indices.

RESULTS

Cell Synchronization. Syrian hamster embryo cells weresynchronized by a modification of the method of Chang andBaserga (14), which combines a growth period in mediumcontaining 1% serum with a subsequent blockade of the cellsat the G1-S boundary by 0.32 [email protected] progression throughthe cell cycle of cells released from HU blockage by theaddition of complete medium is shown in Chart 2. The cellsentered S phase immediately after HU release; [3H]dThd incor

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1;g:@ ::@0 1 2 3 4 5 6 7 8 9 tO 11 12 13 14 15 16 17 18

Hoursafter hydroxyureoreleaseChart 2. Progression of Syrian hamster embryo cells through the cell cycle

following release from HU by the addition of complete medium. 0, [Â°H)dThdincorporation into the acid-insoluble fraction; â€¢,[Â°H)dThdlabeling index obtainedby autoradiography; @o,cell number.

poration and the labeling index reached a maximum after 2 to3 hr; S phase lasted 5 hr. A second peak of [3HJdThd incorporation and labeling index was observed 15 hr after HUrelease. To determine the percentage of cells capable of DNAsynthesis after the HU treatment, a continuous labeling experiment was performed. Cells (2 x 10@)were plated on 15-mmglass coverslips and synchronized. Following removal of HU,complete medium containing 1 zCi [3H]dThd per ml with 1 @tMdThd was added to the cultures. After 2, 4, 5, and 6 hr, thecultures were rinsed with cold 0.1 5 MNaCIfollowed by washingwith 5% cold trichloroacetic acid. The coverslips were autoradiographed to determine the labeling index. As shown in Chart3a, the labeling index reached a maximum of 80%. Withasynchronous cells in logarithmic phase, the continuous labeling index reached 100% after 24 hr (Chart 3b). These resultsindicate that after the synchronization process, 20% of thecells are unable to enter S phase. Together, the data indicatethat Syrian hamster embryo cells which are capable of enteringinto the S phase exhibited about 80% synchrony within the firstS phase following release from HU blockages. Chart 2 alsoshows that the labeling index of SHE cells between the fifth

S.

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EU

0123456 01234567

Hours after hydroxyurea release Hours after tiydroxyureo release

Chart 3. a, continuous labeling index of synchronized Syrian hamster embryocells obtained by autoradiography after release from HU. Cells were continuouslylabeled for 2, 4, 5, and 6 hr with medium containing 1 @sCi[3H]dThd per ml and1 ,.tMdThd. b, continuous labeling index obtained by autoradiography of asynchronous Syrian hamster embryo cells in logarithmic growth. Cells were continuously labeled for 6, 24, and 48 hr with medium containing 0.1 @tg[Â°H]dThdperml and 1 @LMdThd. c, effects of serum concentration and HU dose during thesynchronization procedure on DNA synthesis of Syrian hamster embryo cells.0, 1% serum medium for 36 hr and 0.32 mMHU for 12 hr; â€¢,2% serum mediumfor 36 hr and 0.32 mM HU for 12 hr; L@,1% serum medium for 36 hr and 1 mp@iHU for 12 hr. d. effect of BrdUrd treatment (during the first hr of S phase) onDNA synthesis. Cells (2 x 10@)were inoculated into 25-sq cm flasks andsynchronized. Immediately after removal of HU, cells were treated with 10 @oMBrdUrd for 1 hr. The cells were then washed once with complete medium andcultured in medium containing 10 @MdThd. At 0.5, 1.5, 2.5, . . . and 5.5 hr afterBrdUrd treatment, the cells were labeled with [3H]dThd (1 @tCi/ml)for 30 mm. Incontrol cultures, the cells were labeled with 1 @sCi[Â°H]dThdper ml for 30 mm at0.5, 1.5, 2.5, . . . and 6.5 hr after HU release by replacement with mediumcontaining 10 @MdThd. 0, no BrdUrd treatment; â€¢,BrdUrd (10 toM)treatment.




Dependence of percentage of substitution of BrdUrd for dThd on thedifferent periods in S phase during which BrdUrd was incorporatedDNAdensityof Syrianhamsterembryocellswas 1.6996 Â±0.0005

under our experimental conditions.%

ofsubstitutionPeriod

of BrdUrd labeling 1 @tMBrdUrd 10 @s.iBrdUrdlsthr

12.9a 21.22ndhr 15.2 24.13rdhr 15.2 24.14thhr 15.2 24.45th hr 16.426.3a

Average of 3 independent experiments.

LIK@T


and 12th hr after HU release was approximately 15%, representing the subpopulation not in synchrony. The populationentering into second S phase was small due to contact inhibition of cell replication as the cells reached saturation density.Thedegreeof synchronywas not improvedby the applicationof 2% serum medium or 1 mp@iHU (Chart 3c).

The synchronization by treatment with medium containing1% serum and subsequent 0.32 m@HU resulted in 20% of thecells grownat highcell densitynot enteringS phase.Additionally, the synchronization process reduced the cloning efticiency of cells plated at a low cell density to 50% of asynchronous cells. Increasing the serum concentration from 1 to 2%during the synchronization process improved the cloning efficiency to 70% that of untreated cells. The 2% serum:0.32 m@iHU synchronizationyielded an identical pattern of [3HJdThduptake (Chart 3c) (45). Chromosome analysis of cells in thefirst mitosis after release from HU block demonstrated that thesynchronization procedures induced no change in chromosome number and no gross chromosomal abnormalities.

Effect of BrdUrd Treatment on Cycling Progression ofCells in S Phase. The treatmentof cellswith 10 @tMBrdUrdforthe first hr of S phase did not disturb the normal cyclingprogression of synchronized cells since identical patterns of[3HJdThd incorporation were observed for the BrdUrd-treatedand control cultures in the S phase (Chart 3d).

Effect of BrdUrd plus Irradiation Treatment on MitoticDelay. The treatmentof cellswith 1 @MBrdUrdduringthe first,third, and fifth hr of S phase, followed by irradiation for 30 mm,did not cause mitotic delay. The peaks of mitotic indices underthese conditions occurred at the same time as untreated cultures, and the level of mitotic indices was similar to that of thecontrol (Chart 4). This suggests that there is no differencebetweentreatedand control cultures in the progressionof thecell cycle prior to the first metaphase.

BrdUrd Substitution for dThd. The extent of BrdUrdsubstitution for dThd in newly synthesized DNA during each 1-hrperiod of S phase was determined by CsCl buoyant densityanalysis. In this experiment, [3HJdThd and [3H]BrdUrd wereused to monitor the newly synthesized DNA from the controlcells or from the BrdUrd-treated cells respectively, in the CsCIgradient sedimentation. The percentage of BrdUrd substitution

is calculated from the difference in density of the [@H]dThdpeak and the [3H]BrdUrd peak according to Luk and Bick (27).Therefore, the percentage of BrdUrd substitution is the % ofBrdUrd substituted for dThd in the newly synthesized DNA, notthe total DNA. As shown in Table 1, the extent of BrdUrdsubstitution in DNA replicated was found to be similar duringeach hr of the S phase. When synchronized cells were labeledwith 0.1 , 1.0, and 10 @MBrdUrd during the entire S phase, theextent of BrdUrd substitution for dThd in newly synthesizedDNAoccurred in a linearmannerwith the logarithmof BrdUrdconcentration (7.6, 16.5, and 25.5%, respectively) (Chart 5).In the case of Chinese hamster cells, the logarithm of the % ofdThd replacement was found to be linear with the logarithm ofBrdUrd in the medium (9).

Cytotoxicity Induced by BrdUrd plus Irradiation. BrdUrddose and irradiation time response curves of cell survival aftertreatment with BrdUrd during the first hr of S phase followedby irradiation are shown in Chart 6. Cytotoxicity increased withincreasing BrdUrd dose and irradiation time.

Effect of BrdUrd plus Irradiation Treatment on Chromosomal Aberrations. To examine possibledamage to the genetic apparatus at the chromosome level caused by BrdUrdplus near-UV treatment, treated cells were analyzed for theincidence of chromosomal aberrations (Table 2). In controlcultures, none of the 100 metaphases examined containedchromosomal aberrations. Following 20-mm irradiation ofBrdUrd-treated cells, only 2% of the metaphases containedchromatid gaps. However, a significant increase of chromosomal aberrations was observed in BrdUrd-treated cells irradiated for 30 mm. Chromosomal aberrations were induced atthe chromatid level in every period of BrdUrd labeling during Sphase. The incidence of chromosomal aberrations increased

Table1

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20

10

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U)

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0 1 2 3 4 5 6 7 8 9 1011 12Hoursafter hydroxyurearelease

Chart 4. Effect of BrdUrd plus irradIation treatment on mitotic delay of synchronized cells. Cells (2 x 10@)were plated on 15-mm glass coverslips andsynchronized. At 0, 2, and 4 hr after removal of HU, 1 it,., BrdUrd was added tothe cells. Following 1 hr treatment with BrdUrd, the cells were washed twice withPBS and subsequentlyirradiatedfor 30 mmat roomtemperaturein PBS. Afterirradiation, the cells were reincubated with medium containing 1 @&MdThd, fixedat 4, 6, 7, . . . and 11 hr after release from HU block, and stained withhematoxylin. The time used for washing and irradiation was excluded. Untreatedcultures (0), cultures receiving a 30-mm irradiation alone (ti), and cultures keptat room temperature in PBS for 30 mm (O) were used as controls. 0, A, and U.mitotic indices of cells which were pulsed with 1 @MBrdUrd during the first, third,and fifth hr of S phase, respectively, and subsequently irradiated for 30 mm.Over 1000 cells were scored at each point.

0.1 1.0 10

BrdUDose(SM)Chart 5. % of dThd substitution of newly synthesized DNA in hamster cells

versus concentration of BrdUrd (BrdU) (in logarithm) added to culture.

JULY 1979 2359



Incidence of chromosome aberrations in synchronized hamster embryo cells induced by treaBrdUrd and subsequent irradiationtmentwith1-hr

pulseofPeriod

of Type of aberrations (%)BrdUrd Ia- No. of mets

beling during BrdUrd dose Irradiation time phases anaS phase (@th4) (mm) lyzed G@ ICG B E DAberrant

metaphase(%)0Flsthr

0 0 100 0 0 0 0 01 20 100 2 0 0 0 0

10 20 100 2 0 0 0 01 30 100 8 0 5 5 0

10 30 132 5 0 6 6 00

00000

00000

22

14172ndhr

1 30 100 19 0 3 9 010 30 226 21 0 11 7 00 00 028383rdhr

1 30 100 17 0 7 7 010 30 100 20 0 6 6 00 00 025324thhr

1 30 100 16 0 6 0 010 30 113 18 0 10 0 00 00 019275th

hr 1 30 100 15 0 0 0 010 30 111 7 0 3 0 00 00 01510a

G, gap; ICG, isochromatid gap; B, break; E, exchange; D, Dicentric; 0, 0-ring; F, fragmentation.

T. Tsutsui et al.

with increasing BrdUrd dose and irradiation time except for thefifth hr. The highest incidences were observed in the secondand third hr of S phase, and the lowest incidences wereinduced in the first and fifth hr.

Degradation and Repair of Irradiated-Cell BrdUrd DNA.The effect of BrdUrd plus irradiationtreatmenton sedimentation profiles of DNA on alkaline sucrose gradients was analyzed. This study was divided into 2 phases.The first phasewastheeffectof BrdUrddoseandirradiationtime,by usingsynchronizedcells in the first hr of S phase.Thesecondphaseconcerned the dependence of the DNA damage on the differentperiods of S phase during which BrdUrd was incorporated.

In untreated cultures, the sedimentation profile of DNA fromsynchronized cells was identical to that of unsynchronizedcells, indicating that the synchronizationprocedure itself didnot produce any perturbations in the cellular DNA. By usingalkaline sucrose gradient analysis, the radiosensitivity to thenear UV of DNA from cells labeled was compared with either 2@Ci[3H]dThd per ml or 0.76 @Ci[3HJBrdUrd per ml, plus

nonradioactive BrdUrd at a final concentration of 1 @LM.Thesame degree of DNA damageunder alkaline conditionswas

0>

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In

0)C.)

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â€˜I-'

0a)

observedin both DNA preparations.Moreover,the extent towhich BrdUrd substituted for dThd was found to be identical inDNA labeled with either [3H@dThdor [3H]BrdUrd (data notshown)suggestedthat there was no preferentialutilizationofeither nucleoside for DNA synthesis under these conditions.Therefore, we used [3HJdThdrather than [3H]BrdUrd for labeling newly replicated DNA in synchronized cells.

The solid lines (indicating no subsequent irradiation) in Chart7A show that the sedimentation profiles of DNA from cellstreated with 0. 1 or 1 @MBrdUrd (Curves b and c) were similar

Bottom Fractton Number Top Bottom Fraction NumberTop

Chart 7. Sedimentation profiles of DNA from cells treated with BrdUrd plusnear-UV irradiation. Vertical lines, position of the main peak of DNA from theuntreated cells in the sedimentation profile. A, Dependence of DNA damage onBrdUrd dose. Cells (2 x 10@)were plated on 100-mm dishes, synchronized, andtreated with 0 (a), 0.1 (b), 1.0 (C),and 10 @M(d) BrdUrd, and 2 @Ci[3H]dThd perml for the first hr of S phase. The cultures were then incubated for 1 hr withcomplete medium containing 10 @t.idThd and irradiated for 5 mm with near Uv.â€”â€˜ no irradiation; , 5-mm irradiation. B, Dependence of DNA damage

on irradiation time. Cells (2 x 10@)were plated on 100-mm dishes, synchronized,and treated with 1 @MBrdUrd and 2 @&Ci[3H]dThd per ml for the first hr of Sphase. The cultures were then chased for 1 hr with complete medium containing10 @iMdThd and irradiated for 10 (b), 20 (C), and 30 mm (d) with near [email protected], 30-mm irradiation alone.

Table2

(A) (B)

0.5-

0. 110 20 30

IrradiotLon ttme (mmChart 6. Effects of BrdUrd dose and irradiation time on cell survival. Cells

(1 0@)were plated in 100-mm dishes, synchronized, and treated with BrdUrd forthe first hr of S phase and subsequently irradiated for 0, 10, 20, or 30 mm.Complete medium was then added to the dishes, and the cells were incubated toform colonies for 7 days. The colonies were fixed with methanol and stained withGiemsa. The cloning efficiency of untreated cultures was normalized to 1. 0,near UV alone; â€¢,1 MMBrdUrd; & 10 ;LMBrdUrd.





to that of untreated cells (Curve a). The sedimentation profilesof DNA from cells treated with 10 @MBrdUrd without subsequent irradiation (Chart 7A, Curve d) had a lower peak andslightly broader trailing portion than did those of the control.Thedotted lines in Chart7A showthe sedimentationprofilesofDNAfromcellstreatedwithBrdUrdfor thefirsthr of S phaseand subsequently irradiated for 5 mm. No DNA lesions apparentas single-strandbreaksunderalkalineconditionsweredetectable following 5-mm irradiation without prior incubation withBrdUrd (Chart 7A, Curve a). In contrast, BrdUrd-substitutedDNA was degraded by 5-mm irradiation in a BrdUrd dosedependent fashion (Chart 7A, Curves b, c, and d).

Thedependenceof DNAdamageon near-UVirradiationtimefollowing BrdUrd treatment was examined in cells treated with1 @LMBrdUrd for 1 hr and subsequently irradiated for 10, 20,and 30 mm (Chart 7B). Thirty-mm irradiation alone had noeffecton DNAdamagedetectablein alkalinesucrosegradients(Chart 7B, Curve a), whereas the peaks of DNA profiles fromcells treated with BrdUrd plus irradiation shifted toward thelower sedimentation coefficient values in a manner dependenton the irradiation time (Chart 78, Curves b, c, and d).

The dependenceof DNAdamageon the periodsin S phaseduring which BrdU was incorporated was examined. DNA fromcells treated with 1 @LMBrdUrd during the first hr of S phasefollowedby a 10-mmirradiationsedimentedslowerand with abroader distribution than did DNA from cells treated withBrdUrd but not exposed to near UV (Chart 8, a and b). Treatment during the second hr of S phase produced a furtherdecreasein size. However,BrdUrdtreatmentduring the third,fourth, or fifth hr of S phase produced sedimentation profilesquite similar to that producedby the BrdUrdtreatmentduringthe secondhr (Chart8, c to f).

TheDNAdamageinducedby treatmentwith 1 @sMBrdUrdforthe first hr. followed by 10-mm irradiation, was repaired almostcompletely during 1-hr incubation at 37Â°in medium containing10 @MdThd. The DNA damage induced in the other periods ofS phase was partially repaired at rates which were similar toone another(Chart8, b to f).

In our alkaline sucrose gradient system, the treatment ofsynchronized cells with 1 @sMBrdUrd for the first hr, followedby 5-mm irradiation, caused enough perturbation to allow thedetection of DNA damage. Such DNA damage was repaired ina subsequent 1-hr incubation (Chart 8b). In contrast, 30 mm ofirradiation were required to produce detectable chromosomalaberrations in cells that incorporated BrdUrd (Table 2). Tostudy the relationship between DNA damage and chromosomalaberrations, cells pulsed with 1 fLMBrdUrd and 2 @Ci[3H]dThdper ml for the first hour of S phase, followed by exposure tonear UVfor 20 or 30 mm,were subjectedto repair incubationwith medium containing 1 @sMdThd. At the ninth hr, followingremoval of HU block, 1O@metaphases(arrested by 0.2 @gColcemid per ml and collected by shaking) were assayed forrepair of DNA damageby alkaline sucrosegradient centrifugation. The DNA damage induced by treatment with I @sMBrdUrd plus either 20- or 30-mm irradiation was not completelyrepaired (Chart9). A significant increasein chromosomalaberrations was not observedin cells which were irradiatedfor20 mm following BrdUrd treatment. Thus, no correlation between DNA damage and chromosomal aberrations was found.

Morphological Transformation. To studythe relationshipofa direct perturbation of DNA to the initiation of neoplastic

20ra10

020

0@1@'.0 10 20 30

Bottom . TopFroctton NumberChart 8. 5 phase period dependence of DNAdamage Induced by BrdUrd plus

near-UV treatment and its repair in synchronized cells. Vertical line, position ofthe main peak of DNA from the untreated cells in the sedImentation profile. Cells(2 x 10@)were platedon 100-mm dishesand synchronized.Synchronizedcellsin different periods of S phase were treated with 1 @MBrdUrd and 2 @Ci[3H)dThdper ml for 1 hr, incubated for 1 hr with complete medIum containing 10 @idThdand irradiated for 10 mm with near UV. After Irradiation, 1o@cells were placed Inlysing solution on alkaline sucrose gradients and centrifuged to detect DNAdamage (â€”). Parallel cultures were reincubated for 1 hr wIth complete mediumcontaining 10 fLMdThd after irradiation to examine DNA repair ( ). a, 1@LMBrdUrd alone (treatment for the first hr); b, BrdUrd treatment for first hr; c,BrdUrd treatment for the second hr; d, BrdUrd treatment for the third hr; e,BrdLird treatment for the fourth hr; f, BrdUrd treatment for the fifth hr.

transformation, we examined morphological transformation induced by BrdUrd plus irradiation treatment as described in,MaterialsandMethods.'â€˜Morphologicallytransformedcobflies (Figure1) werenotobservedin the controlcultures(Table3). Chart 10 shows both the relative cell survival calculated byusing untreated cultures as controls and the frequency ofmorphobogicalbytransformed colonies induced by treatment ofsynchronized cells with 10 @tMBrdUrd for 1 hr of S phase andsubsequent irradiation for 5 mm. This chart is a compilation of7 experiments, and the total number of transformed colOniesper surviving colonies is presented. The cytotoxicity was minimal (approximately 20%), and there was no specific period inthe S phase that was more sensitive to the toxic effect. A 1-hour pulse with 10 @&MBrdUrd, followed by 5-mm irradiation,induced morphological transformation. The highest incidenceof morphological transformation was observed when the pulseswere administered in the middle of S phase, particularly in thesecond hr, whereas no transformation was observed in late Sphase, in the G1-S interphase, or in the G2 phase.

As shownin Chart7A, Curved, andChart7B, Curveb,treatment of cells with 10 @iMBrdUrd plus 5-mm irradiationinduced a similar degree of DNA damage as with I @sMBrdUrd

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JULY 1979 2361



Summary of morphological transformation of synchronous Syrianhamsterembryocells in controlexperimentsNo.

of morphologically transformed colonies/totalcoloniesPeriod

of 5 No BrdUrd andphase no UV BrdUrd only UVonlylsthr

0/372 0/342 0/3782ndhr 0/287 0/320 0/2973rdhr 0/417 0/306 0/3614thhr 0/302 0/263 0/3175thhr 0/372 0/402 0/372

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Hours after hydroxyurea reteqseChart 10. Relative cell survival and the frequency of morptiologically trans

formed colonies induced by treatment of synchronized cells with 10 @MBrdUrdfor 1 hr of S phase and subsequent irradiation for 5 mm. Cells (10@)wereinoculated into each of five 100-mm dishes/9roup, synchronized, and treatedwith BrdUrd and irradiation. After treatment, the cells were incubated for 7 daysto allow colony formation. The colonies were fixed with methanol and stainedwith Giemsa, and both the number of colonies and the number of morphologicallytransformed colonies were scored. Data from 7 independent experiments werecompiled. Relative cell survival was calculated by using untreated cultures (noBrdUrd and no irradiation) as controls. Fractions, total number of morphologicallytransformed colonies observed in 7 experiments, divided by the total number ofcolonies scored.

-1 0 1 2 3 4 5 6Hoursafter hydroxyurearelease

Chart 11. Relative cell survival and the frequency of morphologically transformed colonies induced by treatment of synchronized cells with 1 @iBrdUrd for1 hr of 5 phase and subsequent irradiation for 10 mm. Cells (10@)were inoculatedinto each of five 100-mm dishes/group, synchronized, and treated with BrdUrdand irradiationas describedabove.Aftertreatment.the cellswere incubatedfor7 days to allow colony formation. The colonies were fixed with methanol andstained with Giemsa, and both the number of colonies and the number ofmorphologically transformed colonies were scored. Data from 3 independentexperiments were compiled. Relative cell survival was calculated by using untreated cultures (no BrdUrd and no irradiation) as controls. Fractions, totalnumber of morphologically transformed colonies observed in 3 experiments,divided by the total number of colonies scored.

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9. Effect on DNA damage of repair incubation for 8 hr after BrdUrd plusnear-UV treatment. Cells (2 x 10') were plated on 100-mm dishes and synchronized. Immediately after release from HU block, the cells were pulsed with 1 @tMBrdUrdand 2 @Ci[â€˜H]dThdper ml for 1 hr. followedby exposureto near UV for20 and 30 mm. The cells were then subjected to repair incubation in completemedium containing 1 @e.idThd. At the ninth hr after removal of HU block. 1o4metaphases, which were arrested by treatment with 0.2 @gColcemid per ml for4 hr and collected by shaking, were assayed by alkaline sucrose gradientcentrifugation. â€¢,no BrdUrd and no irradiation; 0. 1 @MBrdUrd plus irradiationfor 20 mm; x , 1 @dbiBrdUrdplusirradiationfor 30 mm.

Table3

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not appear to affect the cell cycle or induce any chromosomeaberrations. Hamlinand Pardee(20) and Cress and Gerner(16) observed that HU treatment similar to that used here didnot alter the length of S phase relative to cells selected inmitosis from untreated cultures. Since it is possible, however,that synchronizingcells by chemical(s)producesan artificialcell response,we cannot neglectthe possibleeffect of HUonthe temporal order of DNA replication.

Previously,we demonstratedthat a direct perturbationofDNA by treatment with BrdUrd and near-UV irradiation resultsin neoplastic transformation of Syrian hamster embryo cells(8). The present results strongly support the conclusion thatDNAis a criticaltargetin thesetransformationexperiments.Transformation occurred only when cells were treated withBrdUrd plus near-UV irradiation during DNA replication and not

2362 CANCERRESEARCHVOL. 39

plus 10-mmirradiation.In addition,both treatmentsinducedasimilar degree of cytotoxicity and frequency of morphologicaltransformation (Charts 10 and 11). In these 2 sets of experiments, change in the dosage of BrdUrd was compensated bychange in duration of irradiation.

DISCUSSION

It is difficult to obtain early passage, embryonic cell populations which proceed through DNA-syntheticperiod (S phase)with a high degree of synchrony. Partial synchrony of Syrianhamsterembryo cells has been reported by Kuroki and Sato(26), whoobtained50 to 60% synchronyof the cellsby excessdThd or HU treatment, and by Popescu et al. (33), whoachieved 65 to 70% synchrony with isoleucine or argininedeprivation. This moderate degree of synchrony has beenattributed by Kuroki and Sato (26) to the heterogeneity of theculture, which contains cells with different growth kinetics. Inthis paper, we demonstrated that a combined treatment withmedium containing low serum and a HU blockade inducedabout 80% synchrony of Syrian hamster embryo cells. Inaddition, the applicationof this treatmentto transformedhamster cell lines resulted in >98% synchrony (14, 43). Thus, thismethod appears superior to the previous methods utilized withthesecells.Useof HUblockageto obtainsynchronizationdid



Correctedfrequencyof morphologicaltransformationinducedbypulseof BrdUrdtreatmentplusnear-UVirradiationduringdifferentperiods

in S phase(correctionfromlabelingindex)Correctedmorphologi

cal transformation frequency'(%)Periods

Corrected Iain S Labeling Indexa beling indexb ExperimentExperimentphase

(%) (%) (Chart 10) (Chart11)Before

re- 0 00lease0â€”lhr

47 59 1.11.01â€”2hr58 73 1.81.92-3hr64 80 0.961.23â€”4hr50 63 0.761.24â€”5hr18 23 00After5hr15 20 0


when cells were treated similarly during the G1or G2period, aperiod at which DNA synthesis does not take place. Thesefindings indicate that BrdUrd incorporation into DNA is requiredfor the neoplastic effect of BrdUrd plus near-UV irradiation.

Not only was morphological transformation induced byBrdUrd and near-UV treatment only when this treatment wasadministered during the S phase, but also certain times duringthe synthetic period appeared more susceptible to this induction, as shown in Charts 10 and 11. The results in both chartssuggest that the induced morphological transformation frequency is highest during the 1- to 2-hr period and lowest duringthe 4 to 5 hr in the S phase. However, due to imperfection inthe synchronization, the labeling indices of all these periods inS phase are not exactly the same. To have the quantitativeanswer concerning the propensityto morphologicaltransformation of cells having their DNA perturbed at a different periodof S phase, this cell number in a population must be correctlyidentified by the labeling index. Such an analysis was performed in Table 4. The labeling indices in Table 4 were obtainedfrom experiments reported in Chart 2. These indices werefurther corrected by dividing them with 0.80, the fraction ofcells in the population capable of entering into S phase, mdicated in the experiment of continously labeling (Chart 3a).Therefore, the number of the corrected labeling index identifiedthe fraction of cells in each population which had incorporatedBrdUrd into their DNA and therefore was perturbed by thistreatment in the experiment. For instance, in the experimentsreported in Chart 10, the observed number of surviving coloniesin each period of the S phase (in parentheses) are as follows:2023 (0 to 1 hr); 1821 (1 to 2 hr); 1948 (2 to 3 hr); 1872 (3 to4 hr); and 1869 (4 to 5 hr). The numberof survivingcoloniesin these populations, originating from cells which had incorporated BrdUrd and therefore received the perturbation in theirDNA,shouldbetheobservednumbermultipliedbythecorresponding, corrected labeling indices and are listed as follows:1194 (0 to 1 hr); 1329 (1 to 2 hr); 1558 (2 to 3 hr); 1179 (3 to4 hr); 1179 (3 to 4 hr); and 430 (4 to 5 hr). The correctedmorphological transformation frequency (%) shown in Table 4was obtained by dividing the number of morphologically transformed colonies with the number of the surviving colonies

Table4

which had been perturbed (i.e. , the observed number of surviving colonies multiplied by the appropriate corrected labelingindex). For example, the corrected morphological transformation frequency for the 1- to 2-hr period reported in Chart 10was 24/1329 = 1.8%.

From examining the corrected morphological transformationfrequency in Table 4, the cells having their DNA perturbed atthe 1- to 2-hr period during the early S phase have twice thepropensity to become transformed morphologically comparedto cells having their DNA perturbed at the periods of 0 to 1 hr,2 to 3 hr, and 3 to 4 hr. On the other hand, the cells havingtheir DNA perturbed at the 4- to 5-hr period during the late Sphase do not have any tendency to become transformed.These findings suggest that certain region(s) in the DNA ofSyrian hamster embryo cells, as designated by their specifictemporal relationship in the S phase, are the more sensitivetargets to the perturbation by BrdUrd treatment plus near-UVirradiation for the initiation of morphological transformation.These â€˜â€˜transformation-sensitivegenes'â€m̃ay be replicatedmore preferentially at the 1- to 2-hr period and least preferentially at the 4- to 5-hr period of the S phase.

In contrast to the resultswith morphologicaltransformation,no specific period during S phase of synchronized Syrianhamster embryo cells was markedly more sensitive to treatmentwith BrdUrd plus near-UV irradiation with respect to cell survival, chromosomal aberrations, or DNA damage and repair.The degree of cytotoxicity, incidence of chromosomal aberrations, extent of DNA damage and repair, and the level of BrdUrdsubstitution showed no correspondence with the frequency ofmorphological transformation. Although this does not rule outa role for these general toxic effects of BrdUrd plus near-UVtreatment in the transformation of the cells, it does indicate thata specific portion of the genome may be important and that anonspecific action of the treatment, e.g. , cytotoxicity, is probably not sufficient to cause transformation.

Although only morphological transformation was studied inthis report, many reports (1 1, 17, 18) have suggested that invitro morphological transformation of Syrian hamster embryocells correlate with tumorigenicity. Pienta et a!. (32) recentlydemonstrated a very high positive correlation (90.8%) betweenmorphological transformation and the reported carcinogenicactivity of several chemicals. We have presented evidence thatmorphological transformation is an early event in a progressivedevelopment of neoplastic transformation of these cells inculture (6, 7). In further experiments,5 we have observed thatsynchronized mass cultures treated with a 1-hr pulse of 10@LMBrdUrd within the first 4 hr of S phase and subsequent

irradiation for 5 mm exhibit neoplastic transformation followingsuccessive passages in culture. Cells treated with BrdUrd plusnear-UV irradiation in the absence of DNA synthesis or in lateS phase were not induced to undergo neoplastic transformation. Control cultures treated with BrdUrd alone or near-UVirradiation alone in either nonreplicating or S phase cells werenot transformed to the tumorigenic state.

In conclusion, this report strongly supports our observationsthat a direct perturbation of DNA is sufficient to induce neoplastic transformation. This work demonstrates that DNA canbeonecritical target in chemical-physicalcarcinogenesis.Fur

S T. Tsutsui and P. 0. P. Ts'o. Involvement of DNA replicated during different

periods of S phase in neoplastic transformation, manuscript in preparation.

a Data from Chart 1.

b The continuous labeling index during the entire S phase is 80% (Chart 3a).

Therefore, the corrected labeling index â€”labeling index/0.8.CCorrected morphological transformation frequency (%)

No. of transformed coloniesx100

(Total no. of scored survival colonies x Corrected labeling index)(see text).

JULY1979 2363



Syrian hamster embryo cells by diverse chemical carcinogens. Nature, 235:278â€”280,1972.

19. Giacomoni, D., and Finkel, D. Time of duplication of ribosomal RNA cistronsina celllineofPotoroustridactylis(ratkangaroo).J. Mol.Biol.,70:725â€”728, 1972.

20. Hamlin, J. L., and Pardee, A. B. S phase synchrony in monolayer CHOcultures. Exp. Cell Res., 100: 265â€”275,1976.

21. Hamper,B., Tanaka, A., Nonoyama,M., and Derge,J. G. Replicationof theresident repressed Epstein-Barr virus genome during the early S phase (5-1 period) of nonproducer Raji cells. Proc. Natl. Acad. Sci. U. S. A. 71: 631â€”633, 1974.

22. Hutchinson,F. The lesionsproducedby ultravioletlight in DNA containing5-bromouracil. 0. Rev. Biophys., 6: 201-246, 1973.

23. Kasupski,G. J., and Mukherjee, B. B. Effectsof controlledexposureof Lcells to bromodeoxyundine (BudR). Exp. Cell Res., 106: 327â€”338,1977.

24. Kee, S. G., and Haber, J. E. Cell cycle-dependent induction of mutationsalong a yeast chromosome. Proc. NatI. Acad. Scm.u. S. A., 72: 1179â€”1183.1975.

25. Kikuchi,V., and Sandberg,A. A. Chromologyand patternof humanchromosome replication. 1. Blood leukocytes of normal subjects. J. NatI. CancerInst., 32: 1109â€”1143,1964.

26. Kuroki, T., and Sato, H. Partial synchronization of hamster embryonic cellsin secondary culture. Exp. Cell Res., 61: 210â€”213,1970.

27. Luk, D. C., and Bick, M. D. Determinationof 5'-bromodeoxyuridinein DNAby buoyant density. Anal. Biochem., 77: 346â€”349,1977.

28. Meuth, M., and Green, H. Inductionof a deoxycytidinelessstate in culturedmammalian cells by bromodeoxyuridine. Cell, 2: 104â€”112, 1974.

29. Mueller, G. C., and Kajiwara, K. Early-and-late-replicating deoxyribonucleicacid complexes in HeLa nuclei. Biochim. Biophys. Acts, 114: 108â€”115,1966.

30. Mukherjee, B. B., Wright, W. C., Ghosal, S. K., Burkholder, G. D., andMann, K. E. Furtherevidencefor the simultaneousinitiationof DNA replication in both x chromosomes of bovine female. Nature, 220: 714â€”716,1968.

31. Orkin, S. H., and Littlefield, J. W. Nitrosoguanidine mutagenesis in synchronized hamster cells. Exp. Cell Res., 66: 69â€”74,1971.

32. Pienta, R. J., Poiley,J. A., and Lebherz,W. B., III. Morphologicaltransformation of early passage golden Syrian hamster embryo cells derived fromcryopreserved primary cultures as a reliable in vitro bioassay for identifyingdiverse carcinogens. Int. J. Cancer, 19: 642â€”655,1977.

33. Popescu, N. C., Casto, B. C., and DiPaolo, J. A. Infrequent chromosomeaberrations in Syrian hamster cells following partial synchrony by aminoacid deprivation. J. Cell. Physiol., 86: 599â€”604,1975.

34. RaskÃ³,I., Burg, K., and Dallmann, L. Temporal sequence of mutation for 6-thioguanmneresistance in synchronized Chinese hamster cells. Theor. AppI.Genet., 48: 157â€”162,1976.

35. Roufa, D. J. 5-Bromodeoxyuridine-DNA strand symmetry and the repair ofphotolytic breaks in Chinese hamster cell chromosomes. Proc. NatI. Acad.Sci.U.S.A.,73:3905-3909,1976.

36. Stambrook, P. J. The temporal replication of ribosomal genes in synchronized Chinese hamster cells. J. Mol. Biol., 82: 303-31 3. 1974.

37. Suzuki, N., and Okada, S. Location of ala 32 gene replication in the cellcycle of cultured mammalian cells, L5178Y. Mutat. Res., 30: 111â€”116,1975.

38. Taylor, J. H. Asynchronous duplication of chromosomes in cultured cells ofChinese hamster. J. Biophys. Biochem. Cytol., 7: 455-467, 1960.

39. Taylor, J. H., Myers, T. L., and Cunningham, H. L. Programmed synthesis ofdeoxyribonucleic acid during the cell cycle. In vitro, 6: 309â€”321, 1971.

40. Thirion, J. P., Labrecque, R., and vu, T. P. Selection of Chinese hamstersomatic cell mutants after irradiation of BudR-labelled cells. J. Cell. Physiol.,87: 135-140, 1976.

4 1. Tikhomirova, L. P. Effect of incorporation of 5-bromodeoxyuridine into phagesd DNA on the lethal and mutagenic effects of uv irradiation. J. Mol. Biol.,4: 100-105, 1970.

42. Tsutsui, T., Barrett, J. C., and Tso, P. 0. P. Neoplastic transformationinduced by a direct perturbation of DNA. Proc. Am. Assoc. Cancer Res., 18:52, 1977.

43. Tsutsui, T., Barrett, J. C., and Ts'o, P. 0. P. Induction of 6-thioguanmneandouabain resistant mutations in synchronized Syrian hamster cell culturesduring differing periods of the S phase. Mutat. Res., 52: 255-264, 1978.

44. Tsutsui, T., Enaka, K., and Umeda, M. Dissociation of lipid and protein fromcell-DNA by direct alkaline sucrosegradient analysis.Jpn. J. Exp. Med..45: 215â€”222,1975.

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T. Tsutsui et al.

thermore, these studies suggest the intriguing possibility thatdiscrete regions of DNA replicated during different periods ofSphasehavevaryingdegreesofsensitivitytowardthisperturbation capable of initiating neoplastic transformation. Sincethis perturbationalso leadsto somaticmutation,it is plausiblethat somaticmutation(s)in this regionof genomeis the causeof neoplastic transformation. It should be noted that this investigation does not exclude the possibilitythat other macromolecules insidethe cell, such as RNAand proteins,can also becritical targets for other perturbations which might lead toneoplastic transformation or the possibility that neoplastictransformation can be initiated by nonmutational events.

ACKNOWLEDGMENTS

We wouldlike to thank KathleenMcDonaldfor her excellenttechnicalassistance. We are grateful to Dr. Stephen Lesko, Dr. Sarah A. Bruce, and BrianCrawford for their critical reading of the manuscript.

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16. Cress, A. E., and Gerner, E. W. Hydroxyurea treatment affects the G, phasein next generation CHO cells. Exp. Cell Res., 110: 347â€”353,1977.

17. DiPaolo, J. A., Nelson, R. L.. and Donovan, P. J. Morphological, oncogenic,and karyological characteristics of Syrian hamster embryo cells transformedin vitro by carcinogenic polycyclic hydrocarbons. Cancer Res., 31: 1118â€”1127, 1971.

18. DiPaolo,J. A., Nelson,R. L., and Donovan,P. J. In vitrotransformationof



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1979;39:2356-2365. Cancer Res Takeki Tsutsui, J. Carl Barrett and Paul O. P. Ts'o Perturbation of Synchronized Syrian Hamster Embryo CellsChromosomal Aberrations Induced by a Direct DNA Morphological Transformation, DNA Damage, and

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