[CANCER RESEARCH 27 Part 1, 1805-1812, October 1967]

Effect of Methyl Methaiiesulfonate on the Growth of P388

Lymphoma Cells in Vitro and on Their Rate of Progressthrough the Cell Cycle1

MARGARET FOX AND B. W. FOX

Palerson Laboratories, Chrislie Hospital and Holt Kadium institute, Manchester 20, England

SUMMARY

The effect of methyl methanesulfonate on the growth kineticsof the cloned line P388F of the P388 lymphoma in vitro in suspension culture was studied. The proportion of live and dead cells,mitotic index, labeling index, percentage of labeled mitoses, andgrain counts were examined in an asynchronous population,during the period of departure from exponential growth. Fromthis data, it was shown that the S (DNA-synthetic) period wasthe most sensitive phase of the cell cycle. DXA synthesis fromlabeled thymidine was increased and the duration of the S wasprolonged. The rate of passage of cells from S to Gz was moreaffected than that from Gìto S, but both were reduced relativeto control rates. Cells in G2 at the time of treatment were notdelayed through the first post-treatment mitosis but were delayedthrough the subsequent Gì.Cells in Gj at the time of treatmentwere delayed in their passage through the subsequent S i>eriod.This alkane sulfonate induced periods of increased mitotic activity, each of which was followed by a wave of increased cell death.Recovery to exponential growth at the control rate occurred fourdays after treatment.

INTRODUCTION

Alkyl alkane sulfonates are capable of transferring an alkylgroup in vino to a nucleophilic site within biologic tissue (23).Variation of the structure of this alkyl group has been shown toinduce a variety of biologic responses in sjiermatogenesis (9,13, 21), leukopoiesis (8, 9), bacteria (27), bacteriophage (19),plant tissue (20), and fungi (1). Some of these esters show anti-tumor projierties (9, 12), and the simplest member, methylmethanesulfonate (24), has been shown to exert an anti tumoraction on Lymphoma 8 (26). This ester has further been examinedin some detail with regard to its action on nucleic acids (2, 14)and was shown to react with several bases within the nucleicacid, particularly with the N-7 position of the guanine molecule.Furthermore, systems are normally present, at least in bacteria,which can repair such alkylation (28).

With these observations in mind, it was considered of interestto examine in detail the action of methyl methanesulfonat a onemouse lymphoma, P388F (10), grown in vitro in suspension cul-

1This work was supported by grants from the British EmpireCancer Campaign and The Medical Research Council.

Received February 14, 1967; accepted May 29, 1967.

ture, in an attempt to understand the extent and nature of theinhibition of the growth kinetics, based on parameters derivedfrom measurements made in an asynchronous jwpulation.

MATERIALS AND METHODS

The P388F clone was isolated in 1963 in Dr. G. A. Fischer's

laboratory, Pharmacology Department, Yale University, from aDBA/2 mouse bearing the transplantable lymphocytic leukemiaP388.

The methods of isolation and culture of murine lymphoma lineshave been described previously (7). For all experiments, cellswere grown in the medium described by Fischer and Sartorelliwith the addition of 10% horse serum (Grand Island BiologicalCompany, New York). Cells were normally grown in static suspension culture and were maintained in logarithmic growth at concentrations up to 5 X IO5cells per ml by appropriate dilution

every two to three days. Under these conditions, the populationdoubling time was twelve to fourteen hours. The durations of thevarious phases of the cell cycle have been shown to be as follows:S = 6.5 hr; G2 + JM = 1.5 hr; GÌ= 4 hr (10).

Methyl Methanesulfonate Treatment

Methyl methanesulfonate (AIMS) was dissolved in growthmedium without serum and sterilized by passage through a membrane filter (pore size, 0.22 n, Millipore Filter Corp.). The concentration of the stock solution was adjusted so that the requireddose was given by the addition of between 0.1 and 0.2 ml to the5 ml cultures. Cultures were exposed to the drug for 1 hr, 3 hr,or 12 or more hr. For the short exposure experiments, MMS wasadded to cultures (IO5(«Ilsper ml) which at the end of the treat

ment were either diluted tenfold into fresh medium or centrifuged(800 rpm for 5 min) and the drug-containing medium replacedwith fresh medium. In long treatments, the drug was allowed toremain in cultures with a cell concentration of IO4cells per ml.Since the half-life of hydrolysis of methyl methanesulfonate inaqueous solution is about 9.1 hr at 37°C(24), the cells would be

treated with a mean dose level over the whole cell cycle of 69%of the concentration at the moment of mixing, and 60-70% of the

total MMS will have been destroyed by hydrolysis or reaction bythe end of one cell cycle (12.0 hr). The survival of cells aftertreatment with MMS was determined by the extrapolated growthcurve technic described elsewhere (11).

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Margaret Fox and B. \Y. Fox

Counting Procedures

Cell concentrations in growth rate experiments were determined by counting aliquots from replicate tubes in a standardhemocytometer. Live and dead cells were determined using theexclusion of nigrosin as an index of viability. Two parts of thecell susjjension were mixed with one part of a 0.1 % nigrosin solution in physiologic saline. The wet preparations were counted in astandard hemocytometer ten minutes after the addition of nigrosin.

Slide Preparation

For determination of the fraction of labeled mitoses, labeledcells, and of grain counts, slides were prepared for autoradiog-raphy as described previously (10). The mitotic index was determined at various times after treatment from unlabeled slidesprepared in the same way and stained with acetoorcein. A totalof IO3cells derived from two slides were scored for each point.

Labeling Experiments

Pulse labeling was used for the determination of the mitoticwave. Initiated thymidine (specific activity, 5.0 c i>er mmole;Radiochemical Centre, Amersham, England) was added to cultures to give a concentration of 0.1 MCl>er ml of medium. Thelabeled thymidine was followed thirty minutes later by unlabeledthymidine to give a final concentration of 5 ¿ugl>ei'ml of medium.

Immediately after addition of unlabeled thymidine, MAIS wasadded and left for one hour. At the end of this )>eriod, cultureswere centrifugea and fresh medium added. Samples were takenat one to two hourly intervals over the following twenty-twohours and subsequently prepared for autoradiography. One hundred mitoses were scored for each point, and these were derivedfrom two to three slides. An average of 50 to 60 grains per cellwere obtained after three days of exposure.

Labeling index of cultures (IO6 cells per ml) was determined

after treatment with the drug for one and three hours and dilution tenfold into fresh medium. Tritiated thymidine (0.1 juc |>erml) was added to cultures for thirty-minute periods at one toforty hourly intervals up to 120 hr after treatment, and slideswere prepared for autoradiography as previously described. Thefraction of labeled cells was determined by counting 500 cells foreach point.

Grain count measurements were made on the same slides aswere used for the pulse labeling experiment. At the level oftritiated thymidine used, labeled cells were easily distinguishablefrom background after three days of exposure, and in controlcultures, an average of 50 to 60 grains |>er labeled cell was seen.The number of grains over a minimum of 40 cells was determinedin control and treated cultures, and the change in grain countwith time at different dose levels was determined.

Continuous Labeling. Cultures (IO5 cells per ml) weretreated with M MS for one hour, then centrifuged for five minutes,and the drug-containing medium replaced by fresh medium containing tritiated thymidine (0.1 /jc per ml). Samples were takenat hourly intervals for up to ten hours after addition of thymidineand the fraction of labeled cells determined by scoring 500 cellsfor each point.

RESULTS

Inhibition of Cell Growth

The survival of P388F cells after treatment with M MS for oneor three hours was essentially the same. If the drug remained incontact with the cells for twelve hours or more, however, a muchsteeper survival curve was obtained (Chart 1).

Two doses were chosen for further study, 0.09 mMgiving 75%survival and 0.36 HIMwhich reduced the surviving fraction toapproximately 1%.

The changes in the relative proportions of live and dead cellsduring the i)eriod of departure from exponential growth after adose of 0.36 HIMfor one hour are shown in Chart 2A. During thefirst ten hours after addition of the drug, there was little or noincrease in the live cell population, but between ten and forty-

10

10

0.25 0.5 0.75DRUG CONC (mM)

1.0

CHART1. Dose-survival curve of P338F lymphoma in vitro aftertreatment with methyl methanesulfonate for (O O) 3 hr, or(X X) 12 hr.

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Effect of Methyl M ethanes uIfonate oti Lymphoma Cells

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HOURS AFTER ADDITION OF DRUG

CHART2. A, changes in live cell numbers of P338F lymphomacells during the first 140hr after 3-hr treatment with methyl meth-anesulfoiiate (0.30 HIM).O O, control population; X X,treated cells. B, changes in the numbers of dead cells expressed asa percentage of the total population after a 3-hr treatment withmethyl methauesulfonate (0.30 mm). The method of determiningdead cell numbers is described in the text.

five hours, numbers increased at approximately the same ratein control and treated cultures. This rate of increase in cellnumber in treated cultures was not maintained, however, andfrom 50 to 100 hours, numbers remained stationary.

The changes in dead cell numbers (expressed as a |>ercentage ofthe total population) are shown in Chart 2B. Numbers remainedat control levels (1.1 ±0.023%) over the first 24 hours, but rosesharply to a peak (27%) of the population by 30 hours. Afterfalling to approximately 9% at 55 hours, the numbers againrose sharply to 25% at about 70 hours. Numbers then fell slowlytowards control levels by four to five days.

Mitotic Index Changes

The changes in the mitotic index at various times after treatment at doses of 0.09 IÕIMand 0.36 «IMfor one hour are shown inChart 3 and are expressed as % of the control levels (2.55 ±0.22).

The number of mitoses in the treated cultures began to fallone to two hours after addition of the drug, and minimum valueswere reached for both doses by six hours. Between eight andtwelve hours, the mitotic index in both treated cultures returnedto near control levels. At 0.09 mM, a slight overshoot occurredfollowed by another low value at 20 hours, then by thirty hoursthe mitotic index had again reached control levels. After thehigher dose (0.36 HIM), the overshoot was considerably moremarked and reached higher values, viz. 210% of the controlvalue at 24 hours; after this peak the values fell precipitouslyuntil at 40 hours the mitotic index was some 20% of the controlvalues. Normal values were again evident by 65 hours.

Labeled Mitotic Wave

The results are indicated in Chart 4. The rate of accumulationof the percentage of labeled mitoses was identical in control andtreated cultures during the first 6 hr, even though the mitoticindex in the treated cultures was decreasing at this time (Chart3). Thus cells in 62, i.e., between end of synthetic (S) ]>eriodandmitosis (M), at the time of treatment would ap]>ear to progressinto mitosis at normal rate, while those in S, apart from those atthe very end of S period, are somewhat delayed. Cells from Gìdo not enter mitosis at the time expected from the control experiments, and there was no evidence of these cells appearing duringthe 22 hours of the expriment, as indicated by the near 100%labeled mitoses between 8 and 16 hr. In the meantime, controlcultures showed their second labeling maximum at 16-18 hr,and by 22 hr the number of labeled mitoses had begun to decreaseagain as unlabeled GÌcells began to divide again.

Labeling Index

The changes in the labeling index at various times after twodoses of methyl methanesulfonate are shown in Chart 5.

The labeling index in the control population was 51.2 ±5.1%There was an increase of labeling index up to 8 hours after administration of the drug (0.36 UIM)with a maximum labeling of128% of control. The labeling index then fell to minimum valuesat 18 hours. Recovery to control levels was fairly rapid at thelowest dose (0.09 mM), but after 0.36 IÕIM,control levels were notreached until 90 to 100 hours. The return to control labeling indexcoincided with the return to exponential growth at control rates.

The rate of increase of labeled cells during continuous incubation of cultures with tritiated thymidine is shown in Chart 6.In the treated cultures, the overall rate was slower during theten-hour ])eriod studied. The control cultures reached maximumlabeling (95%) by eight hours, whereas in treated cultures,only 80% of the cells were labeled by ten hours.

Grain Counts

Cultures treated w'ith 0.09 mM showed little change in grain

count, but at higher dose levels, values of 50% of control wererecorded at 18 hours after treatment (Chart 7).

Recovery to normal levels occurred more rapidly at the lowerdose but even at 0.36 mM, was complete by 42 hours.

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Oce

ou

20

MMS I hr

30 40 50 60 70 80

HOURS AFTER ADDITION OF DRUG

90 100

CHART 3. Changes in mitotic index (expressed as percentage of the control mitotic index) of cultures treated with 0.09 mM (X X)and 0.36 mM (O O) methyl methanesulfonate (MMS) for 3 hr.

100

LU80

g 60_lLU

< 40

20

6 8 IO 12 14 16

HOURS AFTER ADDITION OF MMS18 20 22 24

CHART 4. The labeled mitotic wave at various times after a 1-hr treatment of P388F cells in vitro with methyl methanesulfonate(MMS) 0.36 mM. Pulse label of tritiated thymidine was given 30 min prior to addition of the drug. O O, methyl methanesulfonate(0.36 mm); X X, methyl methanesulfonate (0.09 mw).

DISCUSSION

In attempts to determine the nature of the inhibition of growthof cells in culture, synchrony or partial synchrony can be inducedprior to drug treatment by selecting cells in a specific stage of thecell cycle or by arresting the cell cycle at a point for a shortperiod, followed by a release from the block. Both these methodshave the basic disadvantage that unknown biochemical changesmay have occurred which could influence the sensitivity spectrum

to the drug or physical agent under investigation. The use of5-fluoro-2'-deoxyuridine has been described (25) as well as its

special application to the study of the site of action of nitrogenand sulfur mustards (30). From this work, it was shown that theDXA synthetic period of strain L mouse fibroblasta (S) was themost sensitive period of the cell cycle, the GI and GÃŒperiodsbeing less sensitive. In a later paper (29), it was possible to confirm this result by showing that the sensitivity was independent

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Effect of Methyl Methanesulfonate on Lymphoma Cells

20 120HOURS AFTER ADDITION OF DRUG

CHART5. Changes in the percentage of labeled cells at varioustimes after treatment of P388F cells in vitro with methyl methane-sulfonate, 0.09 nw and 0.36 mm. The labeling index in treatedcultures is expressed as a percentage of the control labeling index.O O, methyl methanesulfonate (0.36 HIM); X X, methylmethanesulfonate (0.09 HIM).

of the amount of mustard bound to the cells in both S and 62stages of the division cycle. The actual spectrum of sensitivitymay, however, be influenced by the process of synchronization.

The work described here utilizes several parameters derivedfrom pulse and continuous labeling with thymidine-3H to assess

the site and nature of the damage produced by methyl methanesulfonate in an asynchronous population, and is thus not influenced by any specific effects induced by the S3rnchronizationprocess. Each stage of the cell cycle will be considered in turn.

S (DNA Synthetic) Stage

The reduction of the mitotic index of the P388F cell populationsix hours (representing 4.5 hr of S plus the 1.5 hr of Ga) followingtreatment at two dose levels of methyl methanesulfonate (Chart3) suggests that the primary action of the drug is on cells in Sat the time ot treatment, their rate of progress through thisstage being reduced. The duration of this inhibition, however, isrelatively short since between twenty and twenty-four hours,large numbers of cells enter mitosis, the majority of which arelabeled. The magnitude and duration of this peak in mitoticindex indicates either that partial synchrony has been induced inthe population or that the cells are delayed in their passagethrough mitosis. However, since chromosome abnormalities areseen, the latter factor probably makes the major contribution tothe rise in mitotic index observed. The sudden rise in the numberof dead cells (Chart 2B) observed around twenty-four to thirtyhours post-treatment, seven to ten hours after the mitotic peak,

may be the result of an unbalanced chromosome constitution dueto damage to DNA.

The rise in labeling index during the first eight hours, followedby a sharp fall to a minimum at twenty hours, is thus the result ofa release of cells from the block in S and their passage throughinto mitosis. The pattern of grain count increase over the first tenhour follows closely that of the labeling index, indicating thatthe rate of DXA synthesis from labeled thymidine increases andthe S period is prolonged. This is clear indication, therefore, thatthe primary effect of this alkane sulfonate is not the inhibition ofDNA synthesis from thymidine, but a prolongation of the Sl>eriod.

Transport Passage of Cells from Gìto S and S to G-_>

After the higher dose level of methyl methanesulfonate, thelabeling index increased during the first eight hours (Chart 5),which would be expected if there were an inhibition of the rate ofpassage of cells from S to G¡.Using data from continuous andpulse labeling exj>eriments, the rate of passage of cells from GÌto S and from S to Gz was calculated (cf. Réf.31) and the results

lOOr

10

HOURS AFTER ADDITIONof DRUG

CHAHT6. Rate of increase of labeled cells during the continuouspresence of thymidine-3H (TdR-3H) in control (X X) and methylmethanesulfonate (MMS)-heated (0.36 mm) (0 0).

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160-1

140-

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Ou

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80-

60-

40-

20-

20 40

—i—60 80 100 120

HOURS AFTER ADDITION OF DRUG

CHART 7. Changes in grain count/labeled cell at various timesafter treatment with two doses of methyl methanesulfonate, 0.09mM (X X) and 0.36 HIM(O O). The grain counts in treatedcultures are expressed as a percentage of that in control culturesfixed at the same time.

are shown in Chart 8. The rates of GÌto S and S to G2are identical in control cultures, but in treated cultures both are decreased,the rate of S to G2being more greatly affected than Gìto S.

Effect on G2

The interference by nitrogen mustard with the growth kineticsof human amnion cells (16), guinea pig kidney cells, strain RCP(17, 18), L5178Y murine leukemia cells (11), mouse fibroblasts(L cells) grown in culture (30), and Ehrlich ascites cells grownin vivo (15) has been previously reported. It was observed thatguinea-pig kidney cells in G2 were not prevented from enteringmitosis by the treatment since all mitotic phases dropped to zeroonly after three hours, this being the length of G? in this system(17, 18). This is in contrast to the findings with Ehrlich ascitescells in vivo,where cells in G2 were shown to be the most sensitive(15). In that work, it was considered that cells blocked in G2reverted to DNA synthesis again without dividing since bytwenty-four hours the DNA content of the cells was shown to be50% above normal.

From the present study, cells in G2 at the time of treatmentapi>ear to be unaffected in their rate of passage through G->andthe first post-treatment mitosis, since the rate of increase oflabeled mitoses is the same in control and treated cultures (Chart4). ]5y six hours, all cells which were in G2 at the time of treat

ment should have passed through Gìand into S if their rate ofprogress is normal. However, between four and seven hours, therate GÌto S decreases (Chart 8), which suggests that G->cells aredelayed through GÌ.

Effect on GìCells

Gìcells appear to progress into S at a more or less normal rateduring the first four hours after treatment (Chart 8), but this rateis reduced between four and seven hours, corresponding to actionof the drug on cells in G2and ven' late S at the time of treatment.

The cells in Gìwhich enter into S during the four hours followingtreatment are presumably delayed in S since no unlabeled cellsreach mitosis during the first twenty-two hours after treatment(Chart 4).

Cell Death

Two peaks of mitotic index occurred after each dose level(Chart 3). After the higher dose level, each peak was associatedwith a peak in dead cell numbers (Chart 2B). This suggests that"cell death" occurred following mitosis, and when the cells would

normally be in Gìor S. However, at the time when dead cellnumbers are high (24-40 hr; Chart 2ß),the labeling index of thepopulation is very low (10-30% of control; Chart 5), suggestingthat cells die before reaching S, presumably in the Gìstage of thecell cycle. A similar situation has been described for X-irradiatedL5178Y cells (31).

Biochemical evidence (4, 5), following sulfur mustard treatment of HeLa and L5178Y cells, suggests that DNA synthesis isinhibited two hours after treatment with a dose level required tokill 90% of the cell population. Although this was interpreted asan inhibition of DNA synthesis, the possibility that it could alsobe due to a reduction in the number of cells undergoing DXAsynthesis should also be considered. In the present study, inwhich a dose of methyl methanesulfonate sufficient to kill 99%of the population was given, there was no reduction in graincount and the labeling index fell markedly by twenty hours.There was a relatively rapid recovery of grain counts and amuch slower recovery of labeling index (Charts 5, 7).

It has been suggested (28) that methyl methanosiilfonateinhibits DNA synthesis only after depurination following theinitial alkylation (3, 5), and it seems possible that the depressionof DNA synthesis in the treated lymphoma cells may be relatedto this event. It has further been proposed (22) that differentmechanisms are involved in the repair of damage by HN2 andM MS in Bacillus subtilis. Some of the differences observed between thest two agents in mammalian cells could perhaps !>edueto differences in repair enzyme activity between cell strains andbetween stages of the cell cycle in the same strain, as has beeninferred (6) in a study of the effects of diethyl sulfate and HN2on recombination frequency in Chlamydomonas.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the valuable suggestionsand criticism of Dr. L. G. Lajtha and the technical assistance ofMr. B. Harrison.

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Effect of Methyl Methanesulfonate on Lymphoma Cells

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CHART8. Percentage of cells progressing from one stage to the next at various times between 0 to 10 hr after treatment with methylmethanesulfonate (0.36 mi«)for 1 hr. The data are calculated from the pulse and continuous labeling experiments shown in Charts 5and 6. The difference between the percentage of labeled cells at the time of sampling and the percentage of labeled cells at the beginning of a continuous labeling experiment, gave Gì—»S values. The percentage of cells passing from S to GÕwas calculated by takingthe difference between the percentage of labeled cells in continuous and pulse labeling experiments. The rate of passage of cells betweenthe phases is given by the slopes of the respective lines. X X, Gì—>S, S —>Gs, control; • •Gì—»S, treated; O O,S —»Gj

treated.

REFERENCES

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10. Fox, M., and Gilbert, C. W. Continuous Irradiation of aMurine Lymphoma Line P388F in vitro. Intern. J. RadiationBiol., 11: 339-347, 1966.

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12. Huddow, A., and Ross, W. C. J. Tumour Growth InhibitoryAlkane Sulphonates. Nature, 177: 995, 1956.

13. Jackson,. H., Fox, B. W., and Craig, A. W. Antifertility Substances and Their Assessment in the Male Rodent. J. Reprod.Fertility, 2: 447-465, 1961.

14. Lawley, P. D., and Brookes, P. Further Studies on the Alkyla-tion of Nucleic Acids and Their Constituent Nucleotides.Biochem. J., 89: 127-138, 1963.

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Synthesis in Nitrogen Mustard Induced Giant Cells in Vitro.Exptl. Cell. Res., S3: 207-215, 1964.

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and on Their Rate of Progress throughIn VitroLymphoma Cells Effect of Methyl Methanesulfonate on the Growth of P388

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