regulation of thymidylate synthetase activity in cultured ... · regulation of thymidylate...

J. Cell Sci. 10, 471-486 (1972) 471

Printed in Great Britain

REGULATION OF THYMIDYLATE

SYNTHETASE ACTIVITY IN CULTURED

MAMMALIAN CELLS

ABIGAIL H. CONRAD* AND F. H. RUDDLE

Department of Biology, Yale University, New Haven, Connecticut 06520, U.S.A.

SUMMARY

Changes in thymidylate synthetase specific activity in Don Chinese hamster cells grown invitro have been examined during the culture cycle and after exposure of lag- and log-phasecultures to drugs which inhibit DNA, RNA, and protein synthesis. During the culture cycleenzyme activity was low during lag phase, rose 6- to 8-fold before log phase, fluctuated between5-5 and 9 nmol dTMP/h/107 cells during log phase, and declined to base level duringstationary phase. Puromycin prevented all increases in enzyme specific activity and caused adecrease in enzyme activity when applied to log-phase cultures. Actinomycin D prevented theinitial rise in enzyme activity if applied during early lag phase but caused a pronouncedincrease in enzyme activity above control levels when applied during log phase.

High thymidine concentration (1 min) stopped cell division in log-phase cultures but didnot alter the log-phase plateau level of thymidylate synthetase activity. Fluorodeoxyuridinestopped cell division and depressed enzyme activity to varying degrees depending upon itsconcentration, but at concentrations less than io~' M enzyme activity eventually returned tonormal log-phase levels and cell division resumed if puromycin was not present. Methotrexatestopped cell division and caused a 3- to 4-fold increase in enzyme activity above control levelsif puromycin was not present. This increase occurred in the presence of actinomycin D butwas retarded by addition of thymidine when actinomycin D was not present.

These experiments suggest that the regulation of thymidylate synthetase activity in log-phasecells is complex and may involve thymidine triphosphate.

INTRODUCTION

Thymidylate synthetase catalyses the conversion of deoxyuridine monophosphateto thymidine monophosphate by facilitating the transfer of a methyl group from5,10-methylenetetrahydrofolate to the 5 position of the pyrimidine ring of dUMP(Hartmann & Heidelberger, 1961). As has been documented previously (Conrad,1971; Kit, Dubbs & Frearson, 1965), thymidylate synthetase activity increases whencells proliferate and decreases when they stop dividing. The same relationshipbetween cell division and appearance of enzyme activity has been demonstrated forother enzymes associated with the synthesis of the precursors for DNA synthesis suchas thymidine kinase (Brent, Butler & Crathorn, 1965; Bresnick & Thompson, 1965;Eker, 1968a, b; Hotta & Stern, 1961, 1963, 1965; Kim, Gelbard & Perez, 1967; Kit,deTorres & Dubbs, 1966; Littlefield, 1965; Maley, Lorenson & Maley, 1965;McAualan, 1963; Nagano & Mano, 1968; Stern & Hotta, 1963; Stubblefield &

• Present address: Department of Biology, Kansas State University, Manhattan, Kansas66502, U.S.A.

472 A. H. Conrad and F. H. Ruddle

Murphree, 1967; Wanka, Vasil & Stern, 1964; Weissman, Smellie & Paul, i960),dihydrofolate reductase (Hillcoat, Swett& Bertino, 1967), deoxycytidylate deaminase(Eker, 19686; Maley ei a/. 1965), andthymidylatekinase(Eker, 19686; Kitetal. 1965;Maley et al. 1965).

Attempts to understand the regulation of these enzymes have focused upon therelationship between DNA synthesis and the changes in activities of related enzymes.Inhibition of DNA synthesis and cell division by such drugs as arabinofuranosyl-cytosine, fluorodeoxyuridine, methotrexate, hydroxyurea, or mitomycin C, or by highthymidine concentrations has been shown to elicit a variety of effects on the specificactivities of different enzymes associated with DNA synthesis (Eker, 1966, 19686;Kit et al. 1966; Littlefield, 1965; Stubblefield & Mueller, 1965). These studiessuggest that the activities of the enzymes associated with DNA synthesis may beregulated individually in relation to the continuation of DNA synthesis.

Recent studies of the regulatory mechanisms which control the specific activities ofglutamine synthetase (Kirk, 1965; Kirk & Moscona, 1963; Moscona & Kirk, 1965;Moscona, Moscona & Saenz, 1968), alkaline phosphatase (Cox & MacLeod, 1962,1963; Griffin & Cox, 1966a, b), tryptophan pyrrolase (Caffery, Whichard & Irvin,1968; Feigelson & Greengard, 1962; Garren, Howell, Tomkins & Crocco, 1964;Greengard, Smith & Acs, 1963; Schimke, Sweeney & Berlin, 1964, 1965), tyrosinetransaminase (Caffery et al. 1968; Granner, Hayashi, Thompson & Tomkins, 1968;Kenney, 1967; Peterkofsky & Tomkins, 1967, 1968; Reel&Kenney, 1968; Thompson,Tomkins & Curran, 1966; Tomkins et al. 1969), and several enzymes in the cellularslime mould (Roth, Ashworth & Sussman, 1968) have suggested that while primaryenzyme induction in higher organisms probably does take place at the level of DNAtranscription, normal maintenance of high enzyme activity and subsequent disap-pearance of enzyme activity may involve other epigenetic regulatory mechanismswhich operate at the level of RNA translation or subsequent protein turnover. Maleyet al. (1965) have demonstrated that following partial hepatectomy in the rat thymidy-late synthetase activity was inhibited by amino acid analogues but scarcely influencedat all by actinomycin D or puromycin. This suggests that thymidylate synthetaseregulation may involve complex control mechanisms.

The present study of the regulation of thymidylate synthetase activity in DonChinese hamster cells was undertaken in an attempt to examine the role of proteinsynthesis in changes in thymidylate synthetase activity and to investigate the effectsof the interruption of DNA synthesis on the level of thymidylate synthetase activity.

MATERIALS AND METHODS

Cell culture

Monolayer cultures of Don Chinese hamster cells (Hsu & Zeuzes, 1964) were grown onEagle's medium (Eagle, 1955) fortified with 0 1 % lactalbumin hydrolysate, 10% foetal calfserum, 100 units/ml penicillin, 50/ig/ml streptomycin, and ioo/tg/ml kanamycin. Preparationof medium and conditions for cell culture propagation have been described previously(Conrad, 1971). Cell cultures were tested periodically for PPLO contamination (Conrad, 1971).During the course of these studies no PPLO-contaminated cell cultures were found.

To obtain sufficient numbers of cells for a time-course analysis of changes in thymidylate

Regulation of thymidylate synthetase 473

synthetase activity under varying experimental conditions, 16 x io" Don cells were inoculatedinto 250-ml Falcon tissue culture flasks containing 20 ml medium per flask, exposed to 5 % CO,in air for 15 s, and incubated at 37 °C. Flasks were selected according to a random numbertable for harvesting at the times indicated in Results.

For drug studies, stock solutions of fluorodeoxyuridine (FUDR), thymidine, methotrexate,puromycin, and actinomycin D were prepared in thrice glass-distilled water at 100 x the finalconcentration desired, sterilized by passage through a Millipore Sweeny Adaptor, and appliedin the appropriate amounts to the test cell cultures at the times indicated in Results. Stocksolutions of methotrexate also contained serine (30 mg/1.), glycine (10 mg/1.), and hypoxanthine(i-4mg/l.).

Enzyme assay

For each enzyme sample approximately io7 cells were collected by trypsinization, washed3 times with isotonic saline, pelletted, overlaid with 001 M Tris-025 M sucrose buffer, pH 70 ,to give a final cell concentration of 2-2-2-3 x I 0 ' cells/ml, and sonically disrupted as describedpreviously (Conrad, 1971). The sonicate was centrifuged at 27 coo g, at o °C, for 1 h, and thesupernatant was used for determination of thymidylate synthetase activity.

Thymidylate synthetase activity was determined by the method of Roberts (1966) in whichthe amount of tritium released to water from the 5 position of the pyrimidine ring of deoxy-uridine monophosphate upon its conversion to thymidine monophosphate (dTMP) wasmeasured in a scintillation counter after absorption of all nucleotides by activated charcoal.Duplicate assays of individual samples by this method gave values which varied by less than10 %. The specific activity of thymidylate synthetase was expressed as the number of nmol ofproduct formed per h per io7 cells.

Chemicals

FUDR was a gift from Hoffman-LaRoch, Inc. Methotrexate was obtained from TheAmerican Cyanimide Company, puromycin from Nutritional Biochemical Corporation,thymidine from K and K Laboratories, and actinomycin D from Merck Company.

Tris, tetrahydrofolic acid, and unlabelled deoxyuridine monophosphate were obtained fromSigma Chemical Company. Tritium-labelled deoxyuridine monophosphate (47 mCi/mmol,0 5 mCi/ml) was obtained from Calbiochem Company, Los Angeles, California. ActivatedCharcoal, N. F. Powder was purchased from Merck Company. BBOT, 2,s-bis-[2-(s-tert-butylbenzoxazolyl)]-thiophene, was obtained from Packard Instrument Company, Inc., andwas prepared according to their instructions.

RESULTS

Variation in thymidylate synthetase specific activity during the culture cycle

Characteristic changes in the specific activity of thymidylate synthetase per celloccurred during the culture cycle of Don Chinese hamster cells (Fig. 1). The specificactivity of the enzyme was low, about 1-1-5 n m ° l dTMP/h/107 cells, during the earlypart of lag phase (0-10 h after subculture), but the activity rose sharply during thelatter half of lag phase before any increase in cell number was observed in the cultures,reaching 7 nmol dTMP/h/107 cells by 18 h after subculture. As the cells proceededthrough log phase, the specific activity of thymidylate synthetase oscillated between5-5 and 8 to 9 nmol dTMP/h/107 cells. The apparent rise in enzyme specific activityin late log phase of Fig. 1 is probably not significant. Other late-log-phase culturesshowed enzyme activities between 6-5 and 7-5 nmol'dTMP/h/107 cells. In all cases,however, when the cultures reached confluency and the cells entered stationary phase,enzyme activity declined toward basal level again in a biphasic pattern, lingering inthe lower log-phase range and then decreasing to the early lag-phase level later.


Effect of inhibitors of protein and RNA synthesis on tlie pattern of thymidylate synthetasespecific activity during the culture cycle

The initial late-lag-phase rise in thymidylate synthetase specific activity wascompletely prevented by addition of puromycin (io/ig/ml final concentration) toearly-lag-phase Don cultures (Fig. 2). Moreover, cultures exposed to puromycin inearly lag phase never entered the log phase of cell proliferation.

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Fig. i. Changes in thymidylate synthetase specific activity during the culture cycle ofDon cells. Cultures of Don cells were established at i-6x io" cells/20 ml mediumand incubated as described in Methods. Enzyme preparations were obtained asdescribed in Methods and were assayed as follows. The reaction mixture contained15 fi\ reagent C which contained 102 /tl 10 M NaF, 41 fi\ dUMP (2 mg/ml), 155 [i\o-i M mercaptoethanol, 4-6 /tl 37 % formaldehyde, 252 fi\ i-o M Tris buffer pH 7-5,50 /tl tritiated dUMP (47 mCi/mmol, 0-5 mCi/ml), and tetrahydrofolic acid at178 x io~6 M final concentration; 5 /tl o-oi M Tris-o-25 M sucrose buffer pH 7-5; and20 /tl enzyme preparation. The reaction was incubated for 40 min at 37 °C, andterminated by the addition of 15 /tl 33 % trichloroacetic acid (TCA) followed bythe addition of 5 /tl unlabelled dUMP (5 mg/ml) and 200 /tl activated charcoal(100 mg/ml). Following centrifugation ioo-/tl aliquots of the supernatant weremeasured for radioactivity in a liquid scintillation counter. 0 , number cells/culture;O, thymidylate synthetase specific activity.

When puromycin was added to mid-log-phase cultures cell division continuedat the normal rate for about 4 h, but then it ceased abruptly and did not resume overthe next 18 h. Changes in thymidylate synthetase specific activity paralleled thoseobserved when Don cultures entered stationary phase under normal confluency


conditions (Fig. 2). That is, enzyme activity remained high for 3-4 h while celldivision was continuing at the control rate, but then the activity decreased in abiphasic pattern as cell division ceased in the puromycin-treated cultures.

Addition of actinomycin D (5 /^g/ml final concentration) to Don cells inhibited theuptake of ^HJuridine into acid-insoluble precipitates by more than 99 % during the2 h immediately following drug administration (unpublished results). When thisconcentration of actinomycin D was applied either at the time of cell transfer or 4-5 h

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Fig. 2. Effect of puromycin on thymidylate synthetase activity during the culturecycle of Don cells. This is a composite graph of several separate experiments super-imposed on the control enzyme activity profile taken from Fig. i. Cultures wereestablished aa described in Methods. Puromycin (io/ig/ml final concentration) wasadded to some cultures at the times indicated by the arrows; the remaining culturesbecame untreated controls. Enzyme samples were prepared as described in Methodsand assayed as described for Fig. i. Separate controls were used for each experimentand almost exactly paralleled the composite control shown here. O, control; A, withpuromycin.

after subculture, the subsequent late-lag-phase rise in thymidylate synthetase specificactivity did not occur (Fig. 3), and no cell division took place in the drug-treatedcultures. On the other hand, when actinomycin D was applied to mid-lag-phasecultures after the initial rise in thymidylate synthetase activity had begun, there didfollow a subsequent rise in enzyme activity over the next 12 h in the absence of anycell division. However, in the drug-treated cultures the enzyme activity per cellincreased in a linear fashion at a rate equivalent to that which had been achieved in thecultures at the time of drug administration, whereas enzyme activity per cell in thecontrol cultures increased in a logarithmic fashion over the same 12-h period

(Fig- 3)-On the other hand, when actinomycin D was applied to late-lag-phase or log-phase

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cultures (Fig. 3) cell division stopped, but enzyme activity continued to increase overthe next 12 h, reaching levels twice as high as the normal log-phase plateau in enzymespecific activity. The maximum possible increase in enzyme activity under theseconditions is not known since these experiments were terminated 12 h after additionof actinomycin D to the cultures.

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Fig. 3. Effect of actinomycin D on thymidylate synthetase activity. This compositegraph was compiled from experiments conducted in the same manner as thosedescribed for Fig. 2 except that at the times indicated by the arrows actinomycin D(5/ig/ml final concentration) was added to some cultures. O, control; # , withactinomycin D.

Effect of high concentrations of thymidine on cell growth and thymidylate synthetasespecific activity

Early log-phase Don cultures exposed to high concentrations of thymidine (1 mM)stopped cell division after an increase of about 2 x io6 cells to the population. However,the cells remained attached to the substratum for the next 24 h, and they maintaineda thymidylate synthetase specific activity within the normal log-phase range of6-9 nmol dTMP/h/107 cells (Fig. 4). On the other hand, when high concentrationsof thymidine and puromycin were added simultaneously to Don cultures, cell divisionceased after an increase in cell number of about 1 x io6 cells. The cells began toslough off the substratum by 12 h after drug administration, and thymidylatesynthetase specific activity decreased to 4-0-47 nmol dTMP/h/107 cells, a level belowthe activity range normally observed in log-phase cultures of Don cells (Fig. 4).


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Fig. 4. Effect of high concentration of thymidine on thymidylate synthetase activity inlog-phase cultures of Don cells. Don cells were established in cultures as described inMethods; 16 h after subculture 1 mM thymidine (final concentration) was added tosome cultures, 1 mM thymidine plus 10/ig/ml puromycin (final concentrations) wereadded to others, and controls received no drugs. Enzyme samples were prepared asdescribed in Methods and assayed as described for Fig. 1. O, control; # , with1 mM thymidine; A, with 1 mM thymidine + puromycin.

Effect of FUDR on cell growth and thymidylate synthetase specific activity

FUDR is converted to fluorodeoxyuridine monophosphate when it is taken intogrowing cells (Harbers, Chaudhuri & Heidelberger, 1959). Fluorodeoxyuridinemonophosphate is a competitive inhibitor for thymidylate synthetase and it has a verylow Ki for this enzyme (Hartmann & Heidelberger, 1961; Heidelberger, Kaldor,Mukherjee & Danneberg, i960; Reyes & Heidelberger, 1965). Thus FUDR preventsthe de novo synthesis of phosphorylated thymidine in cells, thereby creating a thymidinetriphosphate starvation which inhibits cell division and DNA synthesis.

Don cultures, innoculated at about 8 x io4 cells/ml of medium, incubated for 24 h,and then exposed to different concentrations of FUDR during early log phase,subsequently exhibited one of 3 different cell proliferation patterns (Fig. 5): (a) atFUDR concentrations of 5 x io"8 M or less the number of cells per culture continuedto increase at a rate comparable to control cultures; (b) at FUDR concentrations ofapproximately io~7 M the number of cells per culture increased by 4X106, thenremained constant for 6—8 h, and finally began to increase again at a rate equivalent tocontrol cultures at a comparable cell density; or (c) at IO~6M FUDR or more thenumber of cells per culture increased by about 4X106, then plateaued at that cell

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number and did not increase again during the course of the experiment. However,addition of 5 x io~5 M thymidine to cultures permanently arrested by treatment withio"6 M FUDR allowed cell division to resume immediately at control rates (unpub-lished results).

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Fig. 5. Kinetics of cell growth in early log-phase cultures exposed to varying amountsof FUDR. This is a composite graph from several separate experiments. Don cultureswere established as described in Methods; 24 h after subculture FUDR was added tosome cultures at the concentrations indicated below, and controls received no drug.The number of cells per culture were determined periodically thereafter, and cellnumbers were normalized to a starting number of 10' cells/flask at the time of drugadministration. • , no FUDR or 5 x io"" M FUDR or less; O, io~7 M FUDR;A, IO-OM FUDR.

The effects of these various concentrations of FUDR on the specific activity ofthymidylate synthetase are illustrated in Fig. 6. At io~6 M FUDR enzyme activitywas immediately reduced to 20 % of its original value where it remained throughoutthe course of the experiment. At io~7 M FUDR there was an immediate but slightlyless severe reduction in thymidylate synthetase specific activity which was maintainedfor several hours, but then the enzyme activity returned to the control level over theensuing several hours. Cell division stopped at about 6 h after io~7 M FUDRadministration but began again when enzyme activity had regained 85 % of thecontrol level (compare time scales in Figs. 5 and 6). At FUDR concentrations of5 x io~8 M or less the initial reductions in thymidylate synthetase activity weresequentially less pronounced than at the higher FUDR concentrations, and thelengths of time necessary for enzyme activity to return to within about 80 % of thecontrol level were shorter than the amount of time necessary to increase the culturepopulations by 4X io6 cells (compare Figs. 5 and 6). Hence, cell division continuedwithout interruption in cultures treated with 5 x io~8 M FUDR or less.

Simultaneous addition of puromycin with 5 x io~7 M FUDR prevented the


subsequent recovery of thymidylate synthetase activity (Fig. 7). In the absence ofpuromycin, however, enzyme activity reappeared at a rate sufficient to raise thespecific activity by 1 nmol dTMP/h/107 cells. Once cell division resumed in recoveredcultures, thymidylate synthetase specific activity did not increase above control log-phase activity levels.

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Fig. 6. Effect of FUDR on thymidylate synthetase activity in log-phase cultures.Enzyme samples were prepared as described in Methods from cell samples obtained inthe experiments described in Fig. 5. Separate controls were run for each experimentand were used for calculation of % control enzyme activity for each FUDR concen-tration. % control thymidylate synthetase specific activity for cultures exposed toFUDR as follows: O, IQ- 'M;5 x io~' M.

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Effect of methotrexate on cell growth and thymidylate synthetase activity

Methotrexate inhibits dihydrofolate reductase activity (Hillcoat et al. 1967), thuslimiting the intracellular supply of tetrahydrofolate, a co-factor in the thymidylatesynthetase reaction. To limit the effect of methotrexate to substrate deprivation forthymidylate synthetase alone, cultures treated with this drug were also supplementedwith serine, glycine, and hypoxathine (see Methods).

In log-phase Don cultures exposed to io~6 M methotrexate the number of cells perculture increased by about 2-3 x io6 cells. Cell division then stopped but the cellsremained attached to the substratum for more than 24 h. Moreover, thymidylate


synthetase specific activity increased to 4-fold the control log-phase level in the 24 hfollowing administration of methotrexate to the cells (Fig. 8). Again the average rateof appearance of new enzyme activity was sufficient to increase the specific activity by1 nmol dTMP/h/107 cells, as it was in the recovery of enzyme activity after exposureto FUDR.

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Fig. 7. Effect of puromycin on recovery of thymidylate synthetase specific activity incells exposed to 5 x 1 o~7 M FUDR. Cultures were established as described in Methods;24 h after subculture 5 x io~7 M FUDR was added to some cultures, 5 x io~7 M FUDRplus puromycin (io/ig/ml final concentration) to others, and controls received nodrugs. Enzyme samples were prepared as described in Methods and assayed asdescribed for Fig. 1. • , no FUDR; O, 5 x io~7 M FUDR; A, 5 x io"' M FUDR +puromycin.

Puromycin prevented this methotrexate-stimulated increase in thymidylatesynthetase specific activity when it was added to cultures concurrently with metho-trexate (Fig. 8). On the other hand, actinomycin D added concurrently with metho-trexate (unpublished results) or 5 h after administration of methotrexate (Fig. 8)neither prevented nor diminished the methotrexate stimulation of thymidylatesynthetase specific activity.

When thymidine was added at various time intervals after administration ofmethotrexate to cells, cell division resumed in the cultures and the methotrexatestimulation of thymidylate synthetase specific activity was diminished in proportion


to the length of time that thymidine had been returned to the cells (Fig. 9). If actino-mycin D was present in addition to methotrexate, the subsequent addition of thymi-dine was not effective in restoring cell division or in reducing methotrexate stimulationof enzyme activity (Fig. 9).

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Fig. 8. Effects of methotrexate, methotrexate plus actinomycin D, and methotrexateplus puromycin on thymidylate synthetase activity in log-phase cultures of Don cells.Cultures were established as described in Methods; 24 h after subculture io~* Mmethotrexate was added to all experimental cultures, and puromycin (io/fg/ml finalconcentration) was added to some of the methotrexate-treated cultures. Five hourslater actinomycin D (5 fig/m\ final concentration) was added to other methotrexate-treated cultures. Controls received no drugs. Enzyme extracts were prepared asdescribed in Methods starting from the time that methotrexate was added to allcultures, and assays were performed as described for Fig. 1. O, controls; # , withmethotrexate; A, with methotrexate + puromycin; A, with methotrexate + actino-mycin D.

DISCUSSIONThe data suggest that the regulation of thymidylate synthetase activity in pro-

liferating mammalian cells is complex. Clearly there is a structural gene for the enzymeitself, since actinomycin D inhibition of transcription during lag phase prevented theappearance of thymidylate synthetase activity. When RNA synthesis was inhibited incells, the messenger RNA for thymidylate synthetase appeared to be stable for up to


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Fig. 9. Effect of thymidine on thymidylate synthetase activity in log-phase culturestreated with methotrexate or methotrexate + actinomycin D. Cultures of Don cells wereestablished as described in Methods; 24 h later some cultures received io~' Mmethotrexate, others io~' M methotrexate + actinomycin D (5 /tg/ml final concentra-tion) and controls received no drugs. At 3, 6, or 9 h thereafter 5 x io"6 M thymidinewas added to some cultures which had received methotrexate alone and to somecultures which had received methotrexate + actinomycin D. Enzyme extracts wereprepared as described in Methods starting from the time that methotrexate was addedto all cultures, and assays were performed as described for Fig. 1. O, controls;t , with methotrexate; A, with methotrexate + thymidine at 3 h; • , methotrexate+thymidine at 6 h; Y, methotrexate + thymidine at 9 h; A, methotrexate + actino-mycin D + thymidine at 3 h; Q, methotrexate + actinomycin D + thymidine at 6 h;V, methotrexate + actinomycin D +thymidine at 9 h.

24 h. However, the half-life of thymidylate synthetase messenger RNA may bedifferent under normal cellular conditions.

All increases in thymidylate synthetase specific activity were puromycin-sensitive,thus suggesting that increases in enzyme activity result from de novo synthesis andsubsequent accumulation of additional enzyme molecules in cells. Similar data andconclusions have been drawn for other enzyme systems (Eker, 1968 a, b; Feigelson &Greengard, 1962; GarreneJa/. 1964; Grannereia/. 1968; McAuslan, 1963; Mosconaet al. 1968; Reel & Kenney, 1968; Schimke et al. 1964). However, puromycin-sensitivechanges in the conformational state of the enzyme molecule, such as have beendemonstrated for alkaline phosphatase (Griffen & Cox, 19666), have not been ruledout as a possible cause of increases in thymidylate synthetase activity.

The evidence regarding thymidylate synthetase turnover is contradictory. Puro-


mycin did not appear to stabilize enzyme activity when administered alone to log-phasecells, but it did cause partial stabilization in the presence of high concentrations ofthymidine and total stabilization in the presence of methotrexate. Further experi-mentation is required to determine whether directed turnover, such as has beendescribed for tyrosine transaminase (Kenney, 1967; Reel & Kenney, 1968) exists forthymidylate synthetase under normal cellular conditions.

Phosphorylated thymidine appears to play a role in the regulation of thymidylatesynthetase activity. Both FUDR and methotrexate stopped cell division underconditions which resulted in a depletion of thymidine triphosphate in cells. Bothdrugs caused puromycin-sensitive increases in thymidylate synthetase activity whichwere abated when the phosphorylated thymidine contents of cells were restored tolevels sufficient to support cell division. On the other hand, when cell division wasstopped by high concentrations of thymidine, a condition which leads to high intra-cellular concentrations of thymidine triphosphate (Cleaver, 1967), thymidylatesynthetase specific activity did not increase. Neither thymidine monophosphate northymidine triphosphate feedback inhibit the biological activity of thymidylatesynthetase (Hartmann & Heidelberger, 1961; Reyes & Heidelberger, 1965). Therefore,thymidine triphosphate must exert its influence on thymidylate synthetase activity bysome other mechanism.

The data are consistent with the possibility that a second regulator gene is involvedin the control of thymidylate synthetase activity. Actinomycin D applied during logphase stopped cell division and caused an increase in thymidylate synthetase specificactivity above control levels. Analogous results have been described for other enzymesystems in higher organisms (Garren et al. 1964; Moscona et al. 1968; Reel & Kenney,1968; Roth et al. 1968), and have been interpreted as evidence for the existence of asecond regulatory RNA species whose transcription is induced concurrently with orsubsequent to the initial induction of the target enzyme messenger RNA. Theactinomycin-D stimulated increase in thymidylate synthetase activity would seem toresult from the inhibition of transcription and not merely from the cessation ofenzyme dilution by cell division. Cell division could be stopped under other conditionswithout causing a concomitant elevation in thymidylate synthetase activity. Since thethymidylate synthetase system locked in the 'on' condition in the presence of actino-mycin D, the RNA product of such a regulatory gene would have to be more labilethan thymidylate synthetase messenger RNA.

Perhaps thymidine triphosphate interacts as an effector molecule with the hypo-thetical thymidylate synthetase regulator RNA. A similar system has been discussedrecently by Tomkins et al. (1969) for tyrosine transaminase regulation, except that inTomkins' model the effector molecule prevents the function of the regulatory RNA,whereas in this thymidylate synthetase model the effector molecule facilitates thefunction of the proposed regulatory RNA system. The mechanism of action of such athymidylate synthetase regulatory system remains to be resolved.

This work was supported by United States Public Health Service Research Grants(T1-HD-32), (1-F1-GM-35, 125-01) and (5-F1-GM-35, 125-02), and a United StatesPublic Health Service Grant from the National Institutes of Health (GM-9966). Presented at


the 8th Annual Meeting of the American Society for Cell Biology, Boston, Massachusetts,November 1968.

Taken from a dissertation submitted by A.H.C. in partial fulfilment of the requirements forthe degree of Doctor of Philosophy in Biology, Yale University.

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BRESNICK, E. & THOMPSON, U. B. (1965). Properties of deoxythymidine kinase partiallypurified from animal tumors. J. biol. Chem. 240, 3967-3974.

CAFFERY, J. M., WHICHARD, L. & IRVIN, J. L. (1968). Induction of hepatic tryptophan pyrrolaseand tyrosine transaminase by histones and other polypeptides. Biochim. biophys. Ada 157,616-626.

CLEAVER, J. E. (1967). Thymidine Metabolism and Cell Kinetics, pp. 70-103. New York: JohnWiley & Sons, Inc. (Interscience Publishers Division).

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(Received 28 May 1971—Revised 20 September 1971)

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