the effect of [email protected] cell viability, … · contained 1 ia naci, 1 mm...

(CANCER RESEARCH 36, 3789-3797, October, 1976]

SUMMARY

Hamster fibrosarcoma cells were synchronized by mitoticselection and exposed to varying concentrations of i-fJ-Darabinofuranosylcytosmne (ama-C)for 2 hr in mid-S phase.There was a direct relationship between DNA synthesis inhibition and cytotoxicity produced by ama-Conce DNA synthesis was decreased by over 85%.The noncytotoxic concentration of 10@M ama-Cproduced little chnomatidbreakage;but extensive chromatid breakage and chromosomal rearrangement were seen in cells treated with the cytotoxicconcentration of 10@M ama-C,thus supporting earlier observations that chromatid breakage is highly correlated withcytotoxicity.

Predominantly small DNA was synthesized when cellswere treated with both 10@ and 10@ M ama-C,and this DNAcould be completely chased into high-molecular-weightDNA after addition of deoxycytidine. Both concentrations ofama-Calso inhibited,to differentdegrees,the joiningofintermediate-size DNA fragments into larger DNA; thus neither parameter appeared directly related to the ama-C-produced cytotoxicity.

INTRODUCTION

ama-C4inhibits DNA synthesis in various mammalian cells(9, 13, 15, 23). It is believed that this inhibition results fromthe conversion of the nucleoside to the tniphosphate, amaCIP, which in turn inhibits DNA replication at the level ofthe polymemase enzyme (8, 10, 18). This hypothesis is supported by studies on isolated Escherichia co!i enzymes,which show that replicative synthesis is more sensitive thanis repair synthesis(7, 24)and that DNApolymeraseI is moreresistant than polymemaseII on Ill to inhibition by ara-CIP(22). ama-Cis also highly cytotoxic to cells in the S phase ofthe cell cycle (5, 14, 26), causing chromatid damage, whichhas been highly correlated with the degree of cytotoxicity

I This work was supported by Grant CA-i 4226 from the National Cancer

Institute, NIH.2 Present address: Department of Medical Biochemistry, Medical School,

Box 63, Tygerberg 7505, Republic of South Africa.S Recipient of Career Development Award CA-70996 from the National

Cancer Institute. To whom requests for reprints should be addressed.4 The abbreviations used are: are-C, 1-@-D-arablnofuranosylcytosine; ara

CTP, i-ft-D-arabinofuranosylcytosine 5'-triphosphate; CdA, 2'-deoxycytidine; PBS. Dulbecco's phosphate-buffered saline [NaCI (8 g/liter), KCI (0.2 g/liter), Na@HPO4(1.15 g/liter), and KH,P04 (0.2 g/liter), pH 7.2].

Received December 15, 1975; accepted July 8, 1976

(12). Furthermore, ama-Ccan produce oncogenic transformation in hamster and rat cells (ii , 16) and morphologicaltransformation in mouse cells (1).

This study was undertaken in an attempt to determinewhat parameterswere relevanttoama-Ccytotoxicity,aswellas to gain some insight into the mechanisms of ama-C-produced transformation. The cell line used in this studywas the hamster fibmosancomaline, A(11)Cl-3,developed inour laboratory. This cell line was chosen for the followingreasons: it can be easily synchronized by mitotic selection(19); it has a diploid chromosomal number (4); and it growsrapidly, forming discrete colonies which can be countedelectronically (3).

The experimental design was to expose cells to ama-Cfora 2-hr period in the middle of S phase. The effects of variousconcentrations of ama-Con the rate of [3H]thymidine incorpomation and on cytotoxicity were then studied under thesedefined conditions. Preliminary results of the experimentsshowed that i0@ M ama-Cmarkedly inhibited DNA synthesisbut was not cytotoxic, in contrast to i0@ M ara-C whichinhibited DNA synthesis to an even greaten extent and washighly cytotoxic.

Further studies were then carried out at these 2 ama-Cconcentrations in an attempt to elucidate the mechanism ofama-Ccytotoxicity. The parameters chosen were: (a) therecoveries of DNA synthesis and cell mitotic ability; (b) theappearance of chromatid breaks and chromosomal rearrangements; (c) the size of DNA synthesized during thetreatment period; and (d) the effect of ama-Con DNA chainelongation.

MATERIALSAND METHODS

Culture Conditions and Synchronization Technique. Thecells used were the cloned line of ama-C-transformed hamstemfibmoblastsA(I1)Cl-3 (4). They were propagated in suspension culture in McCoy's spinner medium (Grand IslandBiologicalCo.,Grand Island,N.Y)containing10% fetalcalfserum (Flow Laboratories, Rockville, Md.) and seeded into75- or 150-sq cm flasks (Corning Glass Works, Corning, N.V.)at6or12x i0@cells/flaskthedaybeforemitoticharvesting. The medium was then changed to McCoy's medium 5Acontaining 10% fetal calf serum after the cells had attached(2 hr at 37Â°),and this medium was used in all subsequentexperiments. Synchronized cells were obtained from thesecultures, using the technique of mitotic detachment as de

OCTOBER 1976 3789

The Effect of [email protected] CellViability, DNA Synthesis, and Chromatid Breakage inSynchronized Hamster Fibrosarcoma Cells1

Peter A. Jones,2Mary S. Baker, and WilliamF. Benedict@Division of Hematology-Oncology, Department of Medicine, Childrens Hospital, Los Angeles, California 90027

on June 28, 2021. © 1976 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

p. A. Jones et a!.

scnibed by Momparler et a!. (19), and seeded into Petnidishes (Falcon Plastics, Oxnand, Calif.).

Exposure to ara-C. ama-Cand CdR hydrochloride werepurchased from Sigma Chemical Co. , St. Louis, Mo. , andwere dissolved at 100-fold final concentration in PBS andfilter sterilized immediately before use. ama-Cwas addeddirectly to the cell culture medium 5 hr after mitotic selection (i.e. , in mid-S phase). The treatment time was always 2hm, after which either the cells were scraped off 60-mmdishes with a rubber policeman or the medium was changedto fresh medium containing 10@ IACdR. In all of the expeniments reported in this paper CdR was added to all culturesat 7 hr after plating. Cell killing was determined by seeding200 mitotic cells into 60-mm dishes (4/treatment) and exposing them to ama-Cexactly as indicated above. Mediumwas changed to 10@ M CdR after the 2-hr exposure period,and the dishes were incubated at 37Â°for 5 days. On the 5thday the colonies were fixed in methanol, stained withGiemsa, and counted electronically with an automatic colony counter (New Brunswick Scientific, New Brunswick, N.J.).

Measurement of DNA Synthesis. Mitotic cells wereseeded at approximately 105/35-mm dish and treated 5 hrafter plating with varying concentrations of ama-Cfor 2 hr.The dishes were also exposed to [methy!-3H]thymidmne, 1

@Ci/ml (20 Ci/mmole; New England Nuclear, Boston,Mass.), throughout the treatment period. The radioactivemedium was then removed; the cells were washed carefullywith 1 ml of PBS and then lysed in the dish with 1 ml of asolution of 1% sodium dodecyl sulfate containing bovineserum albumin , 500 @g/ml.The lysates were treated with0.3 ml of 50% tnichlomoacetic acid and filtered throughWhatman GF/C filters that had been thoroughly washedfirst with 5% tnichloroacetic acid and then 96% ethanol,dried, and counted in scintillation fluid in a Packard InCarb liquid scintillation spectmophotometem(Packard Instrument Co. , Downers Grove, III.).

The inhibition of DNA synthesis by ama-Cwas also studiedby autonadiography. Cells treated with ama-C and[3H]thymidine, 5 pCi/mI, as above were chased for 2 hr withCdR-containing medium, scraped off the dish; and fixedwith methanol:acetic acid (3:1). Slides were made from thecell suspension, stained with acetooncein and dipped inKodak NIB-2 nuclear track emulsion. After standing for 6days in the dark, the slides were developed and scored forlabeling.

Measurement of Thymidine Phosphates. Mitotic cellswere seeded into 100-mmdishes (1 x 10@/dish)and treatedwith ama-Cplus [methy!-3H]thymidine, 2 pCi/mI, 5 hr afterplating for 2 hr. The radioactive medium was then removed,the culture were washed once with 10 ml PBS, and thedishes were placed on ice before the addition of 2 ml of icecold 0.2 N perchlonic acid. The cells were scraped off thedishes and transferred to centrifuge tubes; after standingfor 20 mm in ice, the tubes were centrifuged for 5 mm at2000 x g. The precipitatewas reextractedwith0.5ml ofpenchlonic acid and the combined supemnatants were neutralized to pH 7 with 2 N potassium hydroxide. The precipitated potassium perchlorate was removed by centnifugation, and the supemnatantwas lyophilized. This freeze-dried

material was then dissolved in 0.4 ml of 0.05 M sodiumcitrate, pH 5.0; the small amount of potassium chlorate wasremoved by centnifugation; and 0.1-mI aliquots were analyzed by high voltage paper electrophonesis on WhatmanNo. 3MM paper. The voltage gradient was 50 V/cm, and therun time was 1 hr in 0.05 M sodium citrate, pH 5.0. Thepaper was cut into 1-cm strips after drying, and these werecounted in the scintillation counter.

Studies on Chromatid Breakage and Metaphase Index.Approximately 1 x 1O@cells were cultured in 60-mm tissueculture dishes in a similar manner as described above andexposed for 2 hr to 10@ and 10@ PAama-C5 hr after plating.At various times after treatment, Colcemid (Grand IslandBiological Co.) was added for 30 mm at a final concentration of 1 @g/ml.The cells were then trypsinized, and chromosomal preparations were made (2). Chromatid breakagewas scoredas previouslydescribed(2).

One hundred metaphases were analyzed for each timeperiod for chromatid breaks on gaps. A gap was consideredto be a lesion at least the width of a chromatid, and a breakwas a gap with a different angle than the adjacent intactchromatid arm. All these lesions were reported as â€œbreaksâ€•for simplicity. Metaphases were also scored for the presence of abnormal chromosomal configurations, particularlytninadial and quadmiradial formations.

The same slides used for chromosomal analysis were alsoscored for the percentage of metaphases present. Onethousand cells were analyzed for each time period and thepercentage of mitotic cells was defined as the metaphaseindex.

Alkaline Sucrose GradIents. Cells to be analyzed on alkaline sucrose gradients were exposed simultaneously to[methy!-3H]thymidine, 5 @Ci/ml,and ama-C as indicatedabove. At the end of the 2-hr treatment time, either theywere directly analyzed on gradients onthe radioactive medium was removed and replaced with fresh medium contaming 10-s M CdR for various chase periods. In someexperiments, cultures received a 5-mm pulse with[3H]thymidine, 10 @Ci/ml,immediately before exposure toama-C.The radioactive medium was removed after 5 mm; thedishes were washed with 5 ml of medium and then treatedwith ama-C.The controls were either scraped into cold PBSon chased with freshmedium for2 hr.

Cells were harvested by scraping with a rubber policemanin 1 ml of PBS. They were then counted on a hemacytometen, the cell concentration was adjusted to 25 x 10@cells/mIand made 0.01 M with respect to EDIA, and 0.2 ml of thissolution was carefully layered over the gradients. The gmadient procedure was essentially that described by Petersonet a!. (21) with the exception that a cushion of 2.2 M sucrosewas used.A 30-mIlinear5 to25% alkalinesucrosegradientwas formed over a 4-mI cushion of 2.2 Msucrose in a 2.5- x8.6-cm cellulose nitrate tube. All of the gradient solutionscontained 1 IA NaCI, 1 mM EDTA, and 0.06 M sodium paminosalicylate at a final pH of 12.5. The gradients wereoverlaid with 0.2 ml of 1 N NaOH immediately before thecells (0.2 ml; 5 x 10@cells) were added. The cells wereallowedtolysefor30 mm atroom temperatureon topofthegradient and then centrifuged for 3 hr at 24,000 rpm in theBeckman SW-27 motorwithout the brake. Deceleration time

3790 CANCER RESEARCH VOL. 36



Synchronized cells were treated for 2 hr in mid-S phase with theindicated concentrations of ara-C and [3H]thymidine, 2 pCi/mI.Acid-soluble radioactivity in these cells was then analyzed for thymidine nucleotides by high-voltage paper electrophoresis. Resultsgiven are the averagevaluesof 2 electrophometicnuns.Incorporation

(cpm/lOecells)Treatment

[3H]TMP [3H]TDP[3H]TTPNone

660 868 16,188ara-C(10@M) 820 1224 21,360ama-C(10@M) 604 764 22,744

Effect of ara-C on Hamster Fibrosarcoma Ce!!s

was 30 mm. A 20-gauge needle was inserted to the cushioninterface, and 30 sequential fractions of 17 drops (about 1ml) were collected. Carrier DNA (200 pg/fraction) was thenadded, and the radioactivity precipitable by tnichlonoaceticacid was determined as outlined above.

The S values given are approximate and were obtainedfrom the data of Parkhunst et a!. (20). They were checkedusing 32P-labeled bacteriophage 17 DNA (37 5) which was agift from Dr. John Leavitt, Johns Hopkins University, Baltimore, Md.

RESULTS

Cell Killing and Inhibition of DNA Synthesis. The killingof synchronized hamster fibrosancoma cells by ama-Cin midS phase is shown as a function of dose in Chart 1. For the 2-hr exposure period used, no cell kill was observed until aconcentration of 3.3 x 10@ M ama-Cwas reached. Ihereaften, the cell kill obtained was concentration dependent,reaching a maximum of 75% kill at 10@ M ama-C.Chart 1also shows the inhibition of [3Hjthymidine incorporation byama-Cas a function of concentration in similarly synchmonized cells. Clearly, the cells are able to survive considerable inhibition of thymidine incorporation without death. Aconcentration of 10@ M ama-Ccaused an 88% inhibition ofthymidine incorporation, but it was ineffective in causingcell death. A concentration of 10@ M ama-C,however, killed75% of the cells and inhibited thymidine incorporation by97%. It seems, therefore, that the cells can withstand an88% inhibition of thymidine incorporation for 2 hr withoutdeath but that, above this level of inhibition, the cells die ata matethat may be correlated with the extent of inhibition.Although the difference between an 88 and a 97% inhibitionof DNAsynthesismaynot appear large, cells incorporated 3to 5 times more thymidine in the presence of 10@ M ama-Cthan with 10@ M ama-C.This large difference may thereforebe significant in terms of survival. The differences in incomponation were not due to drug-induced cell detachment,since the cell numbersfound in treated cultures were identical to those in untreated cultures (not shown).

The experiments shown in Table 1 were carried out toinvestigate whether these 2 concentrations of ama-C inhibited the conversion of the 3H-pmecursomto thymidinenucleotides. Cleanly, this was not the case, since a slightstimulation of the level of radioactivity in TIP was found.The patterns of acid-soluble radioactivity in cells treatedwith 2 concentnations of ama-Cwere very similar. Also, sincethe acid-soluble levels of radioactivity in ama-C-treated cellswere similar to those in untreated cultures, the inhibition ofthymidine incorporation observed was not due to an inhibition of precursor uptake. In other studies (not shown), itwas found that the degree of inhibition of thymidine incorporation into DNA was independent of the amount of radioactivity added over a 25-fold mangein concentration (0.2 to 5

@Ci/ml),arguing against large differences in endogenouspoolsizes.

The experiments shown in Fig. 1 and Table 2 were undertaken to confirm that inhibition of DNA synthesis occurredin all treated cells, since it might be argued that the decrease in thymidine incorporation was due to selective inhi

I00

50

5.

\

I

lo@6@5@y-4l0@3C0N@ENTRATI0NARA-C(M)

Chart 1. Cell killing and inhibition of DNA synthesis by are-C. Synchronized cells were treated in mid-S phase for 2 hr with the indicated concentrations of are-C (5 to 7 hr after plating). The effect of this treatment on[3H]thymidine incorporation into DNA per culture during the exposure periodwas measured ( 0). Cytotoxicity (â€¢)was measured by a colony-forming assayin similarly treated cultures that were subsequently exposed to i0@ M CdA.Bars,rangeof resultsfoundin3 separateexperiments.

Table 1

Theconversionof (3HJthymidineto thymidine nucleotides in ara-Ctreated cells

bition in some cells with other cells being more resistant toama-C. Autoradiognaphy showed that the majority of untreated cells were heavily labeled (Fig. 1A), while thoseexposed to 10@and 10@M ama-Cwere moderately on lightlylabeled, respectively (Fig. 1, B and C). Both concentrationsof ama-Cinhibit DNA synthesis in all treated cells in a uniform manner. Following exposure to 10@M ama-C,no heavily labeled cells were seen (Table 2). Table 2 also indicatesthe high degree of synchrony achieved with at least 90% ofthe cells being labeled during the 2-hr pulse.

The recovery of DNA synthesis and mitotic activity in cells

3791OCTOBER 1976

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Iâ€”z0100



P. A. Jones et a!.

slowly and showed little thymidine incorporation 9 hr afterplating (2 hr after removal of ama-C). Subsequently, theincorporation of thymidine increased substantially, but thecurve was broader than that found either for the control onfor 10@M ama-C-treatedcultures. Maximum mitotic activitywas delayed by 9 hr compared with the control cells and by6 hr compared with the 10@ M ama-C-treated cells.

Table 3 contains a summary of data comparing chromosomal damage in cells treated with the 2 concentrations ofama-C.No metaphases were found with 4 or more chromatidbreaks pen metaphase when the A(I,)Cl-3 cells were treatedin mid-S phase with 10@M ama-C.Also no chnomosomalabnormalities such as tniradial or quadminadial configurations were seen at this dose. In contrast, numerous metaphases were found with greater than 4 chromatid breaks penmetaphase at the cytotoxic level of 10@ M ara-C. Of particulamsignificance were the number of metaphases seen withinthe 1st mitotic period following 10@M ama-Cwhich had 5 or.more chromatid breaks together with several abnormal tnradial and quadnimadial chromosomal configurations (Fig.2). After at least 1 additional cell cycle following treatmentwith 10@M ama-C(more than 25 hr after plating the synchronized cells), there was a gradual decrease in the number ofmetaphases with greater than 4 chromatid breaks per metaphase, along with the number of abnormal tnimadial andquadnmnadialconfigurations. However, a few cells with thesechromosomal aberrations still persisted in the population.

Sizeof DNASynthesizedduringara-CTreatment.Inallexperiments we found that the sedimentation profiles obtamed for material sedimenting at less than 160 5 werereproducible from experiment to experiment. However, mesuIts obtained for the profiles of DNA sedimenting wittj an Svalue higher than 160 5 were inconsistent, presumably meflecting problems in aggregation of high-molecular-weightDNA. The results of the gradient experiments are shown inboth chart and table form for clarity.

The sedimentation pattern in alkaline sucrose gradientsof DNA labeled in mid-S phase (5 to 7 hr after plating) isshown in Chart 3a and Table 4. Much of the radioactivity(56%) is found in DNA sedimenting at greater than 160 S.Lighten material found in the upper regions of the gradient,which presumably represents replication intermediates, canbe largely chased into high-molecular-weight material(>160 5) when the [3H]thymidine-containing medium is meplaced with fresh medium (Chart 3b; Table 4).

Chart 4a and Table 4 show the size of DNA synthesized inmid-S phase in the presence of 10@ M ama-C.Although 24%of the DNAsynthesized is >160 5, a large proportion of thelabelisincorporatedintoa broadheterogeneousband witha peakat approximately 64 S. This material was chased intothe medium- and high-molecular-weight regions of the gradient after 2 hr in CdR-containing medium (Chart 4b; Table4). At this time DNA synthesis in cultures treated with 10@Mama-Chad begun to recover (Chart 2a). A further 2 hr inCdR-containing medium caused another shift in the sedimentation profile (Chart 4c; Table 4), making it similar tothat seen in untreated controls.

The DNA synthesized during exposure to 10@ M ara-Cdiffered in size distribution from that in both the control and105 M ama-C-treatedcultures (Chart 5; Table 4). Only 14% of

Table 2

Autoradiographyof cells exposedsimultaneouslyto (3H)thymidineand ara-C

Synchronized cells were treated for 2 hr in mid-S phase with theindicated doses of ama-C and [3H]thymidine, 5 pCi/mI. After 2 hr inCdR-containing medium, autoradiography was performed and thelabeling scored as heavy (Fig. 1A), moderate (Fig. 18), light (Fig.1C),or none(Fig.1C).Resultsgivenwereobtainedbyexaminationof 500 cells in each case.

% oftotalcellsHeavyModerateLightNolabellabellabellabel76143777878013729

Treatment

Noneara-C (10' M)ara-C(10@M)

0

(1)4

0 2 4 6 8 K@ 12 14 IS @20

HR AFTER PLATING

Chart 2. Effect of era-C on the recovery of [â€˜H)thymidineincorporationand mitotic activity of treated cells. Cells were synchronized and treated withno ara-C (â€¢),10@ M ara-C ( 0), or 10' M are-C (U) for 2 hr 5 to 7 hr afterplating. Medium was then changed to 10@ M CdR. a. At the indicated timesafter plating, cultures were pulsed with [â€˜H]thymidine,1 @Ci/ml,for 30 mmand the acid-insoluble radioactivity was determined . b . Cells were harvestedand chromosome preparations made. The percentage of metaphases inthese preparations was determined on a sample of 1000 nuclei.

treated with ama-Care shown in Chart 2. Cultures treatedwith 10@ M ama-Crapidly regained their DNA-synthetic ability. Significant thymidine incorporation occurred by 9 hrafter plating (2 hr after removal of ama-C),and a sharp peakof DNA synthesis was observed at 11 hr. Maximal mitoticactivity was seen 13 hr after plating. The cultures treatedwith 10@ Mama-Crecovered their DNA-synthetic ability more




Comparatiyechromosomal changes following treatment of synchronized A(T,)Cl-3noncytotoxic (1O@M) and cytotoxic (10@ M) concentrations of ara-Ccells

withMeta

phasesNo.of metaphaseswith indicated breaks/cell

â€”

Concen

with triradial or

quadmimaTime aftertration ofdial fommaplating (hr)ama-C (M) 0 1 2 3 4 5-7 8 or>tions9None

97 3 0 0 0 001110@94 5 0 1 0 0001384

11 2 3 0 000159010 0 0 0 00014.510@

32 17 14 3 5 21100153420 12 8 4 14871764

4 4 0 0 2241119424 2 2 0 22281725708 12 4 0 41350960 0 1 0 0 11

Effect of ara-C on Hamster Fibrosarcoma Cells

Table3

cause of our reservations on high-molecular-weight DNA(seeabove),we cannot attach any significance to this.

Since the results outlined above could be explained bydegradation of the low-molecular-weight DNA rather thanby its subsequent appearance in high-molecular-weight matenial, experiments were conducted to determine the conservation of the radioactivity incorporated during pulsing.Cells were pulsed with [3H]thymidmne, 1 @Ci/ml,during the2-hr ama-Cexposure period, and the acid-insoluble madioactivity was determined in duplicate samples at 0, 2, 4, and 6hr after exposure. These experiments (not shown) failed todemonstrate the loss of any [3H]thymidine in either thecontrol on treated cultures. We therefore concluded that allof the DNA made during ama-Cexposure is retained.

Effect of ara-C on Chain Elongation. Chart 6a shows thesize distribution of DNA synthesized during a 5-mm pulsewith [3H]thymidine. Most of the label was incorporated intoa broad band of material with a peak sedimentation coefficient of approximately 56 5. This material was chased intomore rapidly sedimenting DNA after 2 hr in fresh medium(Chart 6b). Chart 6c shows that 10@M ama-Ctreatment for 2hr following the 5-mm pulse strongly inhibited the extensionof these DNA chains into large DNA, although the profile didshift toward a broad peak of 80 to 112 5. ama-C, 10@ M,caused more marked inhibition of this process with thepeak moving to the 72 to 80 S position (Chart 6d). Bothconcentrations of ama-Ctherefore inhibit the conversion ofintermediate size DNA chains into high-molecular-weightcellular DNA.

DISCUSSION

The dose-response curves (Chart 1) that are obtained forcytotoxicity and DNA synthesis inhibition produced by ama-Csuggest that ama-Ckill may be related to the inhibition ofDNA synthesis within certain limits. Themeis a direct nelationship between ama-C-produced cytotoxicity and DNA synthesis inhibition at high ama-Cconcentrations, i.e. , cell killing and DNA inhibition are parallel once the cell-killingresponsebegins, although the cells can withstand a significant threshold level of inhibition without damage. This con

U)I-z:30C)

.J

30TOP

FRACTION NUMBERChart 3. Alkaline sucrose gradient sedimentation profiles of DNA synthe

sized during a 2-hr (3H]thymidmnepulse in mid-S phase in untreated A(T,)Cl-3cells. Synchronized cells were pulsed 5 to 7 hr after plating with 5[3Hjthymidine, MCi/mI, and the DNA was analyzed (a) at the end of the pulseperiod; or (b) following a 2-hr chase in CdA-containing medium (i.e. , 9 hrafter plating).

the labeled DNA was of high molecular weight (>1 60 5) withmost of the DNAsynthesizedbeing found as low-molecularweight DNA at the top of the gradient in a peak of approximately40 5 (Chart5a).Iwo hrafterexposure,the40 S peakbegan to decrease, and the profile shifted to more rapidlysedimenting material (Chart Sb; Table 4). A further 2 hr inCdR-containing medium resulted in a marked shift in thesize of the DNAthat had been synthesized, and 43%of theradioactivity was now found in the midnegion of the gra

dients (Chart 5c; Table 4). At this time, DNA synthesis intreated cultures had increased (Chart 2a). Chart 5d andTable 4 show that, 6 hr after the ama-Cis removed, most ofthe intermediate-size DNA has disappeared and the distnibution of radioactivity is very similar to that of control cultunes.Thus, the small DNAmadeduring exposure to 10@Mama-C later becomes associated with large cellular DNA.Although the profile shown in Chart 5d appears differentfrom the control (Chart 3b) or 10@ M ama-C(Chart 4c), be

3793OCTOBER 1976



of separate experiments.Time

afterChase pe No. of ex

% recovenedcpminTubes1-1011-20

(>8021-30Treatmentplating(hr)nod (hm)periments(>160 5)5,


that this would be an unlikely explanation of our results,because the large differences required would probably havecaused perturbations in the levels of radioactivity in thymidine nucleotides. Thus, if the differences in incorporation ofthyrnidine into the DNA of cells treated with 10@and 10@ Mama-Cwere due to changes in pool size, it would require a 3-to 5-fold increase in endogenous TIP. We therefore feelthat our results, taken together, argue more strongly thatthe inhibitions of radioactive incorporation into acid-insoluble material represent differing levels of DNA synthesis.

The curves shown in Chart 2 illustnate that cells treatedwith the noncytotoxic concentration of ama-Cresume DNAsynthesis and mitotic activity shortly after removal of theama-C.In contrast,lethallytreatedcellsshowed a moreheterogeneous recovery, which presumably reflects thedamage inflicted by ama-C.Since it is possible that residualintracellular ara-CTP following treatment with 1O@M ama-Cmay still significantly inhibit DNA synthesis, the increasedrecoverytime mayalso be partially due to a longer effectiveexposure to the drug. We are currently examining these 2possibilities in more detail.

The results obtained on the size of DNA synthesized during ama-Cexposure (Charts 3 to 5; Table 4) showed that themolecular weight of DNA synthesized at the cytotoxic doseof 10@ M ama-Cwas lower than that at the noncytotoxic 10@IA ama-C dose. This finding may be a reflection of our obser

vation that 10@M ama-Cis a more potent inhibitor of thejoining of preformed DNA pieces than is 10@ M ama-C(Chart 6). However, since significant inhibition of the joining of preformed DNA pieces occurs at both 10@and 10@Mama-C,this effect of ama-Con DNA synthesis does not appearto be related to cytotoxicity. Ihe inhibition by ama-Cof thejoining of preformed DNA pieces is especially interesting inview of the observation of Leeet a!. (17) that 3 x 10@IAamaC has no effect on the rejoining of radiation-induced singlestrand breaks in L1210 cells. Repair of DNA breaks themefore probably progresssesby a different pathway than joining of newly synthesized single-stnand pieces. The increased time required to chase the small DNA pieces madein the presence of 1O@IA ama-Cinto larger DNA may, however, still be significant.

Studies on chromosomal abnormalities following ama-Ctreatment (Table 2) showed that the noncytotoxic dose of10_s IA produced no metaphases with more than 3 chromatid breaks, although DNA synthesis was inhibited by 86%.Following the highly cytotoxic dose of 10@ IA ama-C,however, 50%of the metaphasesexamined were found to havegreatenthan 4 breaks per metaphase19 hr after plating ofthe cells. Numerous tniradial and quadmimadialconfigurations were also found. Previous work in our laboratory withDon-C cells has shown 4 on more chromatid breaks permetaphase to be highly correlated with cytotoxicity (12). Inaddition, a relationship has been found between transfommation in hamstercells and the production of chromosomalimbalances (4, 25). The chromosomal abnormalities observed in the present study in cells treated with 10@IAama-Cmay thus be involved both in the cytotoxicity (chmomatidbreakage) and the transformation produced by ama-C(chromosomal rearrangement). Thus, we believe that knowledgeregarding the molecular events responsible for ama-C-pro

z:300

-J4Iâ€”0Iâ€”

0 10 20 30BOTTOM TOP

FRACTIONNUMBERChart 6. Inhibition of DNA chain joining by are-C. Five hr after seeding,

synchronized A(T,)CI-3 cells were pulsed for 5 mm with (3H]thymidine, 10@Ci/ml,immediately before treatment. The DNA was analyzed on alkaline

sucrose gradients. a, profile of DNA synthesized during the pulse periodwithout a chase; b , as in a but following a 2-hr chase in fresh medium ;C, as inb, but chase medium contained 1Ã˜-aPAare-C; d, as in b but chase mediumcontained 1O@M ara-C.

duced chromosomal breaks and rearrangements is criticalto our understanding of the mechanism(s) of ama-C-produced cytotoxicity and transformation , respectively.

3795OCTOBER1976

REFERENCES

1. Benedict, W. F. , Baker, IA. S. , and Gardner, A. Malignant Transformationwith ara-C, FUdA, MTX and Bleomycin in 1OT'/@CL8Cells. Proc. Am.Assoc. Cancer Aes., 16: 40, 1975.

2. Benedict, W. F., Harris, N., and Karon, IA. Kineticsof i-@-D-Arabinofuranosylcytosine-induced Chromosome Breaks. Cancer Aes. , 30: 2477-2483, 1970.

3. Benedict, W. F., Aucker, N., and Khwaja, T. A New Hamster Fibrosarcoma Model for in Vitro/in Vivo Evaluation of Cancer ChemotherapeuticAgents. Med. Pediat. Oncol., 2: 271-278, 1976.

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Fig. 1. Autoradiography of cells exposed simultaneously to [3H]thymidine and ara-C. Synchronized A(T,)Cl-3 cells were treated for 2 hr in S phase (5 to 7 hrafter plating) with [3H]thymidine, 5 @Ci/ml,and iO@ or 10' PAare-C. Slides were prepared following a 2-hr chase in CdA-containing medium, dipped inemulsion, and developed after 6 days. A, untreated cells heavily labeled; B, cells exposed to 1O@M ara-C, moderately labeled; C, cells exposed to 1O@M areC, lightly labeled with 2 unlabeled cells visible.

Fig. 2. A, metaphase after 2 hr treatment with 1O@M are-C showing numerous chromatid breaks and rearrangements; arrows, chromatid breaks. B,metaphase after ara-C treatment showing triradial configuration (arrow). C, metaphase after treatment with iO@ M ara-C showing extensive chromatid breaksand fragments.





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1976;36:3789-3797. Cancer Res Peter A. Jones, Mary S. Baker and William F. Benedict Hamster Fibrosarcoma CellsDNA Synthesis, and Chromatid Breakage in Synchronized

-d-Arabinofuranosylcytosine on Cell Viability,βThe Effect of 1-

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