[CANCER RESEARCH 27 Part 1, 2134-2239, November 1967}

Effect of Methyl Methanesulfonate on Macromolecular Biosynthesisin P388F Cells

BRIAN W. FOX AND MARGARET FOX

Paterson Laboratories, Christie Hospital, Withinglon, Manchester 20, England

SUMMARY

The effect of methyl methanesulfonate, ethyl methanesulfonate,isopropyl methanesulfonate, and méthylènedimethanesulfonateon the survival of the mouse lymphoma line, P388F, is described.The concentrations of alkanesulfonate resulting in a 507c inhibition of growth after a 3-hr exposure were 0.1 HIM,>16 mM, >1.5imi, and 0.037 m.M,respectively.

A more detailed examination of the action of methyl methanesulfonate with regard to the time of onset of inhibition of nucleicacid and protein synthesis was made. The relative uptakes ofthymidine-3H, uridine-5-3H, glycine-3H, and tyrosine-3H were

investigated and compared. Methyl methanesulfonate affects differentially the ability of the cell membrane to transport differentmacromolecule precursors into the cell pool. The potential causesof this differentia] action are discussed in relation to the knownsensitivity to alkylation of the guanine bases in DNA andRNA. A mechanism involving the codon-anticodon structure ofthe amino acyl transfer RXA and its recognition is proposed.

INTRODUCTION

Two general approaches to the study of the biochemicalchanges induced by alkylating agents have been employed: (a)the labeled drug method, in which the transferred alkyl groupis isotopically labeled and the treated biologic tissue analyzed indetail for labeled products; and (è)the labeled precursor method,which employs an unlabeled alkylating agent and labeled nucleicacid and protein precursors which are administered to the biologic system before, during, of after drug treatment. The resultingtissue is then examined for the disposition of the precursor andits extent of incorporation with reference to control experiments.

The labeled drug approach has been extensively used withcertain alkylating agents, notably sulfur and nitrogen mustards(29) ethyleneimine (21, 27), diazoalkanes (20), and alkanesul-fonates (17). Apart from detoxification data (24, 25), thisapproach has revealed that nucleic acids are preferentially alkyl-ated in X-7 position of guanine and that there is a lesser degreeof alkylation of N-l cytosine, N-l adenine, and N-3 adenine(17). Based on such studies, the primary action of the bifunctionalmustards has been considered to be the cross-linking of DNAbetween two guanine moieties (18). It is clearly not possible tooffer this interpretation for monof'unctional alkylating agents,

and, in view of the wide diversity of biologic action which thealkanesulfonates exhibit in spermatogenesis (8, 15), hemopoiesis

Received March 31, 190Ì7;accepted July 14, 1907.

(7, 8), and animal tumors (8, 14), there must be many potentialsites for primar}' action. In the case of nitrogen mustard, apartfrom effects on DNA itself, a nonhistone protein has been implicated in a reaction concerned with inhibition of the primeractivity of DNA alkylated in vivo (28).

The aim of this communication is to present some dataobtained using the second, labeled precursor approach in relationto the elucidation of the priman- biologic action of methyl

methanesulfonate and related alkanesulfonates in the P388lymphoma system.

MATERIALS AND METHODS

Cell Line. The cells used in these experiments were primaryexpiants of the murine lymphoma line, P388F, derived from theP388 line, previously described (10). The cells were harvestedfrom the peritoneal cavity of DBA2 mice, about 14 days after aparenteral injection of IO6cells. The cells were diluted, immedi

ately after removal from the peritoneum of the mouse, inFischer's medium supplemented with 10% horse serum, to a concentration of 2.5 X IO6cells per ml for the uptake experiments.The suspension was allowed to equilibrate at 37°Cin a water

bath for 2 hr before the commencement of the experiment.Chemicals Used. Alkyl alkanesulfonates were synthesized

by conventional procedures and purified as described previously (15). All solutions were made up immediately before useand kept ice-cold to avoid hydrolysis. Thymidine-3H and labeledamino acids were obtained from the Radiochemieal Centre,Amersham, England.

Dose Response Studies. The general procedure adopted inthe uptake studies in relation to dose of alkyl alkanesulfonatewas as follows. Sterile, glass-stoppered tubes (total capacity, 15ml) with a small protrusion blown outwards about 1 cm belowthe base of the stopper, were used. Two ml of cell suspension(2.5 X 106/ml) were added to each tube. Stock solutions of

alkanesulfonates in growth medium were adjusted so that therequired dose could be given to the cultures by addition of 0.1-ml volumes. A solution of labeled precursor in 0.05 ml was addedto each side bulb and the tubes stoppered. After exactly 30 minof incubation without shaking, the tubes were inverted, mixingthe isotope with the cell cultures. Two hr or an appropriate timelater, the tubes were plunged in an ice bath and ice-cold bufferedsaline, pH 7.0 (2 ml), added. The cells were subsequently cen-trifuged (2000 X g, 7 min) and washed twice with bufferedsaline.

Time Course Studies. Suspensions of cells (2.6 X 10"cellsper ml) (25 ml) were incubated at 37°Cwith appropriate con-

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Methyl Methanesulfonale

centrations of the alkanesulfonates (1 ml). At different timesafter the addition of the alkylating agent, aliquots (2 ml) werepipeted rapidly into 15-ml glass-stoppered tubes containingisotopically labeled precursor in aqueous solution (0.05 ml).After 15-min incubation, the tubes were removed and cooled inice. Ice-cooled buffered saline was then added as before. Thecells were washed and centrifuged twice more before processing.

Isolation of High-Polymer DNA

Detergent Method. Pellets of washed cells were treated withcitrate-buffered saline (0.8 ml) and homogenized in the cone-shaped centrifuge tubes with a Perspex pestle made to fit closelyto the conic end of a centrifuge tube (10 ml). A solution of sodium dodecyl sulfate (0.2 ml, 5% in 45% ethyl alcohol) wasadded and the mixture allowed to stand at room temperaturefor 1 hr with occasional shaking. A solution of 5 M NaCl (0.21ml) was added to each, followed by 1.2 ml of 95% ethyl alcohol.The DNA was spooled off on thin glass rods and dissolved insodium acetate solution (1.5 ml, 0.2%). It was allowed to standfor approximately 2 hr to allow solution, then sodium acetate(0.16 ml, 40%) was added, and the resulting mixture extractedwith «-butylalcohol :chloroform (1.6ml, 1:3). This mixture wasthen centrifuged (2000 X g for 30 min at 5°C)and the top clear

layer removed. Ethyl alcohol (95%, 2.0 ml) was then added andthe DNA spooled off and again suspended in sodium acetatesolution (1.0 ml, 0.2%). In some cases, UNA was removed byincubating with ribonuclease (heat-treated, 100°Cfor 10 min)

and reprecipitated.Phenol Method. A solution of sodium dodecyl sulfate (1%)

and sodium paraminosalicylate (5%) in 4 ml water and phenol:wi-cresol:9-hydroxyquinoline:H2O (500 gm: 75 ml: 0.5 gm:55 ml) (4 ml) was mixed with the washed cell pellet, and thewhole mixture shaken and allowed to stand overnight. Aftercentrifugation (10,000 X g for 30 min at 5°C),the upper layer

was removed and sodium chloride solution (30%, 0.1 ml) wasthen added to make the final concentration 3% with regard tosalt. The solution was then thoroughly mixed, and phenol (88%,0.5 volume) added; after further mixing and centrifugation(13,000 X g, 10 min), the top phase was removed and treatedwith ethoxyethanol (1 volume). The DNA formed was spooledoff, collected in sodium acetate, and reprecipitated by the methoddescribed above.

Analytic Methods. The diphenylamine procedure of Burton(2) was used to estimate the DNA concentration; RNA andprotein contamination was estimated by the methods of Ceri-otti (3) and Lowry et al. (19), respectively.

Trichloracetic Acid Separations. Washed cell pellets weretreated with ice-cold trichloracetic acid (TCA) (5%, 2.0 ml) andsonicated for 25 sec in an MSE ultrasonic generator. The samplewas maintained in an ice bath so that the temperature did notrise above 5°C.Subsequently, tubes were centrifuged (2,000 X

g, 5 min), the residue similarly extracted 3 times, and the washings from each collected together (cold TCA extracts). A solution of TCA (5%, 2.0 ml) was added to the residue from thelast cold TCA extraction and the tube incubated in a boilingwater bath for 8 min, with a glass marble on the opening to reduce evaporation. The mixture was then cooled and again ultra-sonicated (1 min) to break the precipitate into a fine form suit

able for the two further extractions with hot TCA. The washingsfrom each tube were again combined (hot TCA extracts). Theresidue was washed twice with water, 2 washings of 2 ml each(water extract), and the final residue drained by inverting thetubes over filter paper for 5 min.

Residues were treated with Pronase R (0.2 ml of a 1 mg/mlsolution in sodium phosphate buffer; 0.05 M, pH 6.5) and incubated overnight. The entire contents of the tube were then dissolved in a scintillation mixture (10 ml) consisting of naphthalene (100 gm), toluene (700 ml), 2,5-diphenyloxazole (1 gm),l,4[2'(5'phenyloxazolyl)]benzene (25 mg), and dioxane (700 ml).

The TCA extracts were pipeted (0.2 ml) on to glass fiber discs(Whatman GF/A, 2.1 cm) which were then dried for 25 minunder an infrared lam)). For counting, several discs were combined together in the same scintillation solution, By separateexperiment, it can be shown that the total counts measured ona series of discs in the same phosphor is stoichiometrically related to the number of discs present (J. W. Davies, personalcommunication). Where activity was low, TCA was removedby extraction with ether, then the solutions were lyophilized andtransferred to glass fiber discs described above. There was noadvantage in washing the discs with ether before counting toremove TCA. Reliable and reproducible results were obtainedwith the above procedure with the number of cells used.

The instruments used were the Beckman liquid scintillationcounter and both the two-channel external standardizationmethod on this instrument and an internal standard of toluene-3H (25 fie/ml, 0.01 ml) were used to determine the amount ofquenching. In counting treated residues, quenching rarely exceeded 20%.

RESULTS

Effect of Alkanesulfonates on Cell Survival

The relative effects of 4 alkanesulfonates on the survival ofP388 cells were measured by technics described previously (9, 11)and the results are shown in Chart 1. Méthylènedimethane-sulfonate was the most effective of the four drugs and the dosesrequired to reduce survivors to 50% of control in each case wereméthylènedimethanesulf oriate (0.037 min), methyl methane-sulfonate (0.1 HIM),isopropyl methanesulfonate (>1.5 HIM),andethyl methanesulfonate (>16 HIM).The relative hydrolysis ratesof these esters at 37CC in aqueous media were 22 min, 9.1 hr,

13.6 min, and 10.4 hr, respectively (26).

Time Course of Inhibition of Thymidine-3H Uptake into

DNA after Treatment of P388 Cells with Methyl Methanesulfonate

As can be seen from Chart 2, the time of 50% inhibition ofthymidine-3H uptake into DNA during exposure to a concen

tration of 0.91 HIMmethyl methanesulfonate is about 0.75 hr.

Time Course of Inhibition of Protein and RibonucleicAcid Synthesis by Methyl Methanesulfonate

The relative uptakes of labeled amino acids into the proteinresidues of TCA-extracted cells at different times after treat-

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Brian W. Fox and Margaret Fox

ment with methyl methanesulfonate are shown in Chart 3.The uptake of uridine-5-3H is also shown for comparison. It

can be seen that 50% inhibition of RNA synthesis occurs about1.1 hr after addition of the drug (0.91 HIM). The relative uptakes of different amino acids at this dose level are shown inTable 1 together with the environmental amino acid concentration. The influence of the concentration of the amino acid inthe surrounding medium was studied in the case of glycine bycomparing the uptake of a tracer level of glycine in medium withand without glycine supplementation. In unsupplemented medium, the glycine concentration was 12.01 /ig/cell suspension(2.15 ml) and in the supplemented medium, 48.85 Mg/cell suspension. The fourfold difference in concentration failed to affecteither the uptake of labeled amino acid into the free form withinthe cell or into the protein itself.

I 0-

100-0-

T0.2 0.4 06 08

DRUG CONCENTRATION (mM)

CHART 1. Relationship of surviving fraction of P388F lymphomacells exposed for 3 hr to different concentrations of méthylènedimethanesulfonate (MDMS), methyl methanesulfonate (MMS),ethyl methanesnlfonate (EMS), and isopropyl methanesulfonate (IMS) in Fischer's medium (with 10% horse serum).

Ote.

ou'S 50-

UJ<Jce

•O-:O —i i

O 0.5 1.0 1.5

HOURS AFTER ADDITION OF MMS

CHART 2. Effect of methyl methanesulfonate (MMS) (0.91 HIM)on the uptake of thymidine-'H (15-min exposure) into deoxyribo-nucleic acid at different times after addition of alkylating agent(cell concentration 2.5 X IO6cells/ml).

Effect of Methyl Methanesulfonate on the Kates of Incorporation of Labeled Amino Acids into the Free and RNA-

bound Forms within the Cell

The free amino acid within the cell was taken to be relateddirectly to the ice-cold 5% trichloracetic acid fraction of thecells, while the hot TCA extract would give some indication ofthe extent of binding of the amino acid to the hot TCA-extrac-table nucleic acids, i.e., RXA (presumably the aminoacyl transferRNA) and DNA containing traces of amino acids. The relativeuptakes of glycine-3H, tyrosine-'H, and uridine-5-3H were inves

tigated after methyl methanesulfonate. The tyrosine uptake intothe cell pool increased to 135% of the control, while at the samedrug concentration uridine-5-3H uptake into the nucleotide pool

was reduced to 25% of the control values (Chart 4).The hot trichloracetic acid extract radioactivity from the

tyrosine experiment is increased at the lower concentrations butreturns to normal levels at the higher doses of methyl methanesulfonate (Chart 5). The effect of methyl methanesulfonate onthe glycine levels is less marked. The uridine level in this fractionisdecreased and is inversely related to the dose of methyl methanesulfonate re]¡resentingan inhibitory effect on RNA synthesis.

The residue radioactivity (Chart 6), representing proteinsynthesis, shows that uptake of tyrosine is less affected thanthat of glycine. Since the percentage uptake into protein is independent of the specific activity of the amino acid in the medium, this difference may represent a difference in sensitivity ofthe protein-synthesizing system to methyl methanesulfonate forthe two amino acid species.

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Methyl Methanesulfonate

Txrosine

Glycine

^""A— Alanine

\ ••-•Tyrosme

^^_ RNA synthesis

.0p

1.5 2.0 2.5

HOURS AFTER ADDITION OF DRUG

CHART 3. Effect of methyl methanesulfonate (0.91 HIM) on theuptake of glycine-3H, alaniue-'H, tyrosine-'H into protein, anduridine-5-3H into ribonucleic acids, at different times after addition of alkylating agents (cell concentration and precursor exposure times as in Chart 2).

TABLE 1

Effect of Methyl Methanesulfonate (0.01 m\i) on theRate of Inhibition of Precursor Incorporation

into Macromolecule

PrecursorThymidine-'H

TJridine-3HGlycine-3H

Arginine-3HProline-3HAlanine-3HTyrosine-3H50%

inhibitiontime(min)45

6810179

726947Concentration

ofprecursor(mM)0.023

X IO"60.023 X10-«0.017

0.0740.02980.0290.305

DISCUSSION

Alkanesulfonates, both mono- and bifunctional, have beenshown to induce a variety of biologic effects (1) in whole animals(26, 27) and in cell culture systems (4,5).

From comparative work with a number of simple alkyl alkane-sulfonates in spermatogenesis (15), it became clear that thealkylating alkyl group is the main feature which differentiatesone agent from another, and that the composition of the alkanesulfonic acid used in its synthesis played little or no part in determining the character of its biologic activity.

A comparison of the lethality of 4 alkanesulfonates in P388Fcells is shown in Chart 1 and is not related to the hydrolytic half-life of the esters in aqueous solution. The exposure time waschosen to allow about 75% of the cells to take up labeled nucleic

100-**

Glycine

Oce.

OU

111ute111(L

Uridine

1.2 1.6

DRUG CONCENTRATION (mM)

CHART 4. Effect of methyl methanesnlfonate at different concentrations on the uptake of tyrosine-3H, glycine-3H, and uiidine-5-3H into the cold trichloracetic-acid-solvible pool of P388F cells.

Exposure times: alkylating agent 0.5 hr followed by precursorfor 2 hr in the continued presence of alkylating agent (cell concentration 2.5 X IO6 cells/ml).

acid precursor under normal circumstances. Under these conditions, a reduction in precursor uptake would be due to 1 of 4factors: an interference with deoxyribonueleic acid synthesis, areduction in cells synthesizing DXA, a breakdown of labeledDNA after treatment, or enzymatic failure to utilize preformedprecursor present as nucleoside. There is evidence that at thehigher drug concentrations in these studies all these systems maycontribute to the final result. The drug is also effective in producing marked changes in the cell kinetics at lower treatmentlevels (9), and it is clear that other subtle and important changesoccur at such levels which may not be directly related to theaction of the drug on DNA but to some other controlling factorin the general cell metabolism.

To examine the nature of reactions occurring after treatmentwith methyl methanesulfonate, the levels of free precursor,precursor initially bound within the cell, and radioactivity ofthe final macromolecule under consideration were investigatedusing both the phenol extraction procedure and a modifiedSchmidt-Thannhauser method. \Yhen an amino acid precursorwas used in the latter procedure, the "5% cold trichloraceticacid" extract was taken to be a measure of the quantity of freeamino acid present in the cell pool. The "hot TCA extract" was

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Brian W. Fox and Alargaret Fox

0.4 0.8 1.2

DRUG CONCENTRATION (mM)

1.6

CHART 5. Effect of methyl methaiiesiilfonate at different concentrations on the uptake of tyrosine-'H, glycine-3H, and uridine-5-3H into the hot trichloracetic acid extract of P38SF cells following removal of the cold acid-soluble pool. Exposure times and cellconcentration as in Chart 4.

considered to isolate the aminoacyl transfer RNA radioactivitytogether with any small oligopeptides that may be soluble in thisTCA fraction. The "residue" was regarded as protein and poly-

peptide.In Streptococcusfaecalis (22), protein synthesis was found to be

a function of the concentration of membrane-associated poly-ribosomes, and in Bacillus slearothermophilus, the cell membranewas shown to be the most important initial site of amino acidincorporation in the form of an aminoacyl transfer RXA (1).The highest activity after exposure to a labeled amino acid occurred in the lipoprotein fraction, 30 seconds after addition of theamino acid. This was followed very rapidly by a labeling of themembrane-associated aminoacyl transfer RXA.

It is implied, therefore, that the mechanism of uptake ofamino acids into a cell is very carefully controlled at the cell membrane and that the initial mechanism for protein synthesis occurat this site, at least in bacterial cells. There is now an increasingvolume of data accumulating (4,5) concerning the transportmechanisms involved across the cell membrane in mammalian(23, 26), yeast (12, 13), and bacterial (1, 22) cells. Three mainroutes have been recognized (16), and several of the factors involved in each route have been elucidated (6, 16). In the present

ODCK

OU'S 50-

<Jes:LUO.

Tyrosine

\ Glycine

0.4 0.8 1.7 1.6

DRUG CONCENTRATION (mM)

CHART 6. Effect of methyl methnnesulfonate at different concentrations on the residue (protein) uptake of tyrosine-3H andglycine-3!!. Conditions of exposure times and cell concentrationsas in Chart 4.

study, however, no attempt has been made at this stage to identify which of these routes are likely to be affected.

By comparison of the tyrosine-3H, glycine-'H, and uridine-5-3H uptakes into the various fractions of the cell at different dose

levels of methyl methanesulfonate, it is clear that these precursors are differentially affected in their entry into the cell pool(Chart 4). Whereas the free ty rosine-3H within the cell is in

creased at the higher dose levels of methyl methanesulfonate,uridine-5-3H by contrast is decreased. This study suggests that

there is not an indiscriminate increase in permeability to all precursors, but a marked differential sensitivity.

Clearly, many factors operate in determining the nature ofthis differential effect. The simplest interpretation would be adirect effect on one or more of the transport systems across thecell membrane. However, an alteration of the rate of utilizationof the pool precursor would also bring about differences in poolsize as well as a disturbance of the alternative pathways of denovo and preformed precursor utilization. In the particular caseof amino acid uptake, inhibition of the formation of aminoacyltransfer RNA as well as an inhibition of one of the several eventsinvolved in the incorporation from aminoacyl transfer RNA tothe developing protein on the polyribosome could lead to suchan increase. Supplementation of the medium with glycine doesnot alter the rate of uptake inhibition or its equilibration withinthe cell.

In view of the fact that the guanine base is known to be 2 to3 times more sensitive to the action of certain alkylating agentsthan other bases, such as adenine and cytosine, in the nucleicacid (17), it seems reasonable to conclude that the codon-anti-codon combination which determines the type of amino acid

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Methyl Methanesulfonatc

transferred to the protein-synthesizing site may have a sensitivity to these alkylating agents which is largely dependent onthe guanine-cytosine level in the combination. The choice of tyro-sine and glycine for a comparative study was made with this inmind. The triplet requirement for tyrosine transfer is UA (U,C) and that for glycine is GG (U, C, A, G). Although only 2neutral amino acids have been considered in this paper, theirrelative sensitivity to inhibition would support this suggestion.The dose levels for 50% inhibition of the amino acid uptake intoprotein for these 2 amino acids were 1.4 IMI for glycine and 2.2mil for tyrosine. Clearly, other amino acids will require to beinvestigated in a simpler system before this mechanism can beconsidered to be operating.

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1967;27:2234-2239. Cancer Res   Brian W. Fox and Margaret Fox  Biosynthesis in P388F CellsEffect of Methyl Methanesulfonate on Macromolecular

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