Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

Chem.-Bitd. Interactions. 9 (1974) 351-364

351

Else,,Jer ScientificPublishingCompany, Amsterdam--Printed in The Netherlands

ENHANCEMENT BY ALKYLATING AGEN'['S OF CHEMICAL CARCiNOGEN TRANSFORMATION OF EIAMSTER CELLS IN CULTURE*

J. A. DIPAOLO, P. J, DONOVAN A,~l)B. C. CASTO** National Cancer Institute, Bethesda, Md. 20014, and **Bh~Labs. hw., Northbrook. IlL 60062 (U.S.A.)

(Received February 14th, 1974) (Revision receivedJuly 17th. 1974) (Accepted July 19th, 1974)

SUMMARY When Syrian hanlster embryo cells were pretreated with a weak chemical carcinogen, methyl mcthanesulfonate (M MS) or ethyl methanesulfonate (E MS), or with a physical agent such as X-irradiation vrior to being exposed to a potent cancerproducing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DM BA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene(AcAAF), and N-methyI-N'-nitro-N-nitrosoguanidine(MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with av::'lating agents: with both, maximum enhancement occurred approx. 48 h after tr,.atment and lethality attributable to the pretreatment was 10--20,% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 l t M of MMS demonstrated no alteration in normal cell DNA synthesis through 48-h. posttreatment. Analysis of unscheduled DNA synthesis by autora6iography or by alkaline sucrose gradients indicmed that the damaged DNA was rapidly repaired after treat*A portion of thi~ ,.,'~rk was performed pursuant to Contract NIH-NCI-E-C-71-2164with the National Cancer Institute. HEW. Abbreviations:AcAAF. N-acetoxy-2-acetylaminofluorene:BP. benzo(a)pyrene;BSS. balanced salt solution; DMBA. 7.12-dimethylbenz(a~anthracene;EMS. ethyl methancsulfonate;MCA. 3-methylcholanthrcnc: MEM. modified Eagle's medium; MMS. methyl mcthancsulfonate; MNNG. N-mcthyI-N'-nitro-N-nitrosoguanidine;SDS. sodium dodccyl sulfate; "i'CA. trichloroacetic acid; [31t)rdR, triliatcd thy~afidine.

352

J..~,. DiPAOLOet al.

merit. Therefore, repair of DNA damage as it is now understood is p~obably not involved.

INTRODUCTION

The transformation of Syrian hamster cells induced by chemical carcinogens is Poisson in distribution and may be influenced by a variety of fzlctors t. Pretreatt~:ent of cells by X-irradiation has been found to result in an increase in the number of transformations by BP 2. Enhancement of transformation is time-dependent; it increases during the first two days after irradiation, but with additional incubation may subsequently fall to normal or subnormal levels. Maximum enhancement occurred when chemical carcinogen is added 48 h subsequent to pretreatment of the cells with 250 R. These data and the demonstration of an absolute increase in the number of transformed colonies provide evidence that irradiation does not cause selection of irradiation-resistant cells rlore likely to be transformed by BP. Because MM~ ~ and other alkylating agents are known to be relatively weak carcinogens, the influence of MMS on the transformation by BP and by a mm~ber of other known potent carcinogens was determined. MMS is an alkylating agent which induces physicochemical effects similar to those produced by ionizing radiation in cell DNA. in additiona~ t,xperiments, other carcinogens were substituted for BP following treatment of cells with X-irradiation. There is evidence of common steps in the repair of alkylation and radiation damage '~.s. MATERIALS AND METtlODS

Transformation assay Stz,ndard methods and conditions for the transformation assay system have been described previously6. Briefly, cells derived from 12-14-day-old banister fetuses (Lakeview Hamster Colony) were grown as monolayers in Dulbecco's modification of Eagle's medium (MEM) with 100,o fetal bovine serum in a humidified 10°,,,~ CO: incubator kept at 37:'. In all experiments, 2-4-day-old secondary or tertiary hamster cultures were obtained by seeding 5 • 10~ cells/100 null petri dish (Falcon Plastics). Feeder layers were prepared by irradiation of confluent monolayers of hamster cell cultures in 5 ml of medium. A Picker Portable lndustri;d Apparatus (T55-433) was used at 101/kV and 5 mA, 2225 R/rain, adjusted so that a total of 5000 R in air was delivered at a distance of 22.2 cm. Feeder cells were seeded at 6 • 10"~/60 mm dish in 2 ml of medium. Hamster cells, 300 ceils/plate in 2 ml of medium, were seeded on the feeder layers. Cells were treated with a potent carcinogen in 4 ml medium, bringing the final volume in each dish to 8 ml. 8 days after seeding, plates were washed, fixed, and stained for colony counting and for examination of colony m o r p h o l o ~ with a stereoscopic microscope at 40 ×. A minimum of 20 24 plates were used for each experinaental point. Each experiment was repeated 3-4 times but only the results of single experiments are given.

ENHANCEMEN'I" OF CI'IEMICAL CARCINOGI'N TRANSFORMATION OF HAMSTER CELLS

353

The cloning efficiency was determined by dividing the average number of colonies per plate by the number of cells seeded per plate multiplied by 100. Transformation was calculated as percent of transformed colonies found in treated culture compared to the total number of colonies scored ('r/col ';~) or to the number of cells seeded per dish ~T/cell "o). The enhancement factor is the ratio of the transformation obtained from T/col ~,, or T/cell ",] of cells pretrealed with M MS or with X-irradiatio~n, followed by a potent carcinogen, divided by tl.e corresponding results obtained with cells treated with a pott:nt carcinogen only at the same time period. Bydefinition, the enhancement produced by treatment with only the potent carcinogen equals one.

Chemicals Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA), 7,12-dimethylbenz(a)anthracene (DMBA), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS) were obtained from Eastman Organic Chemical Co. N-methyI-N'-nitro-Nnitrosoguanidine (MNNG) was purchased from Aldrich Chemical Co. N-acetoxy2-acetylaminofluorene (AcAAF) was obtained from the Drug Development Branch, NCI. All chemicals were dissolved in acetone, 10 mg/ml, and immediately added to 100 ml of xvarm complete medium. The desired concentration was prepared by diluting the stock solutions with complete medium. The fina~ concentration of acetone was less than 0.02 °,i, Hydrocarbons were preprrcd in a darkened room illuminated by a red light and used as reported, while the other compounds were used immediately after final dilution. Survival cltrve.h~llowhlg M MS treatment The sensitivity of hamster cells to the monofunctional alkylating agent MMS was first determined. Secondary mass cultures of hamster celb, were treated with various concentrations of M MS for I h il~ complete mediurl, washed and seeded for colony formation, with the cell number varied to obtain approximately the same number of colonies per dish. The proportion of cells that survive6 and formed colonies was then assessed and the results normalized by designating as 100",i the cloning efficiency of untreated control cells. Transformation studies For transforn~ation studies, tlamster secondary cell cultures were pretreated with 100 or 250 !~M MMS (11 or 27.5 ug/ml of medium) for I h, washed with. Hanks BSS, trypsinized and seeded for colony formation. Alter incubation for 24, 48 or 72 h, the cells were treated with BP at a final concentration of 2.5 ,ug/ml. In other experiments, hamster secondary cultures were seeded for colony formation, incubated for 24 h, exposed to MMS (I I or 27.5/tg/ml) for I h, and the cells were then washed with Hanks' BSS and reincubated with 4 ml complete medium. To some plates, 4 ml of medium containing BP (final concentration 2.5 , g / m l ) w:as added to the treated ceils (0 h). To the remaining plates, BP in 4 ml complete medium was added 24, 48 or 72 h after MMS treatment.

354

J.A. DiPAOLOet aL

One series ofexperiments was also carried out by pretreating mass cultures with EMS at 12.5/~g/ml for I h and adding a second carcinogen 48 h after cloning of the cells as above. Treatment with other carcinogens Other potent chemical carcinogens (MCA. 2 pg/ml; DMBA, 0.5 ug/ml; MNNG, 1 pg/ml; and AcAAF, 2 pg/ml) were substituted for BP to determine whether the transformation induced by them was also enhanced by pretreatment of cells with X-irradiation or MMS. Hamster secondary cultures were irradiated'with. 250 R as confluent monolayers in 100 mm plastic petri dishes with. a Westinghouse Quandrocondex machine, with two tubes above and below, with the following factors: 200 kV (CP), 0.25 Cu, 15 mA, 54 cm TSD, output 139 R/min or treated with MMS (i I/,g/ml for 1 h) and then seeded onto hamster feeder cells and treated at various times with the above carcinogens. Effect o f M M S treatment on cell DNA synthesis DNA repair synthesis induced by different concentrations of M MS was determined on hamster embryo secondary cells by autoradiography ~ and by alkaline sucrose gradient techniques 8. Cells were incubated for 24 h in complett medium following transfer of 5 ' i0 s cells into dishes containing coverslips, after which the culture medium was replaced by an arginine-free medium containing 0.5~', fetal bovine serum for 48 Ix to inhibit scheduled DNA synthesis. Cells x~ere exposed to MMS (diluted in argininc-free medium) for I or 2 h, washed once, and fed with 2.5 ml of arginine-deficient medium with 0.5 .~o fetal bovine serum. One series of cultures was pulsed with thymidine (New England Nuclear, Boston, spec. act. 20 Ci/mmole) at a concentration of 10 ,,Ci/ml for the first 6 h (0-6 h) and another series for the subsequent 18 h (6-.24 h). The coverslips were rinsed, fixed, and prepared in the standard fashion, treated with Kodak nuclear track emulsion (NTB2), and exposed for 14 days at ,~. After devel(.ping, the cells were stained in ~ 0. i % crystal violet-0. I M citric acid soiution, rinsed, and covered with a second coverslip. The number of grains per nucleus was counted on 100 cells/coverslip (200 to 400 total cells) for each of the controls and chemical concentrations. DNA strand breaks by MMS and subsequent repair of the breaks were analyzed by sedimentation in 5-30°o alkaline sucrose gradients prepared by the method of BAXTER-GABBARt>t°. Hamster secondary cultures, after 24 h incubation, were pulsed with 0.5/~Ci/ml of [aH]TdR for 24 h, changed to complete medium containing 0.5"o bovine serum and incubated for an additional 24 h. Portions of the cell cultures were then treated with MMS for 1 or 2 h, washed and removed from the dish with EDTAsaline (0.05 % EDTA in PBS) at 0 and 6 h after treatment. Following sedimentation at low speed, the cells were resuspended in EDTA-saline to give l0 s cells/0.2 ml. Control and MMS-treated cell suspensions (0,2 ml) were added to ti~c top of a sucrose gradient tube layered with 0.2 ml of lysing solution ( 1.0 N NaOH). The cells were lysed at room temperature for 1 h, placed in a SW-50 rotor and centrifuged for i h at 30 000 rev./ min in a Model L-2 ultracentrifuge at 20 ~. Three-drop fractions were collected directly

ENHANCEMENI" OF CItEI~:I('AL CARCINOGEN TRANSFORMATION OF ItAMSTER CELLS

355

into scintillation vials following bottom puncture, neutralized with 0.5 ml of 0.2 N HCI and prepared for counting by adding 5 ml cf Bray's scintillation fluid to each vial. Counts were made in a Packard Tri-Carb scintillation spectrometer. 10 min per sample and the data plotted as percent of the higltest count in each gr:tdient. Any variation in sedimentation of the cell DNA between separate nltracentrifl~gations was taken iw~to consideration by including lysed+ untreated control cells into each individual run. Alterations in scheduled cell DNA synthesis after treatment with MMS was determined in secondary cultures of hamster embryo cells which were synchronized by incubation for 48 h in medium containing 0.5 ~o] serum. At 4-h intervals, 3 plates each of treated and control cells were pulsec with [ 3 H ] T d R ( I . 0 / + C i / m l ) for 2 It. Following this labeling period, the cells were lysed with 10O~ SDS in EDTA-saline (2 ml/dish) and a 1.0-ml aliquot precipitated with 100,,~cold TCA (iinal concentration 5°o). One-half of the precipitated sample was collected on an 0.45/+ Millipore filter, dried, scintillation mixture added (Packard pre-mix "p"-toluene), and counts determined in a liquid scintillation spectrometer. R~UL~

The survival rate of a variety ofdifferent types ofceUs after treatment with MMS has previously been shown to decrease with increasing time of exposure to the drug. For the present studies, an exposure period of I h was selected, Fig. l shows the survival curve of hamster cells treated with various dose levels of MMS for I h prior to being plated on hamster feeder layers for colony formation. Similar results were ob+oo

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Fig, I. Cloning efficiency (survival) of secondary hamster embryo culture exposed for l h to MMS; cells were cloned subsequently and treated with BP. Results were normalized by considering the cloning et~cicncy with 0.0 (l--1), and 2.5/+g (O) of BP per ml of medium as 100~.

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Fig. 2. Transformation frequency transformed colonies/total colonies obtained with BP following M MS treatment. (A) Transformation frequency with pretreatment with M MS before plating cells for colony tormation and (B) Transformation frequency obtained with prctreatment with MMS of cells plated for colony formation. A, MMS II + BP 2,5 /~g:ml; A, MMS 27,5 -e BP 2.5 /~g/ml: O, control BP 2.5 !lg/ml.

mined with EMS. The results were normalized by designating as 1(20~o tile cloning efficiency obtained with untreated controls or BP 2.5 !lg/ml without ~ M S . The actual cloning efficiencies were 10.9 % and 7.5 % respectively. The t~vo lowest concentrations of MMS consistently produced only 10-20'3o lethality relative to control and were selected for use in combination with the other chemical carcinogens. Curves of similar shape have been reported after exposure of Chinese hamster cells and mouse lymphonut cells to higher concentrations of MMS. in these experiment:,, few colonies with transformed morpho'ogy were seen among the survivors of normal cells exposed to M MS; the frequency never exceeded 0.3 % and consequently was not included in calculations in determination of enhancement by other carcinogens. A temporal relationship in the incidence of transformation between pretreatment with MMS and subsequent treatment .vii'3 BP was found when carcinogen was added at daily intervals after seeding the cells which had been pretreated with MMS (Fig. 2A) or, by first plating the hamster cells a~ ti~r cloning and commencing the pretreatment with MMS 24 h later (Fig. 2B). In this latter situation, it was possible to add BP at the intervals indicated, including period 0 which was I h subsequent to the addition of MMS. it is apparent that whether MMS pretreatment is api~lied to cells in mass culture prior to plating for colony formation or directly to cells that have been

ENHANCEMENT OF CHEMICAL CARCINOGEN TRANSFORMATION OF HAMSTER CELLS

357

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Fig. 3. M M S e n h a n c e m e n t o f transl'ormation o f hamster c m b r y o cultures exposed to two concentrations o f M M S p r i o r to a n d subsequent to cloning follo~ed by t r e a t m e n t with 2.5 ~ g o f BP at the h o u r s indicated. T h e n u m b e r o f t r a n s f o r m e d colonies from M M S treated cells was c o m p a r e d with that f r o m BP treatment only cells cultured for the same length o f time. A , M M S I I -~- BP 2.5/~g/ml; ,'~, M M S 27.5 ! FIP 2.5 l;g/ml; - - , control BP _2.5 ttg/ml,

seeded for colony formation, the maximum transformation frequency occurs when BP is added 48 h subsequently. This period of addition of BP coincides with either 48 or 72 h post-plating the cells for colony formation. It is apparent that in either case a slightly greater increase in the transformation frequency is obtained with. the lesser concentration (11 l~g MMS/ml of medium). The maximum enhancement with the lower concentration of MMS was 5.5- and 5.I-fold, respectively, for cells which had been pretreated before or after being pl,~ted for colony formation (Fig. 3A and B). In no experiment was there greater enha,:cement obtained with. the higher concentration of MMS than with the lower concentration. It is obvious that the enhancement obtained at the other periods tested may be slightly higher or lower than that obtained with BP only, but in no case does it appear to be significantly different. Data obtained from an ~ther experiment are presented (Table I) to show the variability which may occur between experiments as well as within experiments. As has been observed in the past, the cloning etttciency usually increases even with treatment over the first 48 h. The cloning efficiency is different in this experiment from that given in Fig. 1 since in this experinaent the cells were seeded for colony formation prior to addition of MMS. The total number of transformations increases over the first 48 h with :he maximum occurring as in the pre~'ious case when 11 p g MMS/ml of medium

358

J.A. DiPAOLOel aL

TABLE I TRANSFORMATION OF HAMSTER FETAL CEI_L.~ AFTER SEQUENTIAL TRF~T~IENT V*'ITH ~ | Nt~ &ND

BP'

Period

0

72

MMS~g]ml Total number of colonies Cloning efficiency (%) Total T T]Cell % Enhancement T/Col. % Enhancement

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II

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966

840

746

1432

1142

1056

1528

1129

1004

1250

1073

1035

24

15.3 13.3 12.4 32 48 32 0.507 0,762 0 . 5 3 3 1.0 1.5 I. I 3.31 5.71 4.29 1,0 1.7 1.3

48

19.9 16.6 14.7 21,2 17.1 55 43 61 32 238 0,764 0,623 0.847 0,444 3.t,0 1.0 0,82 I. 1 1.0 8. I 3.84 3,77 5,78 2,09 21.1 1.0 1.0 1.5 1.0 l0

14.6 18.1 15,6 171 25 47 2,47 0.362 0,681 5.6 1.0 1,9 1 7 . 0 3 2.00 4.38 8. I 1,0 2.2

15,0 34 0,493 1.4 3.29 2.7

~'Cells were seeded on a feeder layer and prctreated with MMS t'or one hour 24 h later, BP final concentration 2. /.~g/ml was added at periods indicated: period 0 refers to first hour after MMS pretreatment.

was added 48 h before BP. The enhancement factor is significantly increased at 48 it whether it is based on % T/cell or % T/col. These results showed certain similarities to those obtained when the cells had been pretreated with X-irradiation in that maximum enhancement t~curred 48 h following pretreatment. Additional experiments were performed to determine whether pretreatment with X-ray or M MS would also enhance the incidence of transformation with a variet) of other chemical carcinogens. When cells in mass culture were pretreated with X-irradiation or with MMS for I h before being plated for colony formation, and then treated with the chemicals indicated in Fig. 4 at 48 h, significant enhancement was obtained with all the carcinogens used. Furthermore, if the concentration

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Fig. 4. Pretreatment of mass cultures with X-ray or M MS for I h prior to seeding the cells for colo~.v formation, The various carcinogens were added 48 h subsequent to seeding ,'ells for colony formation, The enhancement factor is the ratio of transformed c,~lonics following double treatment compared with that from cells treated with the carcinogen only, m, X-ray, 250 R: E . MMS, I I t~/ml,

ENHANCEMENT OF CttEMICAL CARCINI,'K3EN TRANSFORM.,VIION O1"- IIAMSTER CELLS L~CT

359

OF MMS TREATMENT ON DNA SYNTHESIS

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HOURS AFTEO, TREATk~.~'NT Fig. 5. Etl"cct of M MS treatment on cell I')NA synlhesis. Secondary hamster embryo cells ~vere incubaled in medium 'containing 0.5 ", serum for 48 h, exposed to M MS f,~r I h (27,5 and I 1.0/~g/ml). and oh:raged to medit~m cont;lining I0",, serum. At 4 h intervals, 3 plates of eath concentration and controls x~ere ptdscd for 2 h ~ith ['~H ]TdR t l,O 'd?i roll The cells xxere t,'~cd with I0!',,~ SDS, the D N A precipitated in 5 " , TCA, the precipitate collected on tihcrs and an~otmts of radioacti¥ity retained on the tillers xsas determined by liqt|id sc~millation techniques, Q, control: O, 11.5 /tg;ml; • . 27,5 l, gr'n11,

of the chemical used was doubled, the ~nhancement ratio remained constant. The ratio of enhancement s~ith MMS pretreatment ranged from a low of 4 with M N N G to a high of 7.4 with BP, howe',er, after pretreatment with radiation the enhancement factor was greater than that obtained with MMS pretreatment with a low of 7.5 for M N N G and a high of 8.8 for DMBA. If EMS was substituted for M MS and BP added after 48 I1, the enhancement (7.5) was similar to that obtained with MMS. Treatment of synchronized hamste:" cells with 27.5 or I 1 , g / m l MMS gave no evidence of apparent delay or alteration ~n cell DNA synthesis in the first or second cycle following MMS exposure for I h (Fig. 5). Radioautography of MMS-treated hamster embryo cells demonstrates that cell DNA repair synthesis occurs at both the 0-6 h and the 6-24 h labeling periods at concentrations equal to, or higher than, those used in the enhancement experiments (Fig. 6A and B), The rate of activity after MMS treatment appeared to be higher in the 0-6 h than the 6-24 h period. Most of the repair that occurred ~,ith 25/~g/ml appeared to be completed relatively early and did not coincide with the time of maximum enhancement of carcinogen-induced transformation. Direct evidence for early repair of breaks in cell D N A following MMS treatment was also obtained in several experiments by sedimentation of DNA from M MS treated cells in alkaline sucrose gradients. Cells treated with 27.5/~g/ml (250/~M) of MMS for 1 h demonstrated a marked decrease in the molecular weight of the cell DNA when tested immediately after, treatment. However, incubation for an additional 6 h resulted in a shift of the sedimentation profile to nearly control values (Fig. 7). In two of the three experiments, a 2-h

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Fig. ,~. Alkaline sucrose Gradients o f D N A from cells treated 48 h previously ~ i t h M M S and subsequently treat,,d with A c A A F . Secondary hamster embryo cells were treated for I h with 2"/.5 pg'm~ o f M MS, washed once and labeled osernight with 1.0/~Ci: ml o f [-~t! |Fd R. 4,g h after M MS treatment, treated and control ceils were incubated t\',r 2 h with 5 ygrml u f A c A A F , In'm~ediatcly following treatment. the cells ',,,'ere lysed on alkaline gradients and centrifuged as described in Fig. "/. Fig. 9. Alkaline sucrose gradients o f D N A f r o m cells treated 48 h previously with M M S . S e c o n d a r y h a m s t e r cells v.crc treated for t h v.ith 27.5/~g:ml o f M MS, washed once, labeled with [all )TdR for 18 h a n d c h a n g e d to m e d i u m c o n t a i n i n g 0 . 5 % fetal bovine serum. 48 h after treatment, treated a n d control cells were lysed o n alkaline g r a d i e n t s a n d centrifuged as described in Fig. 7.

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incubation with A c A A F (5/Ig/ml), added 48 h after MMS, resulted in a greater decrease in the sedimentation velocity of the cell DNA when contrasted to that observed with DNA from cells treated only with A c A A F (Fig. 8) or MMS (Fig. 9). DISCUSSION MMS or EMS pretreatment of hamster cells bs vitro results in enhancement or" transformation by BP as well as by other potent chemical carcinogens. The neoplastic property of the morphologically altered clones has been verified by subcutaneous injection of cells into weanling hamsters: progressively growing fibrosarcomas result (unpublished data). Although the alkylating compounds produce a few transformants by themselves and X-irradiation does not, there are similarities between the results with M MS or EMS followed by potent carcinogens and those previously reported with X-irradiation 2. When cells were pretreated with an alkylating agent, maximum enhancement was observed at 48 h subsequent to the exposure to MMS and was independent of whether or not the M MS had been applied to cells in mass culture or to cells which had been previously seeded for colony formation on an irradiated hamster cell layer. The results obtained with MMS or EMS differ front those with X-irradiation in that a higher enhancement ratio is obtained with X-irradiation and enhancement is demonstrable if the second carcinogen is applied 6, 12 or 24 h after treatment, whereas with MMS, no enhancement is obtained at these earlier times. Increasing the concentration of M MS from i ! to 27.5 .ug~ml causes no further increase in the enhancement of transformation, nor do the two concentrations of MMS differ significantly in decreasing cell survival relative to controls. It is not known how MMS treatment enhances transformation by chemical carcinogens, nor is it known why maximal enhancement is obtained 48 h after MMS treatment. The uniqueness of this time period (48 h) does not appear to be related to partial cell synchronization or alterations in the cell cycle, since the rates of DNA synthesis were indistinguishable through 48 h incubation in treated and control cultures. Unscheduled DNA synthesis, while appearing to be relevant to enhancement of viral transformation by M M S ~2, is of relative unimportance when M M S is used at the aforementioned concentrations to obtain increased transformation by potent chemical carcinogen, since dentonstrable D N A repair synthesis induced by 25 .,g/ml or MMS essentially ends within the first 6 h after treatment as analysed by autoradiography and alkaline sucrose gradients. In preliminary experiments, treatment with M MS increased the sensitivity of cell DNA to damage by the addition of a second agent (AcAAF): but additional experiments must be performed to determine whether other carcinogens will produce similar effects. Studies with a variety of different types of cells in culture indicate that MMS may affect the production of new DNA by reducing the relative rate at which it is synthesized '3. Although there is no qualitative difference between the DNA in control and alkylated cells, there are important quantitative distinctions, MMS, as opposed ~o X-ray insult, produces single-stranded DNA in HEp2 cells that persists for longer

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363

periods of time ~'~. Thus, the relative rate of synthesis of D N A is reduced, a c c o m p a n i e d by an increase in the average a m o u n t of single-strandcdness per D N A fragment : the fragments fronlt alkylated cells are also larger, In n o n - m a l i g n a n t h u m a n cells the net effect o f c o n c e n t r a t i o n s of M MS higher than that used in these e n h a n c e m e n t studies results in an increase in the relative length rather than in the frequency of occurrences o f single-stranded regions' s It is interesting to refer to oll~er biochemical work that I~as been done with cells in ctflture a n d in animals. SWAIN ANI~ M('GEI0 s h'tve rcportcd a lack of correlation between th,e a m o u n t o f alkylat:oil o f D N A a n d the p r o d u c t i o n o f tutaors by MMS. Whereas rare brain t u m o r s are found in rats following a single dose o f M M S , consistent with the hypothesis that alkylation of D N A a n d carcinogenesis are causally related, n o kidney t u m o r s were f o u n d and the metl'tylation of kidney D N A is high after M M S treatment, it is suggested that the alkylation o f something othe~ titan D N A may be responsible for the carcinogenic action o f M M S or that alkylation plays n o part a n d some entirely different reaction is involved. Studies on the biochemical effects of irradiation on the D N A of cells in culture have also led to some puzzling resttlts. Fox .',NI) F o x ~* h.ave reported that two lines of lymphoblasts, L 5 1 7 8 v s a n d R., show a large difference in radiosensitivity as indicated by a difference in let!'~'dity a n d in the n u m b e r of bases inserted per break in the cell lines, but b o t h lines have a similar n u m b e r of strand breaks a n d identical levels o f repair replication. As a consequence, it is concluded that single-strand breaks or the rate of repair are not the cause of the lethal event in m a m m a l i a n cells a n d that o t h e r processes must be tire d e t e r m i n a n t s in the ultimate survival of irradiated cells. N o t until the nature of the initial reaction is better u n d e r s t o o d will it be possible to o b t a i n meaningful i n f o r m a t i o n concerning the ability of a cell to repair d a m a g e to D N A , a n d to elucidate the nature o f M M S e n h a n c e m e n t o f t r a n s f o r m a t i o n induced by chemical carcinogens. REFERENCES 1 J. ,,%. DIPAOLO. P. J, D3.~OVA~ aNl~, R. L. NEt.StUN, la vitro transformation of hamster cells by polycyclic hydrocarbor~.: Factors influencing the number of cells transforn:ed, Nature New Biol., 230 (1971) 240-242. 2 J.A. DIPAoLO. P. J. DoNov.~,s ant, R. L, Nvt s(),~, X-Irradiation enhancement of transformation by benzo(a)pyrene in hamst:," embryo cells, Pr~¢'. Natl. Acad, Sci. (U.S.), 68 (1971) 1734-1737. 3 J. V, FRt-I.Tumour induction by low molecular weight alkylating agents. Chem.-Biol. Interactions, 3 (1971) 117-121, 4 M. BR~Nt~EI,.N, A. KHaN ,~N~ R. H. H,XYNES.Common steps in the repair of alkyiatio~ and radiation damage in yeast, Mol. Gen. Gt'net., 106 (1970) 289-295. 5 R, B. SErLOW ANL~J. K. SErcow, Effects of radiation on polynucleotides, Ann. Rev. Biophys. Bhwng,, (1972) 293-346, 6 J.A. DIPAot.o, P. J. DONVOANA N D R. L. NEt.SON,Quantitative studies of in vitro transformation by chemical carcinogens, 3. Natl. Cancer Inst., 42 (1969) 867-874. ~' R.B. P/UNTtR ANDJ. [. Ct, tAVEV~,Repair replication in HeLa cclls after large doses of X-irradiation, Nature, 216 ;,196"/)36.~-370. R. A. McGRATH ASt~ R, W. WILLIAMS. Reconstruction in vivo of irradiated Escheriehia colt deoxyribonucleuc acid; the rejoining of broken pieces, Nature, 212 (1966) 534-545. 9 J.J. FREEr>AND S, A. SCaATZ, Chromosome aberrations in cultured cells deprived ot" a single essential amino acid, EaT~tL Cell Res., 55 (1969) 393-409.

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10 K, L. BAXTER-GABB.*,,~.D,A simple method for the large-scale preparation of sucrose gradients. FEBS Letters, 20 (1972) i 17-119. 11 R.R. BURK, Growth inhibitor of hamster fibroblast cells, Nature. 212 (1976) 1261-.1262, 12 B.C. CASTO,W. J. PZZCZY~SKiAND J. A. DIP^OLO, Enhancement of adenovirus transformation by treatment of hamster embryo cells with diverse chemical carcinogens, Cancer Rcs., 34 (1974) 72-78. 13 M. Fox ^~a S. R. ArAb, Characteristics of repair synthesis i~l P388 cells treated with methyl methanesulfonate, Chem.-Biol. Interactions, 3 (1971) 193-211, 14 D, SCUDJE~O AND B. STn^USS, DNA synthesis by alkylated cells, in P,O.P. Ts'o AND J. A. D~P^OLO (Eds.), Chemical Carcinogenesix, Part A, Marcel Dekker, New York, 1974, p, 381-399, 15 S . N . BUnL AND J. D. RECiAN, DNA replication in human cells treated with methyl methanesulfonate, Mutation Res,, 18 (|973) 191-197. 16 J.J. RoaFnTs, J. E. STUm~OCKAND K. N. W^RD. DNA repair and alkylafion-induced toxicity and mutagenicity in mammalian cells, in P.O.P. Ts'o ^~o J. A. DIPAOLO (Eds.), Chemical Carcinogenesis, Part A. Marcel Dekker, New York, 1974, p. 401--425. 17 J.J. ROnER'rS,J, M. PASCOE. B. A. SMITHAND A. CARTHORN,Quantitative aspects of the repair of alkylated DNA in cultured mammalian cells, il. Non-semicons~rvative DNA synthesis (repair synthesis) in HeLa and Chinese hamster cells following treatment with alkylating agents, Chem,Biol. Interactions, 3 (1971) 49--68. 18 P . F . SWA~ A~D P. N, MAGEE. Nitrosamine-induced carcinogenesis, Biochem. J., 110 (1968) 39-47. 19 M. Fox AND B. W. FOX, Repair replication in X-irradiated lymphoma cePs in vitro, Intern. J. Radiation Biol., 23 (1973) 333-358.