[CANCER RESEARCH 36, 101-107, January 1976]

ence inhibition of division, has a very low incidence ofspontaneous transformation (29), and can be transformedby numerous types of compounds including polycyclic hydrocanbons (28), alkylating agents (6), X-nays (31), cigarettesmoke condensates (4), and some cancer-chemothenapeutic drugs (3, 19).

In this report we show that the C3H/iOT1/2 cells can betransformed by both FUdA and FU and that the transformation is dependent on the dose and time of exposure. Furthermore, since FUdR is a cell cycle-specific agent (13), it mightbe anticipated that the transforming event would also becell cycle dependent. Indeed, we report that the maximumsensitivity to FUdR transformation is in early S phase, unlikethe late G, sensitivity for MNNG transformation (6).

The transformation induced by FUdR isespecially interesting because the drug is not incorporated into DNA (7, 12,13) and is not mutagenic to mammalian cells (18). Wewished, therefore, to determine whether pynimidine nucleoside analogs that are incorporated into DNA (2, 9, 10) andare mutagenic (9, 18) also produce transformation of thesecells. These compounds included BUdR, IUdR, and F3TdR,a clinically effective antiviral drug (32) developed in one ofour laboratories (12—14).These latter compounds werefound to be inactive as transforming agents in theC3H/iOT'/2 cells.

MATERIALS AND METHODS

Chemicals. FUdR and BUdR were obtained from SigmaChemical Co., St. Louis, Mo.; IUdR was from Calbiochem,Los Angeles, Calif. ; FU was from Hoffmann-La Roche, Inc.,Nutley, N. J.; and F3TdRwas from Burroughs Wellcome andCo. , Research Triangle Park, N. C. The drugs were dissolved in calcium- and magnesium-free PBS at 100 timesthe final concentration tested, sterilized by filtration, andthen added to the medium. [methy/-3H]Thymidmne (>20mCi/mmole), [6-3H]FU, and [6-3H]FUdR were purchasedfrom New England Nuclear, Boston, Mass.

Cells and Cufture Conditions. The cell line used was theC3H/iOT1/2 CL8 line between the 12th and 18th passage.The cells were handled as originally described (29) and weregrown in Eagle's basal medium containing 10% heat-mactivated fetal calf serum and penicillin and streptomycin. Seraand medium were obtained from Grand Island BiologicalCo., Grand Island, N. Y. Stock cultures were propagated in75-sq cm plastic flasks (Falcon Plastics, Oxnard, Calif.), andtransformation assays were carried out in 60-mm plastic

SUMMARY

Oncogenic transformation has been induced in vitro inthe C3H/iOT1/2clone 8 line of mouse cells by exposure to 5-fluoro-2'-deoxyuridine (FUdR) or 5-fluorouracil. This tmansformation is both dose and time dependent and can bemarkedly decreased by simultaneous exposure of the cellsto thymidine. The transformation induced by 5-fluonounacilis probably due to its intracellular conversion to FUdR or itsmonophosphate. Transformation by FUdR was found to becell cycle dependent with maximum sensitivity to transfommation occurring in early S phase. Cell lines that producedsarcomas in antithymocyte-treated syngeneic mice were isolated from FUdR-tmansformed cultures. Tnifluorothymidine,5-bromo-2'-deoxyunidine , and 5-iodo-2'-deoxyunidine induced no transformed foci in the C3H/iOT1/2 clone 8 cellline. Thus, not all mutagens produce oncogenic transformation nor does the lack of mutagenicity, as classically measured, completely exclude the possibility that a given agentis oncogenic. Also, there was no evidence of the “switchon' â€of oncomnaviral information in the FUdR-transformedcell lines.

INTRODUCTION

It has been reported by one of us (20) that hamster fetalcells are transformed by treatment with FUdR,―an antineoplastic agent that was developed in one of our laboratories(12, 13). Thattransformation, however, was difficult to quantitate in mass cultures. Hence, it seemed desirable to engage in a joint collaboration for a quantitative study of theabilities of FUdR, FU, and related nucleosides to producemalignant transformation of the C3H/iOT1/2 CL8 cell line,which was also developed in one of our laboratories (28,29). This cell line was chosen because, like our earliermouse prostate cells (8), it is highly sensitive to postconflu

I This work was supported in part by Grants CA-14226 and CA-071 75 and

Contract 72-2022 from the National Cancer Institute, NIH.2 Present address: Department of Medical Biochemistry, Box 63, Tyger

berg 7505, Republic of South Africa.3 Recipient of Career Development Award CA-70996 from the NIH. To

whom requests for reprints should be sent.4 American Cancer Society Professor of Oncology.

a The abbreviations used are: FUdR, 5-fluoro-2'-deoxyuridine; FU, 5-fluerouracil; CL8, clone 8; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; BUdR,5-bromo-2'-deoxyuridine; lUdR, 5-iodo-2'-deoxyuridine; F3TdR, trifluorothymidine; PBS, phosphate-buffered saline [NaCl (8 g/Iiter), KCI (0.2 g/Iiter),Na,HPO4 (1.15 glliter), and KHPO4 (0.2 g/liter), pH 7.2]); MuLV, murineleukemia virus.

ReceivedJuly 21, 1975;acceptedSeptember12,1975.

JANUARY1976 101

Oncogenic Transformation of C3H/10T1/2Clone 8 Mouse EmbryoCells by Halogenated Pyrimidine Nucleosides1

Peter A. Jones,2 William F. Benedict,3 Mary S. Baker, Sukdeb Mondal, UIf Rapp, and Charles HeideIberger@

Divisionof Hematology-Oncology,Departmentof Medicine,ChildrensHospital,LosAngeles,California90027[P. A. J., W.F. B., M. S. B.], and McArdleLaboratoryfor CancerResearch,Universityof Wisconsin,Madison,Wisconsin53708[S. M., U. R., C. H.]

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

P. A. Jones et a!.

dishes obtained from Falcon or Lux Scientific Corp. (Thousand Oaks, Calif.). Cultures were grown in humidified incubators at 37°in an atmosphere of 5% CO2 in aim.

Standard Transformation Assay. The experimental conditions for this assay have been previously described (28).Briefly, 1000 cells obtained from stock cultures in log phasewere seeded into 60-mm dishes. Twenty-four hr after seeding, 12 dishes were treated at each dose for an additional 24hr. The medium was then removed, the dishes were washedonce with 5 ml of PBS, and fresh medium was added.Medium was then changed twice weekly for 4 to 6 weeks,after which the cultures were fixed with methanol andstained with Giemsa. The stained dishes were then examned for dense colonies of crisscrossed cells (type Ill foci)

(28). Cytotoxicity was determined by the reduction in plating efficiency in similarly treated dishes seeded with 200cells. Three dishes/dose were used and the colonies werestained and counted after 9 to ii days. Cytotoxicity wascalculated by comparing the plating efficiency of the treatedcultures with that of untreated controls. The transformationfrequency is defined as the percentage of surviving cellsgiving rise to type III foci.

Cell Cycle Studies. Cells were synchronized either byrelease from postconfluence inhibition of division or byrelease from isoleucine deprivation as previously described(6). Using the former method, several 75-sq cm flasks weregrown to confluence and allowed to stand for 48 hr withoutmedium change. The monolayers were then trypsinized andiO@ cells seeded per 60-mm dish. At various times afterplating, the cells were treated with 1O@ M FUdR (12dishes/group) for 2 hr, after which the medium was memoved, the cells were washed with 5 ml PBS, and freshmedium was added. In order to treat the cultures throughout a 36-hr period, replicate 75-sq cm flasks were trypsinized 12 hr apart, and studies were performed simultaneously on the 2 groups. Dose and time responses to FUdRwere also determined in cells synchronized by this method.Cells synchronized as outlined above received variousdoses of FUdR for either 2- or 4-hr exposure, 24 hr after thecells had been seeded from confluent monolayers.

Cells synchronized by release from isoleucine deprivationwere derived from confluent monolayers and plated in medium containing 10% freshly dialyzed serum and no isoleucine (6). Forty-eight hr after seeding, the medium waschanged to whole medium, and the cultures (12dishes/group) were treated for 2 hr with i0@ M FUdR atvarious times throughout the cell cycle. The medium wasremoved after the drug exposure, the cultures were washedwith 5-mI PBS, and fresh medium was added. Medium wassubsequently changed twice weekly for 4 to 6 weeks toscore for transformation.

These 2 methods used to synchronize the C3H/iOT1/2cellshave previously been shown to yield a high degree of synchrony (6).

Measurement of DNA Synthesis and Cytotoxicity. DNAsynthesis was determined as previously described (6). Cytotoxicity was estimated by an indirect method in which thecells were trypsinized immediately after exposure to thechemical and then plated as follows.

The medium was removed from dishes that had just under

gone treatment, and the cells were washed rapidly withtrypsin. The culture was then trypsinized with 1 ml of enzyme and, after the cells had detached, 1 ml of medium wasadded. Aliquots (50 @l)of the 2-mI cell suspension werethen pipetted into fresh 60-mm dishes containing 5 ml ofmedium (3/treatment) and the cells allowed to grow ascolonies for 9 to ii days. One ml of the remaining cellsuspension was counted on a Coulter counter to estimatethe number of cells seeded. From a knowledge of the plating efficiency it was possible to estimate cytotoxicity.

Tumorigenicity Studies. Several type Ill foci that arose inFUdR-tneated cultures were isolated by the ring isolationtechnique and subsequently necloned. These cells werethen injected s.c. into litters of 4- to 6-day-old C3H miceobtained from the Drug Development Branch, National Cancer Institute, Bethesda, Md. The mice were immunosuppressed with rabbit anti-mouse thymocyte serum obtainedfrom Microbiological Associates, Bethesda, Md. , as previously described (4). Each animal received 2.5 x i0@cellsand was examined weekly for the appearance of tumors.

Metabolism of [6-3H]FUand [6-3H]FUdR. C3H/i 0T1/2cellswere plated at iO@cells/roller bottle. Twenty-four hr later thebottles were treated with the labeled compounds (i0@ M;0.001 MCi/mI) for 24 hr, and the cells were harvested byscraping to yield 2.08 x i0@cells that had been treated withFU and FUdR, respectively. The cells were lysed with distilled water and counted. The extract was then passedthrough a DEAE-cellulose column (1 x 5 cm). FU and FUdRpassed through with the effluent, and the monophosphateswere eluted with 2 M pynidineacetic acid. FU (RF0.2) wasseparated by thin-layer chromatography on silica with chlonofonm:methanol (1:1).

Determination on Oncornaviral Antigens and InfectiousVirus. The attempt to detect the gs-1 structural proteinantigen was carried out by the indirect immunofluorescence test (25). The search for the production of low levelsof infectious MuLV vinions was done by cocultivation of 3FUdR-tnansfonmed C3H/iOT1/2clones with the ecotropic virus indicator cell line Ill 6A (ii), or a mink xenotmopic virusindicator cell line (15) for 9 passages using similar techniques as described in these papers (ii , 15). Culture fluidsfrom these cells were also concentrated 500-fold and testedfor reverse transcniptase activity (1).

RESULTS

Transformation of Asynchronous Cells. The transformation of asynchronous C3H/iOT1/2 cells, which were treatedfor 24 hr with varying doses of FUdR 24 hr after seeding, isshown in Table 1. In these experiments we have only scoredtype III foci as transformed, although type II foci were alsoseen. Type Ill foci are characterized by spindle cells whichgrow with a piled crisscrossed morphology and, in general,have a higher tumonigenicity than type II foci (28). Transformation was induced by FUdR over a wide concentrationrange (iO@ to i0@ M) and was dose dependent. The hightransformation frequency observed with i0@ M FUdR wasof the same order of magnitude as that induced by polycyclic aromatic hydrocarbons in the C3H/iOT1/2 cells (28).

102 CANCERRESEARCHVOL. 36

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Nucleosides added (M)No.

of expen

mentsAv.platingefficiency (%)Total

type Illfoci/totaldishesTransforma

tion frequency―(%)Control

(none)5240/510FUdR(10@)31.2513/303.5FUdR(10@) + TdR(10@)110.71/70.13FUdR

(10@)57.618/430.55FUdR(10―)+ TdR(10@)116.50/60FUdR

(10-a)215.15/190.17FUdA(10@)113.53/110.20TdR(10@)1100/80TdR(104)119.50/90

Nucleosideadded (M)No.of ex

perimentsAv.plating ef

ficiency (%)Total

type IIIfoci/totaldishesTransforma

tion frequency―(%)FU

(10@)31 .255/221.82FU(10@)+TdR(10@)130/90FU

(10―)4153/280.07FU(10―)+TdR(10@)1190/100FU

(10-a)117.50/120FU(10@)119.50/110

Pyrimidine Nucleoside Oncogenic Transformation

Table 1 also shows that both the cytotoxicity and transformation produced by iO@ and i0―M FUdR were markedlydecreased by simultaneous treatment with a 10-fold excessof thymidine. Thymidine itself was somewhat cytotoxic tothe cells but did not induce transformation.

The transformation of asynchronous cells by varyingdoses of FU is shown in Table 2. Transformation was notobserved at iO@ or iO@ M FU, although at these concentrations FUdA induced transformation; the transformation frequencies produced by both iO@ and i0@ M FU were significantly less than those seen with the same levels of FUdA(Table 1). As with FUdR, simultaneous treatment of the cellswith a 10-fold excess of thymidine abolished transformationby FU, although the decrease in cytotoxicity was less dmamatic than that seen with FUdA.

It was of interest to determine whether there was appneciable interconversion of FU and FUdR in these C3H/iOT1/2cells. Accordingly, logarithmically growing cells weretreated with iO_6 M [6-3H]FU or with [6-3H]FUdR (0.001 j.@Ci/ml). One day'Iaten the cells were harvested and processed(see “Materialsand Methods―).As shown in Table 3, FUdR

was converted to FU to a small extent and was extensivelyconverted into the biologically active monophosphates. FUwas also converted to FUdR and to monophosphates. Thisexperiment suggests that the transformation produced byFU, which required higher concentrations than that produced by FUdR, probably resulted from conversion of FUinto FUdA. Similar conversions have been repeatedly demonstrated in other cells and in vivo (12, 13).

Although transformation of cultured cells by polycyclichydrocarbons has been cleanly demonstrated to be a directtransformation and not a selection of preexisting transformed cells (23, 28), the slight possibility existed that thetransformation produced by FUdR resulted from a selectiveprocess. However, as shown in Table 4, FUdR was notsignificantly more cytotoxic to the nontransfonmedC3H/1OT1/2cells than to 2 clones transformed by FUdA.

Cell Cycle Studies. Chart 1 shows the incorporation of[3H]thymidine by C3H/iOT1/2 cells at various times after 10@ceiis/60-mm dish were plated from cultures in a state ofpostconfluence inhibition of growth. No thymidine was incomporated during the 1st 16 hr after plating. Subsequently,

Table 1

Transformation of C3H/10T1/2CL8 cells by FUdRCellsobtainedfrom log-phasemassculture were seededinto 60-mmdishes (1000/dish)

and treated 24 hr later with the indicated doses of FUdR, TdR,a or mixtures of thenucleosides. Exposure was for 24 hr, after which the cultures were washed and maintamed for 4 to 6 weeks. Type Ill foci were then scored in stained cultures. Cell killing wasdetermined in similarly treated cultures containing 200 cells and stained after9 to 11days.

a TdR, thymidine.

b Percentage of transformed type III foci based on the number of cells surviving.

Table 2

Transformation of C3H/10T1/2CL8 cells by FUCells obtained from log phase mass cultures were seeded into 60-mm dishes

(1000/dish) and treated 24 hr later with the indicated doses of FU or mixtures of FU andTdR,a Exposure was for 24 hr, after which the cultures were washed and transformationand cytotoxicity scored as in Table 1.

a TdR, thymidine.

b Percentage of transformed type III foci based on the number of cells surviving.

103JANUARY1976

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

MonoCompoundaddedAmount

mecovered(pmoles)FU (pmoles)FUdR(pmoles)phos

phates(pmoles)FUdR16.41

.58.76.1FU13.02.46.14.5

8 6 24 32 400

ConcentrationCytotoxicity

(% survivors ctocontrols)omparedC3H/1

0T1/2131P2WofFUdR(M)(PE° 24%)(PE 19%)(PE =14%)10-a63794410-―3222710@513

-4 0 4 8 2@

106 iO-@ iO-@

P. A. Jones et a!.

Table3 trations of FUdR for 2 or 4 hr. Under these conditions, noMetabolismof [6-3H]FUand [6-3HIFUdRby C3H/10T1/2cells transformation was seen at 10@ M FUdR, but it was clearly

Logarithmically growing cells were exposed to [6-@H]FU or [6- dose dependent as the dose was increased to i0@ M. In3H]FUdR(0.001@sCi/ml)for 24 hr. Thecells werethen harvestedand creasing the exposure time to 4 hr also increased the transFU,FUdR,and monophosphatesweredetermined in cell lysatesas formation rate. It was again evident that there is no directdescribed in the text.__________________________________________________ correlation between cell killing and cell transformation,

Table4Cytotoxicity of FUdR for C3H/lOr/2 cells and 2 FUdR-transformed

clonesCells derived from log-phase cultures of C3H/10T1/2cells or 2

derivativestransformed by FUdR (13Hand 20J) were seeded into60-nim dishes (200/dish) and treated 24 hr later with the indicateddoses of FUdR.Exposuretime was 24 hr, after which the cuftureswere washedand kept for 11 daysbeforebeingstainedand thenumber of surviving colonies counted.

400

0

Hr After Plating

Chart 1 . Cell cycle dependency of transformation and cytotoxicity in@3H/1OT'/2cells treated with 10@M FUdR. Cultures were treated with FUdR

for 2 hr at various times after release from postconfluence inhibition of celldivision. Transformation frequency (@) and cytotoxicity (is) were scored, andthe incorporation of tritiated thymidine into acid-insoluble material in untreated controls was determined (•).

4000

L&@3000@

02000@

a PE, plating efficiency.b FUdR-transformed clones that were tumorigenic.

as the cells entered S phase, the incorporation of thymidineincreased and reached a maximum 30 hr after seeding.When cultures were exposed to i0@ M FUdR for 2 hr atvarious times during this period, the cytotoxicity and transformation results shown in Chart 1 were observed. It can beseen that there was little cytotoxicity, as defined by inhibition of colony formation, until the cells entered S phase.The cell-killing effect of FUdR then increased to a maximumof 40% cell kill in mid S phase and subsequently declined.No transformation was seen during the 1st 12 hr after plating (G, phase) but was evident as the cells entered S phase.Maximum transformation was found in early S phase andreached a minimum in mid S phase when cytotoxicity wasgreatest. Thus, there is no direct correlation between cellkilling and cell transformation by FUdR. The results shownin Chart 1 were reproduced in a separate experiment, although the transformation frequency obtained was lower.

Chart 2 shows that a similar result was obtained whencells were synchronized by isoleucine deprivation. Cytotoxicity produced by FUdR was maximal at the peak of DNAsynthesis. Transformation, however, was again highest invery early S phase.

The experiments shown in Chart 3 were conducted todetermine whether the transformation induced in synchronized cells was dependent on the amount of FUdR used andthe exposure time. Cells derived from confluent monolayerswere treated 24 hr after seeding with the indicated concen

I000

0

Hr After R&ease

Chart 2. Cell cycle dependency of transformation and cytotoxicity inC3H/1OT'/2 cells treated with 10@ PAFUdR for 2 hr at various times prior to,during, and after release from cell cycle block induced by 48 hr of isoleucinedeprivation . Transformation frequency (@) and cytotoxicity (@)were measured, as well as the incorporation of tritiated thymidine into acid-insolublematerial in untreated controls (•).

60@

40

IDose FudR (M)

Phart 3. Dose@ tim..e d@p@r@@nçyof transformationand cytotoxicityinduced by FUdR. Cultures of C3H/1OT'/2cells derived from confluent monolayers were exposed to the indicated doses of FUdR 24 hr after seeding.Cytotoxicity (A, @)and transformation frequency (I, 0) were determined for2- and 4-hr exposures, respectively.Points, combined averaged values of 3separate experiments.

104 CANCER RESEARCH VOL. 36

200S

as 800@@

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Nucleoside added (M)Av.plating effi

ciency (9k)Totaltype Ill

foci/totaldishesAcetone

controlsBUdR

10-i10-s10-a10@20.0

12.514.521 .022.00/18

0/100/80/7

0/9lUdA

10@10-s10-a3.0

16.019.00/8

0/120/11F3TdR

10@10-s10-a10-i8.0

17.519.020.00/7

0/90/70/7

LatencyorNo.of cellsNo. of tuobservationinjectedmoms/no.

ofperiodCellline(xlOa)animals(days)C3H/1

0T1/22 .02.50/3 0/3911806F2.00/517513H2.53/4702W2.54/6492912.50/2180F32.52/245

Pyrimidine Nucleoside Oncogenic Transformation

Table 5

Attempted transformation of C3H/10T1/2 CL8 cells by BUdR, lUdR,and F3TdR

Cells derived from log-phase cultures were plated in 60-mmdishes (1000/dish) and treated 24 hr later with the indicated dosesof nucleosides. Exposure was for 24 hr, after which transformationand cytotoxicity were scored as in Table 1.

since the transformation rate increased more rapidly thanthe cytotoxicity. Furthermore, no difference in cytotoxicitywas seen between the 2 exposure times, although the 4-hrexposure was more efficient in inducing transformation.

Search for Expression of Oncornaviruses and Attemptsto Transform Cells with IUdR, BUdR and F3TdR. We havepreviously demonstrated in one of our laboratories (27) thatno infectious oncomnavinuses on viral cones are “switchedon―when C3H/iOT1/2 cells undergo malignant transfonmation by polycyclic hydrocarbons. We have similarly exammed 3 type Ill clones produced by FUdR for MuLV expression. By indirect immunofluorescence analysis, none of 2 x10@cells were positive for the MuLV P30 structural protein@5-1 antigen). Similarly, the search for the production of

low on transient levels of infectious MuLV by cocultivationwith ecotropic indicator cells (III 6A cells) on with minkxenotropic indicator cells was entirely negative. In addition,no reverse transcniptase activity was detected in 500-foldconcentrated culture fluids from 10@ecotnopic and xenotropic cocultivations.

In previous work, however, we did observe a transientswitch on of infectious MuLV following treatment by IUdRunder conditions where no oncogenic transformation wasdetected (27). This prompted us to determine more carefullywhether the base analogs, lUdR, BUdR, and F3TdR, whichare incorporated into DNA and are mutagenic in varioussystems (2, 9, 10, 18), produced oncagenic transformationof C3H/10T1/2cells. As shown in Table 5, no such tnansformation could be found.

Tumorigenicity. The tumonigenicities of 5 cell lines denived from type Ill foci are shown in Table 6. The parental

@3H/i0T1/2 line was not tumonigenic, as has been found in

several laboratories (4, 28, 31). Three of the FUdR-transformed lines gave rise to tumors in syngeneic mice. Approximately 60% of the type III foci were therefore tumonigenic, avalue that is slightly lower than that found for hydrocarboninduced transformation (28).

DISCUSSION

Our results demonstrate that the C3H/iOT1/2line of mousecells can reproducibly undergo malignant transformation byFUdR and, at higher concentrations, by FU. This effect isdependent on both the concentration and time of exposure.Our results also suggest that FUdR- and FU-induced transformation is related to thymidine metabolism, since simultaneous exposure oftreatedculturestothymidinecaused amarked decrease in the transformation frequency. There islittle indication from the literature that FU and FUdR arecarcinogenic in vivo. Poinienet a!. (26) found that FUdR, aswell as IUdR and BUdR, did not significantly increase the incidence of lung adenomas in strain A mice, or when administered repeatedly to newborn Swiss mice (L. A. Poinien, J.A. Miller, and E. C. Miller, private communication). Onthe other hand, Llombart and Marcos (21) reported without data that FU is transplacentally carcinogenic in therat. There is no explanation for the apparent discrepancybetween the in vivo and in vitro results.

It appears that the critical cellular events involved in the

Table 6Tumorigenicities of cell lines in immunosuppressed C3H miceThe indicated numbers of cells derived from the parent

C3H/10T1/2 line or 5 FUdR-transformed derivatives were injectedinto immunosuppressed― C3H mice. Animals were examinedweekly for the appearance of tumors.

a All animals were immunosuppressed with rabbit anti-mouse

thymocyte serum as indicated in the text with the exception ofthose mice given injections of F3. These latter animals were 6weeksold and receiveda single dose of 500A as describedearlier(28).

FUdR in vitro transformation process occur during the earlyS phase of the cell cycle. In cultures synchronized either byrelease from postconfluence inhibition of growth on by isoleucine deprivation, FUdR produced no transformation inthe G1 phase and a minimum transformation rate in themiddle of the DNA-synthetic phase when cytotoxicity wasgreatest. FUdR is S-phase specific in its cancer-chemotherapeutic activity (13). This S-phase specificity contrasts withthat observed for MNNG, where the sensitive phase fortransformation of C3H/iOT'/2 cells takes place 4 hr prior tothe onset of DNA synthesis (6). It is similar, but not identical,to the cell cycle dependency of transformation induced bycytoSine arabinoside (3).

JANUARY1976 105

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

p.A.Joneseta!.In all in vitro systems designed to detect malignant trans

formation, the question of selection of preexisting malignant cells versus their induction must be carefully considered . We believe that the evidence that we have obtainedstrongly suggests that FUdR directly induces a malignantchange in the C3H/iOT1/2 cells for the following reasons:(a) these cells have a remarkably low rate of spontaneoustransformation (29); (b) there was no significant differencebetween the cytotoxicity produced by FUdR in the nontransformed clone and in 2 FUdR transformed clones, thusexcluding the possibility that the transformed cells have aselective advantage; and (C) no correlation was found between cytotoxicity and transformation, i.e., significanttransformation was observed under conditions of little cytotoxicity.

In C3H/1OTV2 cells, FU was less active than FUdR inproducing transformation. This finding, as well as the dataon the metabolism of [6-3H]FU and [6-3H]FUdR, suggeststhat FU produced transformation as a consequence of itsconversion into FUdR or its monophosphate.

The mechanism by which FUdR induces malignant tnansformation of C3H/iOT1/2 cells is of interest. It has beendemonstrated that transformation of these cells by polycyclic hydrocarbons and MNNG does not involve the switchon of C-type RNA tumor viruses on their cones (27). Similamly,we have shown that FUdR-transformed clones do notexpress infectious oncomnavinuses, their gs antigens, orreverse tnanscniptase. Thus, the genetic information for virus production that is present in these cells (27) is notovertly expressed as a function of, on because of chemicaltransformation. Transformation by FUdR would thereforeappear to be direct. Moreover, with the exception of bacteriophage PBS2, which contains uracil instead of thymine inits DNA (22), FUdR is not incorporated into DNA (12, 13).Such incorporation into DNA might have provided a mechanism of transforming and mutagenic activity for FUdR. However, BUdA, IUdR, and F3TdR, which are incorporated intoDNA and are mutagenic, do not transform C3H/1OTV2cells.Thus, not all mutagens produce oncogenic transformation,nor does the lack of mutagenicity as measured by classicalmethods (18) completely eliminate the possibility that agiven agent is potentially oncogenic. The lack of transforming activity by IUdR is also of particular interest, since itdoes switch on endogenous oncomnaviruses in these cells(27).

FUdR inhibits DNA synthesis as a result of inhibition ofthymidylate synthetase by its 5'-monophosphate (12, 13).However, it seems unlikely that inhibition of DNA synthesisper se could produce oncogenic transformation. It seemsmore likely that the transformation might result from thechromosomal aberrations that FUdR is known to produce(17, 30). This would be in line with recent evidence thatshows that chromosomal balance may be important in malignant expression (5, 16). Nomura et a!. (24) have alsofound that FUdR induces flat nevertants of munine sarcomavirus-transformed 3T3 cells that, in turn, have differentchromosome numbers than the starting cell population.

ACKNOWLEDGMENTSThe authors thank Anne Johnston Gardner for her technical help.

106 CANCERRESEARCHVOL. 36

REFERENCES1. Baltimore, D., and Smoler, D. Primer Requirements and Template Speci

ficity of the DNA Polymerase of RNA Tumor Viruses. Proc. NatI. Acad.Sci. U. S., 68: 1507—1511,1971.

2. Baughnet-Mahieu, L., and Goutier, A. Mechanisms Responsible for theLow Incorporation into DNA of the Thymidine Analogue, 5-lodo-2'-deoxyuridine. Biochem. Pharmacol., 17: 1017—1023,1968.

3. Benedict, W. F., Baker, M. S. , and Gardner, A. Malignant Transformationwith ara-C, FUdR, MTX and Bleomycin in 1OT'/2CL8 Cells. Proc. Am.Assoc.CancerRes.,16:40,1975.

4. Benedict, W. F., Rucker, N., Faust, J., and Kouri, R. E. MalignantTransformation of Mouse Cells by Cigarette Smoke Condensate. CancerRes., 35: 857—860,1975.

5. Benedict, W. F., Rucker, N., Mark, C., and Kouri, A. E. CorrelationBetweenthe Balanceof SpecificChromosomesandExpressionof Malignancy in Hamster Cells. J. NatI. Cancer Inst., 54: 157—162,1975.

6. Bertram, J. S., and Heidelberger, C. Cell Cycle Dependency of Oncegenic Transformation Induced by N-Methyl-N'-nitro-N-nitrosoguanidinein Culture. Cancer Res., 34: 526—537,1974.

7. Brockman, R. W. , and Anderson, E. P. Biochemistry of Cancer (Metabolic Aspects). Ann. Rev. Biochem. 32: 463—512,1963.

8. Chen, T. T. , and Heidelberger, C. Quantitative Studies on the MalignantTransformation of Mouse Prostate Cells by Carcinogenic Hydrocarbonsin Vitro. Intern. J. Cancer, 4: 166—178,1969.

9. Gottschling, H., and Heidelberger, C. Fluorinated Pyrimidines. xlx.Some Biological Effects of 5-Trifluoromethyluracil and 5-Trifluoromethyl-2'-deoxyuridine on Escherichia coli and Bacteriophage, T4B. J.Mol. Biol. 7: 541—560,1963.

10. Goz, B., and Prusoff, W. H. The Ability of Phage Containing 5-Iodo-2'-deoxyuridine-substituted Deoxyribonucleic Acid to Induce Enzymes. J.Biol. Chem. 243: 4750—4756,1968.

11. Hartley, J. W., and Rowe, W. P. CIonal Cell Lines from a Feral MouseEmbryo Which Lack Host-Range Restrictions for Murine Leukemia Viruses. Virology, 65: 128—134,1975.

12. Heidelberger, C. Fluorinated Pyrimidines. Progr. NucI. Acid. Res. Mol.Biol., 4: 1—50,1965.

13. Heidelberger, C. Pyrimidine and Pyrimidine Nucleoside Antimetabolites.In: J. F. Holland and E. Frei, III (ads.), Cancer Medicine, pp. 768—791.Philadelphia: Lea and Febiger, 1973.

14. Heidelberger, C., Parsons, D. G., and Remy, D. C. Synthesis of 5-Trifluoromethyluracil and 5-Trifluoromethyl-2'-deoxyuridine. J. Med.Chem., 7: 1—5,1964.

15. Henderson, I. C., Lieber, M. M., and Todaro, G. J. Mink Cell Line MvlLu(CCL64):FocusFormationandtheGenerationof“Nonproducer―Transformed Cell Lines with Murine and Feline Sarcoma Viruses. Virology, 60:282—287,1974.

16. Hitotsumachi, S., Rabinowitz, Z., and Sachs, L. Chromosomal Control ofReversion in Transformed Cells. Nature, 231: 511—514,1971.

17. Hsu, T. C., Humphrey, A. M., and Somers, C. E. Persistent Nucleoli inAnimal Cells following Treatments with Fluorodeoxyuridine and Thymidine. Exptl. Cell Res., 33: 74—77,1964.

18. Huberman, E., and Heidelberger, C. The Mutagenicity to MammalianCells of Pyrimidine Nucleoside Analogs. Mutation Res., 14: 130—132,1972.

19. Jones, P. A., and Benedict, W. F. Chemotherapeutic Agents CauseMalignant Transformation. Clin. Res., 23: 163A, 1975.

20.Jones,P. A., Taderera,J. V., and Hawtrey,A. 0. TransformationofHamster Cells in Vitro by 1-f3-D-Arabinofuranosylcytosine, 5-Fluorodeoxyuridine, and Hydroxyurea. European J. Cancer, 8: 595—599,1972.

21. Llombart, A., and Marcos, A. J. Carcinogenic Action of DeterminateCarcinostatics. In: P. Bacalossi, U. Veronesi, and N. Cascinelli (eds.),Proceedings of the XI International Cancer Congress, Florence, Italy,Vol. 2, pp. 45-46, 1974 (Abstract).

22. Lozeron,H.A.,andSzybalski,W.Incorporationof5-Fluorodeoxyuridmneinto the DNA of Bacillus subtilis Phage PBS2 and Its RadiobiologicalConsequences. J. Mol. Biol., 30: 277-290, 1967.

23. Mondal, S., and Heidelberger, C. In Vitro Malignant Transformation byMethylcholanthrene of the Progeny of Single Cells Derived from @@3HMouse Prostate. Proc. NatI. Acad. Sci. U. S., 65: 219—225,1970.

24. Nomura, S., Fischinger, P. J., Mattern, C. F. T., Gerwin, B. I., and Dunn,K. J. Revertants of Mouse Cells Transformed by Murine Sarcoma Virus.II. Flat Revertants Induced by Fluorodeoxyuridine and Colcemid. Virology, 56: 152—163,1973.

25. Nowinski, R. C., Sarkar, N. H., and Fleissner, E. Isolation of SubviralConstituents and Antigens from the Oncornaviruses. In: H. Busch (ed),Methods in Cancer Res., Vol. 8, pp. 237—285.New York: Academic Press,Inc., 1973.

26. Poirier,L.A.,Stoner,G.D.,andShimkin,M.B.BioassayofAlkylHalidesand Nucleotide Base Analogs by Pulmonary Tumor Response in Strain AMice. Cancer Rca., 35: 1411—1415,1975.

27. Rapp,U. A., Nowinski,R. C., Reznikoff,C. A., andHeidelberger,C.Endogenous Oncornaviruses in Chemically Induced Transformation. I.

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Pyrimidine Nucleoside Oncogenic Transformation

Transformation Independent of Virus Production. Virology, 65: 392—409,1975.

28. Reznikott,C.A.,Bertram,J. S., Brankow,D.W.,andHeidelberger,C.Quantitative and Qualitative Studies of Chemical Transformation ofCloned C3H Mouse Embryo Cells Sensitive to Postconfluence Inhibitionof Cell Division. Cancer Res., 33: 3239—3249,1973.

29. Reznikoff, C. A. , Brankow, D. W. , and Heidelberger, C. Establishmentand Characterization of a Cloned Line of @3HMouse Embryo CellsSensitive to Postconfluence Inhibition of Division . Cancer Res. , 33:3231—3238,1973.

30. Taylor, J. H., Haut, W. F., and Tung, J. Effects of Fluorodeoxyuridine onDNA Replication, Chromosome Breakage, and Reunion. Proc. NatI.Acad. Sci. U. S., 48: 190-198, 1962.

31. Terzaghi, M., and Little, J. B. Repair of Potentially Lethal RadiationDamagein MammalianCellsIsAssociatedwithEnhancementof Malignant Transformation. Nature, 253: 548—549,1975.

32. Wellings, P.C.,Awdry,P. N., Bors,F.H.,Jones, B. R., Brown, D.C.,andKaufman, H. E. Clinical Evaluation of Trifluorothymidine in the Treatmentof Herpes Simplex Corneal Ulcers. Am. J. Ophthalmol., 73: 932-942,1972.

107JANUARY1976

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

1976;36:101-107. Cancer Res   Peter A. Jones, William F. Benedict, Mary S. Baker, et al.   Cells by Halogenated Pyrimidine NucleosidesOncogenic Transformation of C3H/10T½ Clone 8 Mouse Embryo

  Updated version

  http://cancerres.aacrjournals.org/content/36/1/101

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/36/1/101To request permission to re-use all or part of this article, use this link

Research. on December 10, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from