cell cycle dependency of oncogenic transformation induced ... · of day when dna synthesis is...

[CANCER RESEARCH 34, 526 537, March 1974]

Cell Cycle Dependency of Oncogenic Transformation Induced byW-Methyl-W-nitro-TV-nitrosoguanidine in Culture1

John S. Bertram2 and Charles Heidelberger3

Me Arale Laboratory for Cancer Research. The Medical School. University of Wisconsin. Madison. Wisconsin 53706

SUMMARY

Malignant transformation has been induced by /V-meth-yl-/V'-nitro-/V-nitrosoguanidine (MNNG) in synchronized

cultures of the C3H/10T1/2 CL8 line of mouse fibroblasts.This transformation shows marked cell cycle dependency,with the sensitive phase for malignant transformationlocated somewhere between 4 hr prior to S and the Gi-Sboundary. Cells were synchronized by arginine or isoleucinedeprivation for 48 hr, by 2 mM thymidine for 24 hr, or byrelease from postconfluence inhibition of cell division.Replicate cultures were treated with MNNG, 4 Â¿Â¿g/ml,atvarious times prior to, at, or after release of the block. DNAsynthesis began 4 hr after release of cells from arginine orisoleucine deprivation, and about 90% of the cells doubledafter 10 to 14 hr. A peak in transformation frequency (TF)occurred at the time of release in arginine-deprived cells,and 4 hr after release of the block in isoleucine-deprivedcells. Synchrony induced by 2 mM thymidine was poorlydefined; DNA synthesis began within 2 hr and cell divisionbegan within 4 hr after removal of the thymidine. The peakin TF occurred at the time of release of the block. Whencells were released from postconfluence inhibition of celldivision, a peak of TF was observed 4 hr prior to S phase,and a second peak of TF was located shortly before thesecond round of DNA synthesis. MNNG killed 99.9% ofcells at the time of maximal TF, and toxicity increased ascells entered S phase. Arginine deprivation did not cause analteration in chromosome number. Morphologically transformed colonies from MNNG-treated dishes were cloned,cultured, and injected into X-irradiated syngeneic mice.Nine of 10 transformed lines gave sarcomas, but theuntreated line did not.

INTRODUCTION

In a previous paper (43) we described the initiation of anew C3H mouse embryo-derived fibroblastic cell line thatexhibits a high degree of postconfluence inhibition of celldivision. This cell line, designated C3H/10T1/2 CL8, has

' This work was supported in part by Grant BC-2C from the AmericanCancer Society and Grant CA07I75 from the National Cancer Institute,NIH. A preliminary account of this work has been presented (4).

2Holder of a research training fellowship awarded by the International

Agency for Research on Cancer, Lyon. France.3American Cancer Society Professor of Oncology.Received September II, 1973; accepted December 3, 1973.

been shown to undergo malignant transformation in response to oncogenic hydrocarbons (42). Furthermore, thetransformation frequency, calculated as the percentage ofmalignantly transformed colonies to total surviving colonieswas, within limits, linearly related on a log-log plot to theconcentration of oncogen. Malignant transformation is heredefined as the possession by morphologically transformedcells of the ability to produce malignant tumors on inoculation into suitable animals, under conditions whereby untreated cells do not produce such tumors.

In this paper we report the use of this cell line toinvestigate the cell cycle phase specificity of malignanttransformation in response to the short-acting chemicaloncogen, MNNG,4 and the induction of cell cycle synchrony

by means of 4 procedures. To our knowledge, there havebeen no previous reports of cell cycle effects on chemicallyinduced malignant transformation in culture, nor havetransformable cells previously been synchronized. However, polyoma virus has been reported to be twice as efficient in transforming G2 cells than those in other phases ofthe cell cycle (2). The current status of chemical oncogene-sis in culture has been recently reviewed by Heidelberger(23).

This investigation of cultures was prompted largely by therepeated finding in vivo, with mouse skin, that the tumoryield after chemical oncogen treatment was related to thenumber of cells in the growth phase at the time of treatment.Thus, an increase in tumor yield results from either stimulation of the rate of cell division by croton oil prior to oncogen treatment (20, 32, 40) or oncogen treatment at the timeof day when DNA synthesis is maximal (21). Conversely,either inhibition of cell division by cantharidin (32) orinhibition of DNA synthesis by actinomycin D (3, 24)reduces the tumor yield after oncogen treatment. It is alsobecoming increasingly clear that cell division plays animportant role in liver carcinogenesis (48, 49) and that thedifferences in response to chemical oncogens cannot beexplained in terms of differences in binding of oncogen toreplicating or nonreplicating liver (27) or skin (9) DNA.Possibly related to these in vivo findings is the observationin cultures that at least 1 round of cell division is required to"fix" a mutational (10, 15) or a malignant event (6, 14)induced by chemicals or X-rays.

Thus, previous studies have indicated the importance of

'The abbreviations used are: MNNG, iV-methyl-vV'-nitro-A'-

nitrosoguanidine; TCA, trichloroacetic acid; TF, transformation frequency.

526 CANCER RESEARCH VOL. 34

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MNNG-induced Oncogenic Transformation

the growth cycle in the production of the ultimate malignantevent but have not dissected the cycle into its constituentphases. It is hoped that this report, which identifies asensitive phase for MNNG-induced malignant transformation near the Gi-S boundary, will provide leads into theintracellular site of action of chemical oncogens at thebiochemical and functional level and will increase ourunderstanding of the early events in chemical oncogenesis.

MATERIALS AND METHODS

Chemicals and Radiochemicals

MNNG, L-arginine hydrochloride, thymidine, deoxycyti-dine, and bovine serum albumin (Fraction V) were obtainedfrom Sigma Chemical Co., St. Louis, Mo. Reagent gradeacetone (Mallinckrodt Chemical Works, St. Louis, Mo.)was redistilled before use. All other chemicals were ofreagent grade or better. RPI scintillator PPO:POPOP(Research Products International Corp., Elk Grove, 111.)diluted with toluene was used for scintillation counting andautoradiography. Tritiated thymidine-methyl-3H, 3 Ci/

mmole, was obtained from Schwarz/Mann, (Orangeburg,N. Y.) and deoxycytidine-5-3H, 21.2 Ci/mmole, was from

Amersham/Searle Corp. (Des Plaines, 111.).

Cells

Fibroblastic cell line C3H/IOTI/2 CL8, recently derivedin these laboratories from C3H mouse embryos (43), wasused exclusively in these experiments. Maintenance of thestock cultures has been previously described (43). Toprovide sufficient cells for the transformation studies, glassroller bottles (BÃ©licoGlass, Inc., Vineland, N. J.) wereseeded with IO6cells in 100 ml of complete medium, gassedwith 5% CO2 in air, sealed, and incubated at 37Â°in a roller

bottle assembly (Forma Scientific, Inc., Marietta, Ohio).Medium was changed 2 to 3 days prior to harvesting. Allother cells were grown in 60-mm plastic Petri dishes (FalconPlastics, Oxnard, Calif.) or loosely stoppered 75-mm T-flasks (Falcon) in humidified incubators (Forma) in anatmosphere of 5% CO2 in air. Cell counts were made with aModel B Coulter counter (Coulter Electronics, Hialeah,Fla.).

Culture Media

All media and sera were obtained from Grand IslandBio-logical Co., Grand Island, N. Y. Eagle's basal mediumsupplemented with 10% heat-inactivated fetal calf serumwas used in all cell culture experiments. Stock cultures werenot exposed to antibiotics. All other cultures receivedpenicillin (100 units/ml) and streptomycin (50 /ng/ml)routinely. After 1 to 2 weeks, the transformation experiments received amphotericin B (Fungizone: Squibb and Co.,New Brunswick, N. J.), 1 /ig/ml. Arginine- or isoleucine-free Eagle's basal medium was supplemented with 10%dialyzed heat-inactivated fetal calf serum. Because of

variations in the free amino acid composition of thecommercial serum, heat-inactivated fetal calf serum wasdialyzed in cellulose dialysis tubing (Union Carbide Corp.,Chicago, 111.)against 3 changes of a 10-fold excess ofphosphate-buffered saline, pH 7.4, at 4Â°over a 3-day

period. The tubing was cleaned before use by boiling in 0.01M EDTA and was sterilized by autoclaving.

Cell Cycle Studies

Cells used in the synchrony and transformation experiments were, in all cases, derived from roller-bottle culturesgrown to a state of postconfluence inhibition of cell division.Cells were harvested with 0.1% trypsin (Difco Laboratories,Inc., Detroit, Mich.), and the trypsin was quenched withserum. Following centrifugation, the cell pellet was resus-pended in the appropriate complete medium, and a cellcount was performed. Cells were then diluted to a concentration of 2 x 104/ml in complete medium, in medium

lacking arginine or isoleucine, or in medium to which 2 m.Mthymidine had been added. Cells were then seeded in 60-mmplastic Petri dishes at 5 ml/dish or, for autoradiography, in2-chamber tissue culture chamber-slides (Lab-Tek Products, Westmont, 111.)at 2 ml/chamber. Cells were subjectedto amino acid deprivation for 48 hr or to 2 mMthymidine for24 hr before being released from the block by replacementof the deficient medium with complete medium. In cellsreleased from postconfluence inhibition of cell division, thetime of release was taken as the time of plating. In order tofollow the cell cycle throughout a 24-hr time period,replicate roller bottles were harvested and plated at 9 p.m.and at 9 a.m. the following day, and studies were performedin unison on these 2 groups.

At 2-hr intervals after release from the block, and for aperiod of at least 1 cell cycle, groups of 2 dishes weremonitored for extent of DNA synthesis and for total cellnumber. Chromosome spreads were performed by themethod of Rothfels and Siminovitch (44).

Measurement of DNA Synthesis. Cells synchronized byamino acid deprivation or by release from contact inhibitionwere treated with a 30-min pulse of tritiated thymidine, 1/uCi/ml, followed by a 10-min chase with cold thymidine,10 jug/ml, in complete medium. The cells were washed with0.9% NaCl solution and lysed with 2 ml of 2% sodiumdodecyl sulfate containing bovine serum albumin, 50 pg/m\.The solution was rapidly transferred to 2 ml of ice-cold 20%TCA and the mixture was allowed to stand in ice for 30 min.It was then filtered on glass fiber discs (Gelman InstrumentCo., Ann Arbor, Mich.; Type E), which were washed with20 ml of 2% cold TCA. The discs were dried under a radiantheater, transferred to a PPO : POPOP :toluene solution, andcounted by liquid scintillation. Counting efficiency wasdetermined by means of an internal standard of DNA ofknown specific activity. Results are expressed as dpm/103

cells.Autoradiography.Cells were cultured as described above

and labeled with either tritiated thymidine or, in the case ofcells blocked with 2 mvi thymidine, with tritiated deoxycyti-dine, 2.5 /Â¿Ci/ml,for 30 min, followed by a 10-min chase

MARCH 1974 527



John S. Bertram and Charles Heidelberger

with cold deoxycytidine, 20 jig/ml, in complete medium.The glass slide was washed twice with 0.9% NaCl solutionand the cells were fixed with methanol: glacial acetic acid(1:3) for 10 min, followed by washes in methanol, waterand, finally, methanol. After air drying, the slides weredipped in Kodak (Eastman Kodak Co., Rochester, N. Y.)nuclear emulsion NTB3 which was allowed to gel for 4 hr atroom temperature in the dark. They were then placed inCoplin jars, covered with scintillation fluid, and storedovernight in a light-proof box at -70Â°.After approximately

16 hr of exposure, the slides were removed from the toluene,washed for 15 to 20 min in cold running water until toluenefree, and developed and fixed in the usual manner. Thismethod of scintillation autoradiography, adapted from atechnique developed by Randerath (41) for chromatograms,results in a greatly reduced exposure time for tritium. Slideswere lightly stained with hematoxylin.

A total of about 500 nuclei were counted for each timepoint, and the labeled nuclei were scored as lightly labeled (5to 50 grains/nucleus) or heavily labeled (above 50 grains/nucleus). Only heavily labeled cells are included in the data,and the results are expressed as percentage heavily labeledcells in the population counted.

Transformation

Replicate dishes, seeded with IO5cells/dish as describedabove, were treated at 2-hr intervals throughout the cellcycle with 25 n\ of MNNG in acetone to yield a finalconcentration of 4 ng of MNNG per ml and 0.5% acetone.Controls received 0.5% acetone, which was nontoxic.Twelve dishes were treated per time point. In all cases zerotime (/ = 0) refers to the time of release of the block. Thus,cells treated at / = -4 were treated 4 hr prior to release and,at / = +4, were treated 4 hr after release. At / = 0, cellswere released from the block by medium changing and weretreated immediately. In view of the 90-min half-life ofMNNG in medium, the medium was not changed until 3 to4 days after treatment, and thereafter twice weekly for 3 to 4weeks until the dishes were fixed and stained as describedpreviously (42). All the experiments reported were repeatedat least twice and representative experiments are shown.

Scoring for Malignant Transformation and for Toxicity

The fixing and staining of dishes, the morphologicalcriteria for malignant transformation and the classificationof the transformed colonies have been previously described(42). However, in the experiments reported here, type IIcolonies are rare, and consequently only type III colonieshave been scored (Fig. 1).

In this system, dishes are fixed and stained 3 to 4 weeksafter treatment, when the surviving colonies appear asreadily discernible discrete foci amid a monolayer ofmorphologically aberrant cells killed by the carcinogen, butnevertheless still attached to the culture dish. All coloniescontaining more than about 50 cells are counted. Thus,scoring for transformation and for total surviving coloniescan be done in the same dish. Results are expressed as: (a)

TF, defined as the percentage of type III malignantlytransformed colonies to total surviving colonies at each timepoint and (b) total surviving colonies per dish for each timepoint. The in vivo verification of the morphological transformation as malignant transformation was performed aspreviously described (42).

Rate of Decomposition of MNNG

The stability of MNNG dissolved in complete medium,maintained at 37Â°and pH 7.4 to 7.5, was determined by

monitoring the decrease in absorbance at 400 nm (29).Under these conditions, the half-life of MNNG was 90 min.A solution in acetone showed no decrease in absorbanceover a 4-hr period.

RESULTS

Synchrony

Cells grown to a state of postconfluence inhibition ofreplication and subsequently plated in arginine- or isoleu-cine-deficient medium, attach normally to the culture dishbut fail to initiate DN A synthesis, which occurs in comparable cells plated in normal medium (Chart 1). The low levelof DNA synthesis detected by autoradiography 48 hr afterplating in arginine- and isoleucine-free medium (zero heavily labeled cells, 20% and 7% lightly labeled cells) is incontrast to the situation in logarithmically growing cells inwhich approximately 45% are heavily labeled under thesame labeling conditions. After cells were refed withcomplete medium, approximately 4 hr were required forreinitiation of DNA synthesis, indicating that cells wereblocked 4 hr prior to S phase (Chart 2). The maximalnumber of labeled cells occurred 8 to 12 hr after release ofthe arginine- and isoleucine-deficient block. The exacttiming of the peak of DNA synthesis appeared to vary withthe method of determination. When the incorporation of

175-

te.m 150-

=)z_l 125-

CELL NUMBER

600 Ã›J

0 5 IO 15 20 25 30 35 40 45

HOURS AFTER PLATING

Chart I. The degree of synchrony obtained by releasing cells frompostconfluence inhibition of cell division and plating at 10s cells/dish in

complete medium. Cell number (â€¢)and the extent of incorporation oftritiated thymidine (TdR) into acid-insoluble material (A) were determinedat 2-hr intervals; 10 3 x cell number, the number on the ordinatemultiplied by IO3(i.e., 100on ordinate means 100,000cells).





tritiated thymidine into acid-insoluble material was measured following arginine restoration, the peak was at 10 hr(Chart 3), while autoradiography indicated a maximallabeling index at about 12 hr after release (Chart 2). It is notclear whether these variations are a consequence of themethodology of determining DNA synthesis, or of thedifferent culture conditions (plastic versus glass, respectively). In cells synchronized by isoleucine deprivation, awave of cell division occurred between 10 and 14 hr afterrelease, during which time 92% of the attached cells divided(Chart 4). Arginine-deprived cells behaved similarly. Theincreased tritiated thymidine-labeling index beginning at 16hr indicates the start of a 2nd division cycle (Chart 2).Chromosome analysis performed on cells undergoing their1st mitosis after 48 hr of arginine deficiency indicated nochange in number from the control (Chart 5).

Chart 6 shows the degree of synchrony following a 24-hrexposure to 2 mMthymidine. Although cells were originally

O 4 8 12

HOURS AFTER RELEASE

Chart 2. Tritiated thymidine labeling index obtained by autoradiography of cells blocked for 48 hr in arginine- (â€¢)or isoleucine- (O) deficient

medium, and released from the block by the addition of complete medium.Groups of cells were pulse labeled for 30 min with tritiated thymidine at2-hr intervals after release, and autoradiographs were prepared as de

scribed. Lightly labeled cells have been excluded from the data.

-1500 â€ž¿�S

-4 -2 0 '2 4 6HOURS AFTER RELEASE

Chart 3. Transformation frequency (â€¢)and number of survivors perdish (O) were obtained in replicate cultures treated with MNNG, 4 Mg/ml,at various times prior to, at, and after release from cell cycle block inducedby 48 hr of arginine deprivation. Progression of cells through the cell cyclewas monitored in blocked but otherwise untreated cells by measuring theuptake of tritiated thymidine (TdR) into acid-insoluble material (A) at 2-hr

intervals after release.

e 10 12HOURS AFTER RELEASE

Chart 4. Progression through the cell cycle in cells released from 48 hr ofisoleucine deprivation by the addition of complete medium. Tritiatedthymidine (TdR) incorporation into acid-insoluble material (A) and cellnumber (â€¢)were measured at 2-hr intervals after release. For explanation

of exponential on ordinale, see legend to Chart I.

CU ARGININE BLOCK

E23 CONTROL

so 81 160 16282 " 158

CHROMOSOME NUMBER

Chart 5. Assay for chromosome numbers per cell performed on cells intheir 1st division cycle after release from 48 hr of arginine deprivation, andon normal untreated logarithmic phase cells.

0 2 4 6 8 10 12 14 16HOURS AFTER RELEASE

Chart 6. Cell cycle progression in cells released from 24 hr of exposureto 2 mM thymidine by replacement of thymidine-containing medium withcomplete medium. The labeling index after exposure to tritiated deoxycyti-

dine [determined by autoradiography, as described (A)] and cell number(â€¢)were determined at 2-hr intervals after release. For explanations of

exponential, see legend to Chart I.

in a state of postconfluence inhibition of division, the highinitial labeling index, its immediate increase after removalof thymidine, and the increase in cell number only 4 hr after

MARCH 1974 529




release of the block, all indicate that many cells had enteredS and were progressing slowly through S in the presence of 2m\i thymidine. Nevertheless, it is clear from the autoradio-graphie studies that only 7% of the cells were in S phase atthe time of release, and that 12 to 14 hr were again requiredfor all the cells to divide, as was observed in cells synchronized by isoleucine deprivation (Chart 4). A certain proportion of the cells, therefore, must be assumed to be in late dat the time of release of the thymidine block.

In order to confirm the results obtained with cellsartificially synchronized, it was decided to investigate thedegree of natural synchrony that can be obtained byreleasing cells from postconfluence inhibition of division. Itwas found that a moderate degree of synchrony could beobtained in the 1st cell cycle after release by trypsinizingconfluent monolayers and plating in complete growthmedium (Chart 1).

Transformation

Synchrony Induced by Arginine Deprivation. In culturesblocked in the cell cycle by arginine deprivation and treatedwith MNNG, 4 Mg/ml, prior to, at, and after release of theblock, it was found that a very substantial increase in TFoccurred when cells were treated at the time of release of theblock (Chart 3). Cells treated 4 and 2 hr before releaseexhibited a very low TF. In cells treated after release of theblock, the TF declined progressively with time and almostreached zero by 4 hr after release, before appreciable DNAsynthesis had occurred.

Synchrony Induced by Isoleucine Deprivation. Substitution of arginine deprivation by isoleucine deficiency resultedin a distinct shift in the peak of TF to 4 hr after release fromthe block (Chart 7), which was consistently observed in 3experiments. When treatment was continued on replicatedishes at 2-hr intervals as the cells progressed through the

60

5 Â«0-

2O -

O

*

O 1-4 8 I2

HOURS AFTER RELEASE

-ZO

-o

Chart 7. Transformation frequency (â€¢)and number of survivors perdish (O) obtained in replicate cultures treated with MNNG. 4 Â¿ig/ml,atvarious times prior to, at, and after release from cell cycle block induced by48 hr of isoleucine deprivation. Tritiated thymidine incorporation fromnon-MNNG-treated controls (A) (same data as Chart 4).

1st division cycle and into the next, a small 2nd peak oftransformation occurred 14 hr after release, just prior to thenext round of DNA synthesis. The TF in cells treated inboth S phases was low but did not decrease to zero.

Synchrony Induced by 2 HIMThymidine. A pronouncedpeak in TF was observed in cells treated with MNNG, 4Mg/ml, at the time of release of the block (Chart 8).Qualitatively, the results are similar to those obtained withcells synchronized by arginine deficiency (Chart 3). However, in the thymidine synchrony experiments, the TF was 5to 7 times lower.

Synchrony Induced by Release from Postconfluence Inhibition of Cell Division. When individual groups of cells weretreated at 2-hr intervals from 4 to 44 hr after release fromthe confluent state, 2 peaks of transformation were observed(Charts 9 and 10). The 1st occurred approximately 4 hrprior to the onset of DNA synthesis and coincided with thepeak of transformation seen in the amino acid deficiencyexperiments. A 2nd peak of approximately equal magnitudewas located apparently in late S phase. However, it will benoted that a 2nd round of DNA synthesis was scheduled tobegin within 3 to 5 hr [derived by extrapolating the tritiatedthymidine incorporation curves back to the origin (Charts 9and 10)].

As has already been noted, the timing of the onset ofDNA synthesis in these 2 confluency experiments differedby about 6 hr. Similar shifts in timing of the peaks in TFwere also observed (Charts 9 and 10). When the results fromthese experiments were combined and normalized for thetiming of S. coincident peaks of TF were obtained, againdemonstrating the cell cycle dependency of oncogenictransformation (Chart 11).

Toxicity

In an attempt to avoid the high toxicity caused byMNNG, 4 Mg/ml, and yet obtain transformation, experiments were carried out with various permutations ofMNNG concentration and number of cells seeded (Table 1).Oncogenic transformation was observed only in dishesplated at IO4 and IO5 cells/dish and treated with MNNG,either 1, 2, or 4 Mg/ml. Since treatment of IO5 cells with

doses of MNNG of 1 or 2 Mg/ml resulted in too manysurviving colonies to be counted, which makes accurateassessment of TF impossible, and since treatment of IO4

cells gave a very low yield of transformants, the higher doseof 4 Mg/ml has been used in all experiments requiringquantitation of oncogenic transformation. It was consideredthat the high degree of lethality seen in S-phase cells treatedwith MNNG, 4 Mg/ml, might mask transformation in thatphase of the cycle. Thus, several experiments were carriedout in which groups of cells synchronized by argininedeprivation were treated with MNNG 2 Mg/ml, at 2-hrintervals after release. Transformation was observed only incells treated at or close to the time of release.

Toxicity was consistently found to be minimal at the timeof maximal TF (Charts 3 and 7 to 10) and then to increaseas cells entered S phase. An accurate determination ofpercentage cell survival is impossible to obtain at these high





3.0 r 60

2.0 2

TRANSFORMATIONFREQUENCY

% CELLS LABELED

- 1.0 -20

L0

_

0

IO 244 6 B

HOURS AFTER RELEASE

Chart 8. Transformation frequency (â€¢)and number of survivors per dish (O) obtained in replicate cultures treated with MNNG, 4^g/ml, at varioustimes prior to, at, and after release from cell cycle block induced by 2 mvi thymidine. Labeling indices determined by autoradiography ofnon-MNNG-treated controls (A) (same data as Chart 6).

-300O

TRANSFORMATIONFREQUENCY

O 5 IO 15 20 25 30 35 40HOURS AFTER PLATING

Charts 9 and 10. Transformation frequency (â€¢)and survivors per dish (O) obtained in replicate cultures treated with MNNG, 4 >/g/ml, at varioustimes after release from postconfluence inhibition of cell division. Incorporation oftritiated thymidine (TdR) into acid-insoluble material (A) was determined in non-MNNG-treated controls.

plating densities; however, an estimate can be made. Fromthe cell synchrony studies, IO5plated cells are known to giverise to approximately 6 x IO4 attached cells; of these,

approximately 92% will divide within the 1st cell cycleperiod (Chart 4). If each of these dividing cells is assumed tobe capable of producing a colony, then the plating efficiency

MARCH 1974 531




can be estimated as 57%. At the time of maximal TF,approximately 60 surviving colonies per dish existed in theisoleucine (Chart 7) and arginine (Chart 3) deprivationsynchrony systems. Thus, MNNG at 4 Mg/ml kills 99.9% ofthe cells at the time of maximal TF. To investigate whetherthe surviving colonies possessed increased resistance toMNNG compared with the parent clone, 2 malignantlytransformed and 1 morphologically normal colonies werecloned from dishes previously treated with MNNG, 4Â¿ig/ml.Two of the clones, 1 normal and 1transformed, wereonly slightly more resistant than the parent, untreated cellsto MNNG-induced lethality (Chart 12).

Cytology

Transformed colonies in these synchronous systems canfirst be visualized in the living state approximately 10 days

15 20 25 30EXPERIMENT I oâ€”o1 1 1 r~

15 20 25EXPERIMENT 2*-

10 35 40

HOURS AFTER PLATINGChart 11. Transformation I'rcquencies obtained in duplicate experiments

(Exp.) (Charts 9 and 10) in which groups of cells released frompostconfluence inhibition of cell division were treated with MNNG. 4/ig/ml, at various times after release. The results have been normalized forthe timing of S phase.

after carcinogen treatment, and are usually fixed andstained about 2 weeks later. At this time, cells killed by thecarcinogen are still attached to the dish and form a thinmonolayer of large vacuolated cells with pyknotic nuclei.Interspaced among these reproductively dead cells are smallcolonies of survivors with a normal morphology (Fig. 2),and among these may be found malignantly transformedcolonies, which are usually larger than the untransformedcolonies (Figs. 3 and 4). As determined by morphologicalcriteria defined in a previous paper (42), approximately 90%of the oncogenically transformed colonies were of the typeIII variety, and the remaining 10% were type II. Because ofthe low incidence of type II colonies and the occasionaldifficulty in distinguishing between types II and I, type IIcolonies were not scored as malignantly transformed. TypeI transformed colonies were frequent but were not scoredsince they are not believed to be malignant (42).

The morphological transformation scored in these experiments has been proved to be malignant by demonstration ofthe ability of transformed clones to induce tumors oninjection into X-irradiated syngeneic animals. Ten type III

100

I.O 2.O

MNNG>ig/ml

Chart 12. Comparative toxicity of MNNG toward 3 cell lines derivedfrom colonies surviving MNNG. 4 fig/ml, and toward the parent cell line.TA and TB, malignantly transformed lines; N A, survivor with normalmorphology: IOTI/2, parent C3H/10TI/2 CL8 cell line.

Table I

Variations in transformation frequency with dose of MNNG and number of cells treatedCells were seeded and maintained in arginine-deficient medium for 48 hr. Complete medium was

then added and groups of 10 dishes were treated with MNNG.

MNNG(fig/ml)00.51.02.04.0Platingefficiency"(%ofcontrol)100X4374O.IIO30/8Â»0/90/80/7N.D.rCell

no.treatedIO40/71/10(0.01)0/70/9N.D.IO50/80/92/9

(0.001)26/8(0.13)15/10(2.5)

" Expressed as a percentage relative to control acetone treated dishes. The results were obtainedusing cells seeded at I03/dish and are extrapolated. Control plating efficiencies were typically 20%at lOVdish and 60% at I05/dish.

* No. of malignantly transformed colonies/no, of dishes stained. Numbers in parentheses, trans

formation frequencies calculated as the percentage of transformed to total surviving colonies.' N.D., not determined, due to high toxicity.





colonies were cloned and cultured as previously described(42). The growth rate of the transformed cells approximatedthat of the parent cell line; however, the transformed cellscontinued to divide postconfluence, and reached saturationdensities 2 to 10-fold higher than the parent cell line. Cellsderived from 9 of the original 10 transformed colonies gavetumors after the s.c. injection of approximately 2 x IO6cellsinto X-irradiated syngeneic mice. This incidence of tumorformation is comparable to the 85% incidence for type IIIcolonies reported previously (42). The latent period fordevelopment of palpable tumors ranged between 40 and 270days. A further 60 to 90 days of growth was required tothreaten the life of the host. Histologically, the tumors wereall undifferentiated fibrosarcomas, and all were encapsulated. When the tumors were given time to enlarge,infiltration of tumor cells into muscle and skin was observed. MÃ©tastaseswere never found. Injection of theuntransformed cell line did not give rise to tumors.

DISCUSSION

We have demonstrated, by 4 synchrony-producingmethods, that MNNG induces malignant transformation inC3H/10T1/2 CL8 mouse embryo cells and that thistransformation shows cell cycle dependency. The periodmost sensitive for chemical oncogenic transformation appears to extend from a point about 4 hr prior to S phase tothe G,-S boundary. Due to the 90-min half-life of MNNGin this system and uncertainties about the position of cells inthe cycle, the location of this sensitive phase cannot bestated with greater accuracy. Moreover, the different conditions of synchrony did not give identical results. In thesynchrony experiments, with arginine deprivation (Chart 3)and release from confluence (Chart 11), the peak in TF islocated in late d; however, in cells released from isoleucinedeficiency, the peak in TF occurs close to the Gi-Sboundary (Chart 7). The poor degree of cell synchrony afterrelease from the single 2 mMthymidine block reported here(Chart 6) and by other authors (5, 7) does not allow thetiming of the peak of transformation to be located withmore precision than late GÃ¬to early S phase (Chart 8).

We are unable satisfactorily to explain the differences intiming of TF and toxicity induced in cells by MNNG afterrelease from arginine and isoleucine deprivation (Charts 3and 7). Of possible significance is the higher labeling index 6to 8 hr after release of arginine-deprived cells, comparedwith isoleucine-deprived cells (Chart 2), which may indicate that arginine-deficient cells are arrested closer to Sphase than are isoleucine-deficient cells. Our value of 4hr for initiation of DNA synthesis following addition of theamino acid in the 2 cases is in good agreement with previous studies on Chinese hamster cells released from arginine (19) or isoleucine (47) deprivation. It has been shownthat lack of these 2 essential amino acids does not produce identical results in S phase cells. Whereas isoleucinedeprivation allows cells already in S slowly to continueDNA synthesis (18), arginine deficiency causes an immediate halt to normal DA synthesis (19), while allowing

repair synthesis to proceed (45). Cells not in S phase at thetime of deprivation are in both cases arrested in late GÃ¬.To circumvent these differences in response to lack of arginine and isoleucine, all cells used in these experimentswere grown to a state of postconfluence inhibition of division prior to plating and amino acid deprivation. Chromosome studies performed on cells released from argininedeprivation failed to demonstrate any major damage(Chart 5), such as has been reported in Chinese hamstercells deprived of arginine or isoleucine (19).

The timing of resumption of DNA synthesis and celldivision following release from postconfluence inhibition ofdivision in the IOTI/2 C18 cell line used is very similar tothat reported for 3T3 cells (34). The 1st peak of TF is clearlylocated prior to the onset of DNA synthesis (Charts 9 to 11).However, due to the decay in synchrony, the 2nd peakcannot definitely be located within the cell cycle. Evidencethat places this peak in late GÃ¬(and not S phase) of the 2ndcell cycle includes: (a) the 2 TF peaks are apart 1 cell cycletime of 16 hr (43), (b) a 2nd round of DNA synthesisoccurred shortly thereafter, and (c) a 2nd peak of TF in cellssynchronized by isoleucine deprivation is located after the Sphase (Chart 7).

The cytotoxicity induced by MNNG was inversely relatedto the TF in all synchrony experiments (Charts 3 and 7 to 9).The increase in toxicity seen after the period of maximal TFappears to coincide with the onset of S phase and confirmsprevious results with Chinese hamster cells ( 1). The lack of adirect relationship between cytotoxicity and oncogenicityhas been repeatedly demonstrated for many chemicaloncogens in this (13, 14, 42) and other laboratories (16, 26).

Comparatively little is understood of the molecular eventsthat occur during the 3- to 4-hr period immediately prior tothe onset of DNA synthesis, which corresponds to the timeof maximal TF reported here. However, during this period,the production of specific proteins presumably required forDNA synthesis takes place (25, 46). Fujiwara (22) hasrecently identified a regulatory protein synthesized in lateGÃ¬which is required for the initiation but not progression ofDNA synthesis, while Mueller (33) has proposed thatmembrane changes are responsible for the triggering of Sphase.

Many, if not all, of the known activated forms ofchemical oncogens are also mutagens (17, 31, 35), a factthat has supported the somatic mutation theory of cancer asoriginally proposed by Boveri (8) and as restated byBurdette (11). MNNG has been shown selectively to mutatethe replicating fork of bacteria, and consequently gives riseto higher mutation yields in bacteria containing replicatingforks (dividing) than in bacteria in which DNA synthesiswas complete (nondividing) (12). Conversely, inParamecium, MNNG induces a maximal mutational yieldwhen treatment is given just prior to the onset of DNAsynthesis (28), and the graph of mutation yield against timeof treatment in the cell cycle closely resembles the malignant transformation data presented here (Charts 9 and 10).However, Orkin and Littlefield (36) were unable to demonstrate any consistent change in MNNG-induced mutationsthroughout the cell cycle of Chinese hamster cells, although

MARCH 1974 533




the variations they obtained in mutational yield makes anyconclusion only tentative.

Interpretation of the results reported here is madedifficult by the profound lack of knowledge of the fundamental processes controlling DNA replication and theuncertainty as to the primary intracellular site of action ofchemical oncogens. However, several possible interpretations can be proposed. (It should be noted that the termsused in these proposals, i.e., "the lesion," "the damage,""the cellular site of action," etc., are not directed exclusively

at DNA, but are intended to include all other potentiallyrelevant target molecules.)

1. Cells treated in early or mid-Gi phase are capable ofrepairing prelethal radiation-induced damage (30), whereasthe damage becomes fixed in S phase (38). The 1stinterpretation would be that our cells treated in late Gt orearly S phase may be unable to repair MNNG-induceddamage and are transformed, while cells treated later in thecycle are able to repair the damage during the succeeding GÃ¬phase. An extension of this proposal would be that delays incell cycle progression, in response to chemical injury, couldoccur at specific stages of the cell cycle. Such delays arethought to have profound effects on cell survival in Chinesehamster cells treated with /V-methyl-/V-nitrosourea (39).

2. At the time of maximal TF, a crucial site of action forMNNG oncogenesis becomes available or exposed. Thissite could be (among others) a specific regulator, messenger,or gene. The requirement and production of specific proteins (22, 25) and the increased binding of actinomycin D toDNA as cells traverse GÃ¬and enter S phase (37) make thisinterpretation plausible.

3. Reaction of MNNG with the critical cellular site isidentical throughout the cycle, but the competence of thecells to respond may be phase specific.

4. Local changes within the cell or in cellular permeabilityat the time of maximal TF may enhance the decompositionof MNNG to the chemically reactive species. Since thedecomposition is base and thiol catalyzed (29), localchanges in pH or thiol concentration could substantiallyaffect the degree of methylation of crucial sites. In preliminary studies on the binding of labeled MNNG to cellularmacromolecules, no significant differences have been notedwithin sensitive and insensitive phases of the cell cycle(unpublished observations).

In future work we will examine the interpretations listedabove and will investigate the oncogenic effects of chemicalsof differing structure and reactivity in synchronized cells.These studies should increase our understanding of the earlyevents involved in chemical oncogenesis.

ACKNOWLEDGMENTS

We would like to thank Janice Lovett for invaluable assistance with the cell cultures and Amy Miller for the chromosome preparations.

REFERENCES

I. Barranco, S. C., and Humphrey, R. M. The Response of ChineseHamster Cells to /V-Methyl-A"-nitro-/V-nitrosoguanidine. Mutation

Res., //.-421 429, 1971.

2. Basilico, C., and Marin, G. Susceptibility of Cells in Different Stagesof the Mitotic Cycle to Transformation by Polyoma Virus. Virology,28: 429 437, 1966.

3. Bates, R. R., Wortham, J. S., Counts, W. B., Dingman, C. W., andGelboin, H. V. Inhibition by Actinomycin D of DNA Synthesis andSkin Tumorigenesis Induced by 7,12-Dimethylbenz[a]anthracene.

Cancer Res., 28: 27 34, 1968.4. Bertram, J. S., and Heidelberger, C. Cell Cycle Dependency of

Chemical Oncogenic Transformation in Culture. Proc. Am. Assoc.Cancer Res., 14: 70, 1973.

5. Bootsma, D., Budke, L.. and Vos, O. Studies on Synchronous Division of Tissue Culture Cells Initiated by Excess Thymidine. Exptl.Cell Res., 33: 301 309, 1964.

6. Borek, C., and Sachs, L. Cell Susceptibility to Transformation byX-irradiation and Fixation of the Transformed State. Proc. Nati.

Acad. Sei. U. S., 57: 1522 1527, 1967.7. Bostock, C. J., Prescott, D. M., and Kirkpatrick, J. B. An Evaluation

of the Double Thymidine Block for Synchronizing Mammalian Cellsat the G,-S border. Exptl. Cell Res., 68: 163-168, 1971.

8. Boveri. T. The Origin of Malignant Tumors. Baltimore: The Williams& Wilkins Co., 1929.

9. Bowden, G. T., and Boutwell, R. K. The Binding of 7, 12-Dimethyl-benz[a]anthracene (DMBA) to Replicating and Non-replicating DNA

in vivo. Proc. Am. Assoc. Cancer Res., 14: 28, 1973.10. Bridges, B. A., and Huckle, J. Mutagenesis of Cultured Mammalian

Cells by X-irradiation and UV Light. Mutation Res., 10: 141 151,

1970.11. Burdette, W. J. The Significance of Mutation in Relation to the Origin

of Tumors: A Review. Cancer Res., 15: 201-226, 1955.12. Cerda-Olmedo, E., Hanawalt, P. C., and Guerola, N. Mutagenesis of

the Replication Point by Nitrosoguanidine. Map and Pattern ofReplication of the Escherichia coli Chromosome. J. Mol. Biol., 33:705-719, 1968.

13. Chen, T. T., and Heidelberger, C. In Vitro Malignant Transformationof Cells Derived from Mouse Prostate in the Presence of 3-Methyl-cholanthrene. J. Nati. Cancer Inst., 42: 915-925, 1969.

14. Chen, T. T.. and Heidelberger, C. Quantitative Studies on theMalignant Transformation of Mouse Prostate Cells by CarcinogenicHydrocarbons in Vilro. Intern. J. Cancer, 4: 166 178, 1969.

15. Chu, E. H. Y., and Mailing, H. V. Mammalian Cell Genetics. II.Chemical Induction of Specific Locus Mutations in Chinese HamsterCells in Vilro. Proc. Nati. Acad. Sei. U. S.. 61: 1306-1312, 1968.

16. DiPaolo, J. A.. Takano, K., and Popescu, N. C. Quantitation ofChemically Induced Neoplastic Transformation of BALB/3T3Cloned Cell Lines. Cancer Res., 32: 2686 2695, 1972.

17. Duncan, M. E., and Brookes, P. The Induction of Azaguanine-resist-

ant Mutants in Cultured Chinese Hamster Cells by Reactive Derivatives of Carcinogenic Hydrocarbons. Mutation Res., 21: 107-118,

1973.18. Everhart, L. P. Effects of Deprivation of Two Essential Amino Acids

on DNA Synthesis in Chinese Hamster Cells. Exptl. Cell Res., 74:311-318, 1972.

19. Freed, J. J., and Schatz, S. A. Chromosome Aberrations in CulturedCells Deprived of Single Essential Amino Acids. Exptl. Cell Res., 55:393 409, 1969.

20. Frei, J. V., and Harnsono, T. Increased Susceptibility to Low Doses ofa Carcinogen of Epidermal Cells in Stimulated DNA Synthesis.Cancer Res., 27: 1482 1484, 1967.

21. Frei, J. V., and Ritchie, A. C. Diurnal Variation in the Susceptibilityof Mouse Epidermis to Carcinogen and Its Relationship to DNASynthesis. J. Nati. Cancer Inst., 32: 1213-1220, 1964.

22. Fujiwara, Y. Effect of Cycloheximide on Regulatory Protein forInitiating Mammalian DNA Replication at the Nuclear Membrane.Cancer Res., 32: 2089-2095, 1972.




MNNG-induced Oncogenic Transformalion

23. Heidelberger, C. Chemical Oncogenesis in Culture. Advan. CancerRes. 18: 317 366, 1973.

24. Hennings, H., Smith, H. C., Colburn, N. C., and Boutwell, R. K.Inhibition by Actinomycin D of DNA and RNA Synthesis and of SkinCarcinogenesis Initiated by 7,12-Dimethylbenz[a]anthracene or ÃŸ-Propiolactone. Cancer Res., 28: 543 552, 1968.

25. Highfield, D. P., and Dewey, W. C. Inhibition of DNA Synthesis inSynchronized Chinese Hamster Cells Treated in G i or Early S Phasewith Cycloheximide or Puromycin. Exptl. Cell Res., 75: 314 320,1972.

26. Huberman, E., and Sachs, L. Cell Susceptibility to Transformationand Cytotoxicity by the Carcinogenic Hydrocarbon Ben/o[a]pyrene.Proc. Nati. Acad. Sei. U. S., 56: 1123 1129, 1966.

27. Jackson, C. D., and Irving, C. C. The Binding of /V-Hydroxy-2-acetylaminofluorene to Replicating and Non-replicating DNA in RatLiver. Chem.-Biol. Interactions, 2: 261 265, 1970.

28. Kimball, R. F. Studies on the Mutagenic Action of JV-Methyl-/V'-

nitro-/V-nitrosoguanidine in Paramecium aurelia with Emphasis onRepair Processes. Mutation Res., 9: 261 271, 1970.

29. Lawley, P. D., and Thatcher, C. J. Methylation of DeoxyribonucleicAcid in Cultured Mammalian Cells by /V-Methyl-/V'-nitro-yV-

nitrosoguanidine. Biochem. J., 116: 693 707, 1970.30. Little, J. B., and Hahn, G. M. Life-cycle Dependence of Repair of

Potentially Lethal Radiation Damage. Intern. J. Radiation Biol., 23:401 407, 1973.

31. Miller, E. C., and Miller, J. A. The Mutagenicity of ChemicalCarcinogens: Correlation. Problems and Interpretations. In: A. Hollander (ed.). Chemical Mutagens. Principles and Methods for TheirDetection, Vol. I, pp. 83 119. New York: Plenum Publishing Corp.,1971.

32. Mottram, J. C. A Developing Factor in Experimental Blastogenesis. J.Pathol. Bacteriol., 56: 181 187. 1944.

33. Mueller, G. C. Biochemical Perspectives of the G! and S Intervals ofthe Replication Cycle of Animal Cells: A Study in the Control of CellGrowth. In: R. Baserga (ed.). The Cell Cycle and Cancer pp. 269 308.New York: Marcel Dekker, Inc., 1971.

34. Nilausen, K., and Green, H. Reversible Arrest of Growth in Gl of anEstablished Fibroblast Line. Exptl. Cell Res., 40: 166 168, 1965.

35. Ong, T., and de Serres, F. J. Mutagenicity of Chemical Carcinogens inNeurospora crossa. Cancer Res.. 32: 1890 1893, 1972.

36. Orkin, S. H., and Littlefield, J. W. Nitrosoguanidine Mutagenesis inSynchronized Hamster Cells. Exptl. Cell Res., 66: 69 74, 1971.

37. Pederson, T., and Robbins, E. Chromatin Structure and the Cell

Division Cycle. J. Cell Biol., 55: 322-327, 1972.38. Plant, J. E., and Roberts, J. J. A Novel Mechanism for the Inhibition

of DNA Synthesis following Methylation: The Effect of N- Methyl- N-nitrosourea on HeLa Cells. Chem.-Biol. Interactions, 3: 337 342,1971.

39. Plant, J. E., and Roberts, J. J. Extension of the Pre-DNA SyntheticPhase of the Cell Cycle as a Consequence of DNA Alkylation inChinese Hamster Cells: A Possible Mechanism of DNA Repair.Chem.-Biol. Interactions, 3: 343 351, 1971.

40. Pound, A. W. The Influence of Preliminary Irritation by Acetic Acidor Croton Oil on Skin Tumour Production in Mice after a SingleApplication of Dimethylbenzanthracene. Benzopyrene or Dibenzan-thracene. Brit. J. Cancer, 22: 533 544, 1968.

41. Randerath. K., An Evaluation of Film Detection Methods for Weak/3-Emitters, Particularly Tritium. Anal. Biochem., i-/: 188 205, 1970.

42. Reznikoff. C. A.. Bertram, J. S.. Brankow, D. W., and Heidelberger,C. Quantitative and Qualitative Studies of Chemical Transformationof Cloned C3H Mouse Embryo Cells Sensitive to PostconfluenceInhibition of Cell Division. Cancer Res., 33: 3239 3249, 1973.

43. Reznikoff, C. A., Brankow, A. W., and Heidelberger, C. Establishment and Characterization of a Cloned Line of C3H Mouse EmbryoCells Sensitive to Postconfluence Inhibition of Cell Division. CancerRes., 33: 3231 3238, 1973.

44. Rothfels, K. H., and Siminovitch, L. An Air-drying Technique forFlattening Chromosomes in Mammalian Cells in Vitro. Stain Tech-nol., 33: li-li, 1958.

45. Stich, H. F., and San, R. H. C. DNA Repair and ChromatidAnomalies in Mammalian Cells Exposed to 4-Nitroquinoline-l-oxide.Mutation Res., IO: 389 404, 1970.

46. Taylor, E. W. Control of DNA Synthesis in Mammalian Cells inCulture. Exptl. Cell Res., 40: 316 332, 1965.

47. Tobey, R. A., and Ley, K. D. Isoleucine-mediated Regulation ofGenome Replication in Various Mammalian Cell Lines. Cancer Res..5/:46 51. 1971.

48. Warwick, G. P. The Covalent Binding of Metabolites of Tritiated2-Methyl-4-dimethylaminozaobenzene to Rat Liver Nucleic Acids andProteins, and the Carcinogenicity of the Unlabeled Compound inPartially Hepatectomized Rats. European J. Cancer, 3: 221 233,1967.

49. Warwick, G. P. The Covalent Binding of Metabolites of 4-Di-methylaminoazobenzene to Liver Nucleic Acids in Vvio. In: E. D.Bergmann and B. Pullman (eds.), Physico-Chemical Mechanisms ofCarcinogenesis, pp. 218 225. New York: Academic Press, Inc., 1969.

Fig. I. Control 60-mm Petri dish fixed and stained 3 weeks after seeding.Fig. 2. Morphologically normal colony fixed and stained 3 weeks after administration of MNNG, 4 Mg/ml. Synchrony induced by arginine

depriviation. x 60.Fig. 3. Malignantly transformed focus in a dish treated 3 weeks previously with MNNG, 4 Â¿Â»g/ml.Synchrony induced by arginine deprivation, x 20.Fig. 4. As Fig. 3 above, corded variant, x 20.

MARCH 1974 535



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1974;34:526-537. Cancer Res John S. Bertram and Charles Heidelberger

-nitrosoguanidine in CultureN-nitro-′N-Methyl-Nby Cell Cycle Dependency of Oncogenic Transformation Induced

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