(CANCER RESEARCH 51, 2291-2295, May 1, 1991)

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid Effects on Nucleotide Metabolismin CCRF-CEM Human T-Lymphoblast Leukemia Cells1Giuseppe Pizzorno,2 Barbara A. Moroson, Arlene R. Cashmore, and G. Peter Beardsley1

Departments of Pediatrics, Pharmacology, and Medicine, Yale University School of Medicine, .\'e»'Harén,Connecticut 06510

ABSTRACT

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid |(6R)DDATHF| is afolate antimetabolite with activity specifically directed against de novopurine synthesis, primarily through inhibition of glycinamide ribonucle-otide transformylase. This inhibition resulted in major changes in thesize of the nucleotide pools in CCRF-CEM cells. After a 4-h incubationwith l ¿IM(6R)DDATHF, dramatic reductions in the ATP and GTPpools were observed, with almost no effect on CTP, DTP, and deoxyri-bonucleotide pools. When the incubation was continued in drug-freemedium, recovery of ATP and GTP pools was protracted. ATP did notreturn to normal until 24-36 h, and GTP pools were only partiallyrepleted by 48 h. The ATP and GTP pools were not affected when theinitial 4-h incubation with (6R)DDATHF was conducted in the presenceof 100 /J.Mhypoxanthine. Addition of hypoxanthine to the medium aftera 4-h incubation with (6R)DDATHF caused rapid recovery of the ATPand GTP pools. Similar effects were seen when the purine precursoraminoimidazole carboxamide was used in place of hypoxanthine. Theeffect of (6R)DDATHF on nucleotide pools and the capability of hypoxanthine or aminoimidazole carboxamide to prevent or reverse thisphenomenon correlated directly with the inhibition of cell growth. Presumably as a consequence of the decrease in purine nucleotide triphos-phate levels, the conversion of exogenous!) added uridine, thymidine, anddeoxyuridine to nucleotides was markedly decreased. These effects wereprotracted for almost 48 h and were also reversed by hypoxanthine.Differential repletion of ATP and GTP pools after (6R)DDATHF pretreatment demonstrated that diminished precursor phosphorylation isprimarily a consequence of GTP rather than ATP starvation.

INTRODUCTION

5,10-Dideaza-5,6,7,8-tetrahydrofolate is a representative of anew class of antimetabolites designed as inhibitors of folate-dependent enzymes other than dihydrofolate reducíase (1).DDATHF4 is a potent inhibitor of cell growth in culture (2)

and also displays broad spectrum activity against transplantablemurine solid tumors and human tumor xenografts (3). The 6Rdiastereomer of DDATHF (Lometrexol) is currently undergoing phase I clinical trials. Previous biochemical studies havedetermined the primary site of DDATHF action to be inhibitionof glycinamide ribonucleotide transformylase, an early step inde novo purine biosynthesis. These studies also demonstratedthat inhibition of neither dihydrofolate reducíasenor TS wasinvolved in the action of DDATHF (2). Depletion of inlracel-lular purine ribonucleotide pools was also shown to result fromthe action of DDATHF. In this report we explore more fullythe relationship between the effects of (6R)DDATHF on denovo purine biosynthesis, the consequent changes in intracel-

Reeeived 2/26/90; accepted 2/22/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by NIH Research Grants CA 42367 and CA 57320.2Special Fellow of the Leukemia Society of America.3 Recipient of an American Cancer Society Faculty Research Award. To whom

requests for reprints should be addressed, at Department of Pediatrics, YaleUniversity School of Medicine, 333 Cedar St.. P. O. Box 3333, New Haven. CT06510-8064.

4The abbreviations used are: DDATHF, 5,10-dideaza-5,6,7,8-tetrahydrofolic

acid; TS, thymidylate synthase; PCA. perchloric acid; TCA. trichloroacetic acid.

lular nucleotide pools, and the effects on cell metabolism andproliferation.

MATERIALS AND METHODS

DDATHF diastereomers, differing in configuration about C-6, werekindly supplied by Dr. Chuan Shin, Lilly Research Laboratories (4, 5).A solution of the sodium salt was used and concentrations weredetermined by using an extinction coefficient of 9.15 x IO3 M ' cm '.[5-3H]- 2-Deoxyuridine (23 Ci/mmol) and [2,8-'H]dATP (24 Ci/mmol)were purchased from Moravek Biochemicals (Brea, CA). [methyl-3H]Thymidine (40 Ci/mmol). [6-'H]deoxyuridine (16.6 Ci/mmol), and[2-'4C]uridine (50 mCi/mmol) were purchased from Amersham (Arlington Heights, IL). [methyl-*H]dTTP (21 Ci/mmol) was obtained

from NEN Research Products (Boston, MA). Mycophenolic acid wasa gift from Dr. A. C. Sartorelli (Yale University, New Haven, CT). Allother chemicals were from Sigma (St. Louis, MO) or Schwarz-Mann(Cleveland, OH) and were the highest purity available.

Media, sera, and antibiotics for tissue culture were purchased fromGIBCO (Grand Island, NY). Plasticware was purchased from CorningGlass Works (Corning, NY).

Cell Culture. A cloned subline of the human T-lymphoblast cell lineCCRF-CEM was used (6). Cells were grown and treated in suspensionculture in RPMI 1640 medium supplemented with 10% horse serum,penicillin (100 units/ml), and streptomycin (100 Mg/ml) at 37°Cin a

5% CÛ2atmosphere. Every 3 months stocks were checked for Myco-plasma contamination by using the gene probe method (Gene-Probe,San Diego, CA) and were found Mycoplasma free.

Ribonucleotide Pool Determination. Cells (5-10 x IO6)were collectedand extracted with l M formic acid saturated with «-butylalcohol onice (7). The extract was lyophilized and reconstituted with mobile phasebefore high performance liquid chromatography analysis. Separationof nucleotides was performed on a Whatman Partisi!-10-SAX aniónexchange column (Whatman, Woburn, MA), using 0.4 M ammoniumphosphate isocratic elution at a flow rate of 1.5 ml/min (8). Elutednucleotide triphosphates were monitored with a Model 153 Altex(Berkeley, CA) detector set at 254 nm and the retention peak areaswere determined with a Shimadzu C-RIA Chromatopac Integrator(Kyoto, Japan).

Deoxyribonucleotide Pool Determination. Cells (2 x 10) were washedtwice with ice-cold phosphate buffered saline and extracted with 0.5 NPCA. The supernatant was then neutralized with l M KH2PO4, pH 7.5,and 5 N KOH and stored at -70°C until analysis. Pool sizes were

determined by using a DNA polymerase assay as described by Williamset al. (9).

I'HJNucleoside Uptake. For the determination of transport and up

take into acid soluble fraction, aliquots of cell suspensions were placedin rapid sampling tubes consisting of 400-iil microfuge tubes containing120 M' of oil (a 16:84 mixture of light paraffin oil and Dow Corning550 silicone fluid) layered over 50 ^1 of 15% TCA. The cells werepelleted into the TCA layer by centrifugation for 2 min at 10,000 x g(10). The cell pellet was washed twice with 15% TCA and the super-natants were counted for radioactivity together with the supernatantobtained from the initial centrifugation.

|3H)Nucleoside Incorporation. Cells, at a density of 4-6 x IO5cells/

ml, were incubated with 1 i¿\i(6R)DDATHF for 4 h. After treatment,the cells were harvested by centrifugation ( 1000 x g for 5 min at 37°C),

washed twice with complete medium, and resuspended in drug-freemedium, with or without other agents (hypoxanthine, adenine, guanine,mycophenolic acid) at approximately the initial density. At appropriatetimes, 50 ml of the cell suspension were centrifuged and the cells were

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(6R)DDATHF EFFECTS ON NUCLEOTIDE POOLS

resuspended in 2 ml of medium, after which [m«/r>>/-3H]thymidine,[6-3H]deoxyuridine, or [2-'4C]uridine was added.

At intervals duplicate aliquot portions were removed, washed, andthe macromolecular species were precipitated by adding 2% PCA. Theprecipitate was washed twice with 2% PCA and resuspended in 10%PCA. The suspension was boiled for 15 min, centrifuged, and theradioactivity was determined (11). Results were expressed as cpm/106cells. Both the inhibition by (6R)DDATHF and the rescue by hypoxan-thine, adenine, and guanine caused changes in the specific activity ofthe nucleotide pools compared. Specific activities of these pools werecalculated by using the measured values for radioactivity in the acidsoluble fraction and the total size of the nucleotide pool. These adjustedspecific activities were then used to calculate corrected values for thesynthetic rates of the macromolecular species. The extent of inhibitionof nucleoside incorporation produced by (6R)DDATHF was calculatedby comparing the slopes obtained from the least squares linear regression analysis of the inhibited and control incorporation data.

[5-3H|-2'-Deoxyuridine Tritium Release Assay. Inhibition of TS in

intact cells was measured by estimation of tritium released after cellularuptake of [5-3H]-2'-deoxyuridine conversion to [5-3H]dUMP, and thesubsequent release of 3H2O during the TS reaction which replaced the5-3H with a methyl group (12). Cells were collected by centrifugationand suspended in 1 ml of medium at a concentration of 0.5-1.0 x IO6cells/ml. After addition of [5-3H]deoxyuridine (0.1 ^Ci/ini), aliquots of

100 //I were taken at intervals and the reaction was stopped by additionof 200 ill of a suspension of 10% activated charcoal in 4% trichloroaceticacid. After centrifugation, 200 n\ of supernatant were measured forradioactivity. Results were analyzed as described above.

RESULTS

Our previous studies (2) measured ATP and GTP levels onlyat a single time point after prolonged exposure (6 h) of cells toDDATHF. We therefore conducted a more thorough analysisof the time course of the effects of (6R)DDATHF on intracel-lular nucleotide pools. As shown in Fig. 1, when CCRF-CEMcells were incubated with 1 ^M (6R)DDATHF, an effect onnucleotide pools became evident after 2 h in the presence of thedrug. At the end of 4 h of incubation, ATP and GTP poolswere decreased by 40-50%. There was a slight drop of CTPlevels, limited to 10-15%, and some increase in UTP pools.Cells were then transferred to drug-free medium and, after anadditional 4-h incubation in drug-free medium, both ATP andGTP dropped further to 30-35% of control. The ATP levelremained stable for at least 24-36 h, while there was a slowrecovery of the GTP pool to 50-60% of the size of an untreatedcontrol after 24-48 h.

This dramatic effect on adenine and guanine nucleotide poolswas completely reversed by the addition of 100 pM hypoxan-thine. This was true whether hypoxanthine was added simultaneously with (6R)DDATHF or when, after 4 h of exposureto (6R)DDATHF, cells were resuspended in drug-free mediumwith added hypoxanthine (Fig. 2, A and B). In addition to theaugmentation of ATP and GTP pools by hypoxanthine, decreases in the UTP and CTP pools were also observed. Thisdecrease, as shown in Fig. 2C, was due only to the effect ofhypoxanthine. The reversal of (6R)DDATHF effects by hypoxanthine was seen not only in the size of ATP and GTPpools (Fig. 3) but also in cell growth inhibition (Fig. 4). Thepurine precursor aminoimidazole carboxamide also protectedagainst (6R)DDATHF suppression of nucleotide pools and cellgrowth (Figs. 3 and 4). For both aminoimidazole carboxamideand hypoxanthine, the concentration dependence for reversalof the nucleotide pool effects was concordant with that forprotection from the growth inhibitory effects of (6R)DDATHF.

Deoxyribonucleotide pools were determined after 4 h of

160

140

120o•g100

? «Oojj 60

40

20

O 4 20 40 60

Time (h)Fig. 1. Effects of (6R)DDATHF on nucleotide pools in CCRF-CEM cell lines.

CCRF-CEM cells were incubated for 4 h in the presence of l »M(6RJDDATHFand then resuspended in drug-free medium at the time indicated by the arrow.Incorporation was measured as described in "Materials and Methods." (•)CTP;

(T) UTP; (•)ATP: and (A) GTP.

180

160

140

120

~ 100oo,- 80

60

40

20

24 48 O 24 48 O 24 48

Time (h)Fig. 2. Effects of (6RJDDATHF and hypoxanthine on nucleotide pools. CCRF-

CEM cells were incubated with l UM(6R)DDATHF for 4 h and then (Time 0)washed and resuspended in drug-free medium. .1. medium containing 100 ><Mhypoxanthine following a 4-h exposure to (6R)DDATHF; B, I ^M (6RJDDATHFsimultaneously with 100 e M hypoxanthine; C, 100 n\t hypoxanthine alone. (O)CTP; (V) UTP; (A) GTP; and (D) ATP.

incubation with 1 ^M (6R)DDATHF and additional 4 h in drug-free medium, a time when ATP and GTP reach a minimal level.dATP and dGTP pools were not decreased at this time, rather,their size was increased approximately 50% compared to anuntreated control. The addition of hypoxanthine following(6R)DDATHF doubled the size of dATP and dGTP pools(Table 1).

(6R)DDATHF at concentrations up to 100 ^M, did not haveinhibitory effects on TS in vitro using either Lactobacillus caseipurified enzyme, as previously reported, or a crude extract fromCCRF-CEM cells as assayed according to the isotopie methodof Roberts (13). This finding was consistent with the observation that thymidine did not prevent DDATHF cytotoxicity inthe LI210 and CCRF-CEM cell lines (2). However, CCRF-CEM cells assayed for in situ TS activity after a 4-h treatmentwith 1 MM(6R)DDATHF showed markedly diminished 3H2Orelease from [5-3H]deoxyuridine (Fig. 5). This effect was max

imum 4 h after completing (6R)DDATHF exposure, falling to2292

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(óR)DDATHF EFFECTS ON NUCLEOTIDE POOLS

sïoo»*;a«12010080604020n:•

7:

/////«¡.-v_o-^o-*^—

l_».J 1 1 ..J—0 O.I 1.0 IO 100 0 O.I 1.0 IO 100

Fig. 3. Effects of hypoxanthine and aminoimidazole carboxamide on nucleotidepools after exposure to (6RJDDATHF. Cells were incubated 4 h with l ¡IM(6R)DDATHF. then washed and resuspended in drug-free medium in the presenceof different concentrations of hypoxanthine or aminoimidazole carboxamide. .1.ATP pool; B, GTP pool; (•)hypoxanthine; (O) aminoimidazole carboxamide;(A)(6R)DDATHFalone.

100 -

80 -"5

p"o 40

20

0 o ai i.o 10 ico

Fig. 4. Effects of hypoxanthine and aminoimidazole carboxamide on cellgrowth inhibition after exposure to (6R)DDATHF. Cells were treated for 4 hwith 1 JIM (6RJDDATHF, then washed and resuspended in drug-free mediumwith hypoxanthine or aminoimidazole carboxamide at the indicated concentrations. After 48 h, the cells were counted and the results are expressed as percentageof untreated control. (•)hypoxanthine; (O) aminoimidazole carboxamide; (A)(6R)DDATHF alone.

about 25% of the untreated value, and was followed by a slowrecovery after 8-24 h in drug-free medium. The same treatmentcaused even more dramatic suppression of [methyl-3H]thymi-dine and [6-3H]deoxyuridine incorporation into macromole-

cules (Fig. 5). By 4 h following drug exposure, thymidine anddeoxyuridine incorporation were reduced to 8 and 4% of untreated control values, respectively. These results differed fromour short term (30 min) exposure studies, in which no suppression of thymidine or deoxyuridine incorporation was found asan immediate consequence of incubation with (6R)DDATHF.At this time intracellular purine nucleotide pools are minimallyperturbed (Fig. 1). In the present study cells were incubatedwith (6R)DDATHF for 4 h before being analyzed for nucleotideincorporation. The marked diminution in intracellular ATPand GTP pools which takes place after this degree of drugexposure could alter the apparent rates of a number of processesinvolving nucleotide metabolism.

A marked decrease in the uptake of [3H]thymidine and [14C]-deoxyuridine into the intracellular acid soluble species (nucleo-tides) was seen after 4 h of exposure to (6R)DDATHF, withlevels falling to 25 and 34%, respectively, of untreated controlcells. The early time course of uptake (<2 min) in CCRF-CEMcells of either [3H]thymidine or [14C]uridine in the presence of

1 pM DDATHF did not show any alteration in the transport ofthese precursors of nucleic acids (data not shown). The dimin

ished formation of nucleotides at later time points, however,strongly suggested decreased nucleoside phosphorylation as theactual cause of the apparent suppression of 3H2O release and

decreased macromolecular incorporation. This prompted further investigation into the comparative effects of the sizes ofthe ATP and GTP pools on the overall process of nucleosidephosphorylation.

The incubation of CCRF-CEM cells after 1 MM(6R)DDATHF with 100 UM adenine restored both ATP andGTP pools to normal values with concomitant recovery of theapparent rate of pHlthymidine incorporation into DNA and['4C]Urd incorporation into RNA. DNA and RNA incorpora

tion were also returned to normal when the rescue agent was100 pM guanine. Under these conditions, only GTP returnedto control levels and ATP did not show any change comparedto cells treated with (6R)DDATHF and then left in drug-freemedium (Table 1).

In order to replete only ATP pools, the cells were incubated,after (6R)DDATHF treatment, with medium containing 100/¿Mhypoxanthine and 50 fiM mycophenolic acid, an inhibitorof inosinate dehydrogenase. In this case, ATP reached nearlynormal values, GTP did not increase, and nucleic acid incorporation remained completely inhibited (Table 1).

DISCUSSION

The data presented demonstrate that a major consequence ofexposure of CCRF-CEM cells to (6R)DDATHF is a markeddiminution in the size of purine nucleotide pools. This depletionis time dependent and directly correlates with (6R)DDATHFcytotoxicity. As shown in Fig. 1, both ATP and GTP poolsbecome greatly depleted after 4 h of drug exposure. Purinenucleotide pools depletion deepens further and is protracted formore than 48 h after removal of the drug. This suggests eithera high affinity of (6R)DDATHF for the enzyme target and/orprolonged retention of the drug into the cells as might resultfrom formation of (6RJDDATHF polyglutamates (14).

The results also demonstrate the capacity of hypoxanthine toblock the effects of (6R)DDATHF on adenine and guaninenucleotide pools with a dose response correlating directly withits ability to prevent the growth inhibitory effects of(6R)DDATHF. This strongly implicates the decrease in purinenucleotide pools in the mechanism of (6R)DDATHF action.One consequence of this is demonstrated in the effects of(6R)DDATHF on the incorporation of DNA and RNA precursor nucleosides into acid soluble pools. This almost certainlyresults from a limitation in the supply of ATP and/or GTP toserve as phosphate donors in the kinase reactions necessary forconversion of the nucleosides to nucleotides. An effect due to afeedback inhibition of the kinase reactions by increased pyrim-idine nucleotide pools is also possible, but seems unlikely, asthe magnitude of the increase amounts to only 20-30%. It ishighly likely that other ATP or GTP dependent reactions wouldbe suppressed as well.

The diminished observed incorporation of radiolabeled precursors into DNA and RNA seems to reflect not only a lessenedphosphorylation of labeled nucleosides to activated precursors,but also a diminished rate of macromolecular synthesis overall,a strong indication of the above mentioned effect is reported inTable 1, where there is a marked decrease in the macromolecular synthesis associated with a decrease in ATP and GTPpools even after correction for the specific activity changes inthe nucleotide pools. Surprisingly, no decrease in the purine

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(6R)DDATHF EFFECTS ON NUCLEOTIDE POOLS

Table 1 Ribo- and deoxyribonucleotide pools, and DNA and RNA synthesis in CCRF-CEM cellsThe results are the average of three experiments conducted in duplicate, with SD less than I5°o.Cells were incubated for 4 h with I JIM(6R)DDATHF, then

washed and resuspcnded in drug-free medium containing the above mentioned compounds. Ribo- and deoxyribonucleotide pools, and DNA and RNA synthesis wereexamined 4 h later as described in "Materials and Methods." In control cells, the nucleotide concentrations were: CTP, 0.48 nmol/10' cells; DTP, 1.63 nmol/106cells; ATP, 5.34 nmol/10" cells; and GTP, 3.12 nmol/106 cells; in the case of deoxyribonucleotides dATP, 60 pmol/10'cells; dGTP, 36 pmol/10'cells.

% of controlofHvpoxanthine.

100 /<M(6R)DDATHF, 1 „M(6R)DDATHF + hypoxanthine(6R)DDATHF + adenine. 100 /IM(6R)DDATHF + guanine. 100 /JM(6R)DDATHF + hypoxanthine -I- MA

(6R)DDATHF + MA. 50 pMCTP69.8

80.190.975.095.873.685.6UTP54.2

128.868.760.6

103.1104.4204.5ATP123.5

30.3143.1121.532.582.844.3GTP139.6

35.1167.696.1

461.615.514.9dATP215.0

165.0193.1ND

169.6NDNDdGTP161.8

143.1180.7ND

141.1NDND|3H|dThd°-

*

incorporatedinto DNA

(% ofcontrol)117.4

8.0118.7109.2105.3

3.75.4rqurd*

incorporatedinto RNA

(% of control)72.0

13.295.098.7

108.68.76.8

" dThd. th>niiiliiu-: ND. not determined.* Corrected for specific activity as described in "Materials and Methods."

oo

100

80

60

40

20

8 12 16 20 24

Time (h)Fig. 5. Effects of (6R)DDATHF on |3H]nucleoside metabolism in CCRF-CEM

cells. Cells were pretreated with (6R)DDATHF(1 pM for 4 h), and then incubatedin drug-free medium (Time 0). Control represent untreated CCRF-CEM cells.(•)3H2O release from |5-JH]dUrd; (•)|'H]thymidine incorporation; (A) |6-JH]-

dUrd incorporation.

deoxynucleotide pools was found even when ATP and GTPpools were down to 30-35% of the control levels.

Differential repletion of ATP and GTP pools was used todetermine which pool was primarily responsible for the effectson DNA and RNA incorporation. The addition of adenine tothe media after exposure to (6R)DDATHF repleted both ATPand GTP pools and consequently DNA and RNA incorporation. When guanine was used, there was a complete recovery ofthe GTP pool, but the ATP pool remained depressed. BothDNA and RNA incorporation were completely resumed. Restoring only the ATP pool, using hypoxanthine and mycophen-olic acid (15) resulted in nearly complete recovery in the size ofthe ATP pool, but the GTP pool remained depressed. Therewas still a complete inhibition of DNA and RNA incorporation.These results demonstrate that the depletion of guanine ribo-nucleotide pool is primarily responsible for the inhibition ofprecursor phosphorylation and presumably DNA and RNAsynthesis.

Further indications of a connection between the apparentrate of DNA synthesis and size of the GTP pool are apparentin the timing of recovery of thymidine and deoxyuridine incorporation into DNA after cessation of (6R)DDATHF exposure(Fig. 5). These are better correlated with restoration of the GTPpool, which had returned to nearly 60% of the control value by24 h, then with the ATP pool which returned toward normalmore slowly (Fig. 1).

Recent reports have also noted a specific relationship betweenGTP pool size and DNA synthesis (16-19). In one case (18),the differing effect of adenine and guanine nucleotide pools onDNA synthesis were attributed to differences in the relative size

of the functional nucleotide precursor pools. In another report(19), it was found that preceding the exponential phase of cellgrowth an expansion of the GTP pool occurred with a corresponding decrease in the ATP pool. Adenylates were morepredominant in the plateau phase of growth.

Recent evidence has shown the important relationship between GTP levels and the ability of G proteins to function astransducers of intracellular signals, particularly their controlon adenylate cyclase and cyclic guanosine 3',5'-monophos-

phate-specific phosphodiesterase (20-21). Disruption of these

signal transduction pathways could certainly be part of theaction of (6R)DDATHF.

In summary, the present results demonstrate the importanceof depletion of purine nucleoside triphosphates as a consequence of inhibition of de novo purine biosynthesis in thesecells. There is an absolute requirement for net purine synthesisin order to supply precursors for the new DNA and RNAsynthesis necessary for cell growth and division. In the absenceof a sufficient supply of purities via salvage pathways and alimited supply from de novo synthesis, we presume that purinetriphosphate pools become depleted as a result of attemptedDNA and RNA synthesis as well as ongoing irreversible oxi-dative purine catabolism. Intracellular purine economy eventually reaches a new equilibrium where there is little or no netDNA and RNA synthesis and the purine triphosphate poolsare markedly diminished. A further consequence of this seemsto be a lessened ability to carry out critical phosphorylationreactions as indicated by the decreased conversion of thymidineand deoxyuridine to their corresponding nucleotides. All theabove provide a rational basis for understanding the mechanismof growth inhibition by inhibitors of de novo purine biosynthesissuch as DDATHF.

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1991;51:2291-2295. Cancer Res   Giuseppe Pizzorno, Barbara A. Moroson, Arlene R. Cashmore, et al.   Leukemia CellsNucleotide Metabolism in CCRF-CEM Human T-Lymphoblast (6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid Effects on

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