effects of 5-mercapto-2'-deoxyuridine on the incorporation ...determination of cell number....

[CANCER RESEARCH 36, 3284-3293, September 19761

Exogenous guanosine incorporation into lymphocyteacid-insoluble material is increased by MUdR. This increased utilization of exogenous nucleoside is apparentlythe result of MUdR inhibition of conversion of adenosine toguanine nucleotides within the lymphocytes and a consequent diminution of the total intracellular guanine nucleotide pool size.

The active inhibitory compound is the deoxymibonucleoside or deoxynibonucleotide. Comparison with the nibosideanalog 5-mercaptoumidine showed that MUdR was a moreefficient inhibitor of nucleoside incorporation.

INTRODUCTION

The isostemic substitution of the mercapto group for themethyl group at the pymimidine 5-position was the initialrationale of Baranski et al. (3) for the synthesis and testingof MUdR4 as a thymidine analog and inhibitor of DNA synthesis. It was found that MUdR effectively blocks DNA synthesis in bacterial cells (3, 19). The mechanism of thisinhibitory effect of MUdR involves competition at the deoxynibonucleoside level for thymidine kinase and activation ofthe analog to the corresponding monophosphate, which inturn is a potent inhibitor of thymidylate synthetase. Recentresults in this laboratory show that MUdR is an effectivechemotherapeutic agent against both transplanted and carcinogen-induced animal tumors (Z. F. Chmielewicz, A.Carter, and T. J. Bardos, unpublished observation) andthat MUdR has significant inhibitory effects on human skinneoplasms (28).

PHA-stimulated primary lymphocyte culture has beenproposed as an effective screening system for antimetabolites (11). In particular, DNA biosynthetic enzymes such asthymidine kinase, thymidylate synthetase, and DNA polymerase show elevated activities following mitogenic stimulation that are similar to the changes seen in vimus-transformed cells (21) on regenerating tissue (4). We undertookthese studies to determine the effectiveness of MUdR inblocking DNA biosynthesis in this mammalian system. Inparticular, the inhibition of thymidine kinase and thymidylate synthetase activities were focused upon, since the previous work cited indicates that these are primary loci ofMUdR action.

4 The abbreviations used are: MUdR, 5-mercapto-2'-deoxyuridine; PHA,

phytohemagglutinmn; RPMI, Roswell Park Memorial Institute; MUR. 5-mercaptouridine.

I

3284 CANCERRESEARCHVOL. 36

Effects of 5-Mercapto-2'-deoxyuridine on the Incorporationof Nucleosides into RNA and DNA in a Primary LymphocyteCulture System1

D. Bogyo,2 T. J. Bardos, and Z. F.

Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Buffalo, New York 14214

SUMMARY

The effects of 5-mercapto-2'-deoxyuridine (MUdR) onDNA synthesis in a primary munine spleen lymphocyte cultune system stimulated by phytohemagglutinin (PHA) werestudied. Inhibition of thymidine incorporation into acidinsoluble nucleic acid material was 50% at 0.5 mM MUdRconcentration, while inhibition of deoxyuridine incorporation into acid-insoluble nucleic acids was 50% at 0.01 mMMUdR. Time course studies, at 0.5 and 0.05 mM MUdR,showed that the magnitude of inhibition of incorporation forthymidine and deoxyuridine, respectively, increased from atime point after PHA stimulation when increased synthesisof thymidine kinase and thymidylate synthetase had leveledoff.

At 1 mM MUdR, total cellular DNA in cultures was decreased 43% at 42 hr after PHA stimulation. Neither the totalnumber of cells nor the percentage of PHA-transformedcells was decreased in comparison to that of controls.MUdR therefore blocks the increase in DNA content oflymphocytes that is initiated during the S phase of the cellcycle.

Millimolar levels of MUdR inhibited incorporation of undine, adenosine, and cytidine into acid-insoluble material inPHA-stimulated primary munine lymphocyte cultures. Totalcellular RNA synthesis was inhibited at these levels ofMUdR, with no differential effects on 4, 18, or 28 S RNAspecies observed. Uptake of these nucleosides into the totalcellularacid-solublematerialwas not blocked.

Uptake of different labeled nucleosides into cellular, acidsolublepools occurs at differentmates.Thus, choice of asuitable minimum pulse time to achieve saturation for different labeled nucleosides must relate to this consideration.

Thymidine kinase from whole-cell sonic extracts of PHAstimulated lymphocytes was inhibited 65% by 1 mM MUdRat 24 and 48 hr after stimulation. Uridine kinase extractedfrom the PHA-stimulated cells was also significantly inhibited by 1 mM MUdR at 24 hr (56%).

, Thisworkwas partlysupported by NationalCancer InstituteGrantCA06695 and American Cancer Society Grant CH-20C.

a Trainee under USPHS Training Grant PHS-5T01GM00555, which partlysupported this work. Part of this work is from a dissertation submitted toFaculty of State University of New York at Buffalo. in partial fulfillment of therequirements for the Ph.D. degree. Present address: Department of Experimental Therapeutics and Grace Cancer Drug Center, Roswell Park MemorialInstitute, Buffalo. N. Y.

3 To whom requests for reprints should be addressed.

Received September 16, 1975; accepted May 25, 1976.

Research. on January 22, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Effect of MUdR on Lymphocyte RNA and DNA Synthesis

Our preliminary studies using the PHA-stimulated muninelymphocyte system indicated that MUdR, at higher concentrations, also inhibited RNA synthesis. Further work wascarried out to investigate the effects of MUdR on mibonucleoside on nibonucleotide synthesis and interconversion.

MATERIALS AND METHODS

Materials

Unlabeled nucleosides and nucleotides were obtainedfrom Sigma Chemical Co. , St. Louis, Mo. (unidine, guanosine, 3'-UMP, UMP, 3'-CMP, CMP, 3'-GMP, GMP, 3'-AMP,and AMP). The following radioactively labeled precursorswere purchased from ICN Chemical Aadioisotopes Division(Irvine, Calif.): [methy/-3H]thymidine (16.7 Ci/mmole), [6-3H]deoxyunidine (21.2 Ci/mmole), [5-3H]umidine (21.7 Ci/mmole), [8-3H]guanosine (16.3 Ci/mmole, and [5-3H]cytidine (22 Ci/mmole). [2-3H]Adenosine (10.3 Ci/mmole) was obtained from New England Nuclear, Boston,Mass. , as was Aquasol. Calf thymus DNA was obtained fromSigma Chemical Co. Purified PHA was acquired from Bunroughs-Wellcome, Research Triangle Park, N. C. NCS solubilizer was purchased from Amensham-Seanle, ArlingtonHeights, III. The RPMI 1640 medium and all supplementswere obtained from Grand Island Biological Co. , GrandIsland, N. Y., as was the trypsin-EDTA solution. Giemsastain type B was obtained from Hanleco, Philadelphia, Pa.

Methods

Lymphocytes were isolated from the spleens of 6-weekold male BALB/c mice bythe method ofAdlemetal. (1). Theywere suspended at a concentration of 5 x 106 cells/mI inRPMI 1640 med ium with N â€˜-2-hydmoxyethylpipemazine-N'-ethanesulfonic acid supplemented with 10% fetal calf semum, penicillin (100 units/mI), streptomycin (100 j@g/ml),and L-glutamine (1%). PHA was added (1 @g)to each ml ofthe cell suspension. Aliquots of 1 ml of the cell suspensionin 15- x 45-mm glass screw-top vials were incubated in ahumidified aimatmosphere containing 10% CO2.Amounts ofMUdR listed in the test refer to final concentrations in the 1-ml lymphocyte cultures.

Incorporation of Labeled Nucleosides

Radioactive nucleosides were added (3 @Ci/cultume)6 hrbefore the harvesting of cells, except in the uptake studieswhere they were added for the time periods indicated.

The cells were processed for liquid scintillation countingas described previously (25). NCS solubilizer was used instead of Hyamine hydroxide. Aliquots (0.2 ml) of the 1sttnichlomoacetic acid supemnatant were dissolved in 3 ml ofAquasol and used for acid-soluble incorporation studies.

Determination of Cell Number. PHA-agglutinated cellcultures were centrifuged at 600 x g for 10 mm at roomtemperature. To the cell pellet was added 0.5 ml of a 0.05%trypsin-0.02% EDTA solution with gentle mixing. After 10mm of incubation at 37Â°,2.5 ml of RPMI 1640 medium with10% fetal calf serum were added to each culture. The vials

were centrifuged for 10 mm at 600 x g. Tothe cell pellet wasadded 1 ml of the fully Supplemented medium with gentlemixing. A quantity of cell suspension (0.1 ml) was mixedwith 0.5 ml of trypan blue dye (0.4%), and cell counts weredone on a Neubauer grid hemacytometer at x 10 magnification.

Determination of Percentage of Blasts. Cultures wereincubated with 0.01 ml of 106M colchicine for 2 hr beforeharvesting to produce metaphase arrest. Cells were fixed bythe method of Hastingseta!. (15), and drops were placed onglass covenslips to aim-dry. Covenslips were stained for 10mm with Giemsa stain (pH 6.8), washed, aim-dried, andmounted on glass slides with Canadian balsam. The criteriaof Cooper et al. (6) were used for grading cells, and bothtype 2 and type 3 cells were considered blasts.

DNA determinations were carried out on washed cell pellets of 5 x 106cells by the diphenylamine method of Burton(5).

RNA content was determined by a modified oncinolmethod (22), and cellular protein was assayed by themethod of Lowry et a!. (23).

Nucleoside Kinase Assay. Twenty cultures containing 5x 106 cells/mI were grown with PHA for the appropriate

time and washed , and cells were collected by centnifugationfor 10 mm at 600 x g in 3 ml of 0.9% NaCI solution perculture. To each culture pellet was added 1 ml of 0.1 M Tnis,pH 8.0, at 0Â°.The cells were pooled, collected by centnifugationat 1500 x g for10 mm, and suspended in3 ml of theTnis buffer. The cells were sonically extracted on ice with aBranson sonicatom, microtip, using four 15-sec bursts at apower setting of 0.4 maximum alternating with 15-sec restintervals. The sonic extract was centrifuged at 3500 x g for10 mm, and the supemnatant was used for the kinase assay.

The nucleoside kinase assay was carried out as describedby Ives et al. (18). Incubation time was 90 mm for the assay.DEAE discs were washed with 6 x 10 ml of 1 mM ammoniumfommateand , similarly, with distilled water. The reaction ratewas linear over the 90-mm incubation period, and reactionvelocity increased linearly with addition of increasing enzyme.

Isolation of Spleen Lymphocyte RNA

Two sets of 24 cultures (120 x 106cells) were established,1 with PHA and the other with PHA and 10@@M MUdR fromzero time. Each culture received a 6-hr pulse of tnitiatednucleoside (3 MCi) starting at 18 hr after initiation of incubation. All cultures were then centrifuged at 600 x g for 10mm. The cell pellets were washed with 3 ml of 0.14 M NaCIand then were suspended in 1 ml of ice-cold acetate buffer(10 mM), pH 5.1 , pen vial, with 0.1 M NaCI and 5% bentonite.RNA was extracted from the pooled cell cultures by themethod of Cooper (7). Bentonite was purified by the methodof Frankel-Conmat et al. (13). Rat liver RNA was used as amarker and coprecipitating agent for labeled RNA was prepared by the method of Hiatt (17).

Sucrose Density Centrifugation

The total labeled RNA extract from both the PHA and thePHA plus MUdA cultures was dissolved in 2 ml of the ace

SEPTEMBER1976 3285



D. Bogyo et a!.

tate buffer (10 mM), pH 5.1 , and layered oven the tops of 2linear 5 and 20% sucrose gradients (33 ml) prepared with aBuchler gradient marker. Over a 3rd 5 to 20% gradient waslayered 1 ml of the rat liven RNA marker (600 @g)plus 1 ml ofthe acetate buffer. The gradients were centrifuged at 25,000rpm (113,000 x g) for 14 hr with the use of a Spinco SW-27head. The sucrose density gradients were placed in a Plexiglas piercing apparatus, and the cellulose nitrate tubeswere tapped by gravity flow from the bottom. One-mI fractions were collected . The tubes were read for sucrose concentration on a Bausch and Lomb refractometer. Themarker gradient fractions were read at 260 nm to determinethe position of 4, 18, and 28 S RNA peaks. Aliquots of 0.2 mlof the labeled RNA fractions were removed, mixed with 3 mlof Aquasol in glass screw-cap vials (15 x 45 mm), andcounted in 10-mI plastic scintillation vials in a Packard TnCambliquid scintillation counter.

Separation of Nucleotides

Total labeled RNA, extracted from PHA and PHA plus 10@M MUdR cultures, as described for the sucrose density

centnifugation, was hydrolyzed at a final concentration of0.5 M KOH for 15 hr at 37Â°.This digest was neutralized withperchlonic acid and centrifuged for 10 mm at 1500 x g.Aliquots of the supemnatant were spotted on Whatman No. 2chromatography strips along with the 3' and 2' standards ofAMP, CMP, UMP, and GMP. Descending phase paper chromatogmaphy was carried out with the solvent system, 80%satu matedammonium sulfate-2% isopropyl alcohol-18% sodium acetate, 1 M (v/v/v). The chromatogmam was developed for 16 hr. Spots were identified with a Mineralight UV12 scanner, measured for AF values, and cut out from thechromatognam. Ten ml of Aquasol were added to eachsample in glass scintillation counting vials, and radioactivitywas determined.

RESULTS

Effects of Different Concentrations of MUdR on Nucleoside Incorporation. When munine spleen lymphocytes arecultured in the presence of PHA, a marked increase in themateof unidine incorporation is seen starting at approximately 12 hr, followed by an increased rate of thymidineincorporation starting at approximately 24 hr after stimulation.

Incorporation of thymidine remains high after 48 hn, butSasaki and Norman (27) have shown that, after this time, anincreasing proportion of PHA-stimulated cells show labeledmitoses indicative of a 2nd cell division. Our initial studieson MUdR inhibition of labeled nucleoside incorporationwere carried out only up to 48 hr after PHA stimulation toeliminate this component of net cellular increase.

The dose-response curves for MUdR inhibition of thymidine, deoxyunidine, and uridine incorporation (Chart 1) mdicate 50% inhibitory levels of MUdR to be 0.5 mM for thymidine, 0.01 mM for deoxyuridine, and 1 m@ifor umidine.

Effects of MUdR with Increasing Time. Time course studies of MUdR effects were carried out at several concentrations in the 10 to 90% inhibitory mange.The thymidine incon

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66 72

Time After Addition of PHA (hr)

Chart 2. Effect of MUdR on [3H]thymidine incorporation into PHA-stimulated spleen lymphocytes at various times following drug addition. Cells (5 x10'/ml) were cultured in the presence of PHA and various concentrations ofdrug from 0 hr. Six hr before sampling, cultures were pulse-labeled withthymidine (3 MCi/culture). Cells were harvested and radioactivity counted asdescribed in Materials and Methods.

3286 CANCER RESEARCH VOL. 36

! :@Deoxyuridine

A...â€¢@@

. 0 @.,Thymidine

@ Uridine

l0@ 5@l0@ 0' 5xl0@ 10' 5,i0@ I0@ 5x103

Concentration,MUdR(N)

Chart 1. Effects of MUdR on [3H]uridine, [3H]deoxyuridine, and[3H]thymidine incorporation into PHA-stimulated spleen lymphocytes. Cells(5 x 1O'/mI) were cultured in the presence of PHA and drug from 0 hr.Labeled nucleosides were added at either 18 hr (uridine) or at 42 hr (thymidine or deoxyuridine), at the level of 3 MCi/culture. Six hr later, the cells wereharvested and radioactivity was counted as described in Materials andMethods. â€˜Inhibition is expressed as the percentage of uridine incorporatedinto cultures containing PHA and no drug.

poration time course (Chart 2) indicated that, until 48 hrafter PHA stimulation, there was increased thymidine incomporation at all levels of MUdR tested. From that time pointon, no increase in incorporation was seen at the 2 highestlevels of MUdA, and the absolute magnitude of inhibitionthus increased. Determination of acid-soluble thymidinelevels within the lymphocytes indicated that, at all levels ofMUdA tested, thymidine levels in total soluble pools increased over the whole time course. The deoxyunidine incorporation time course (Chart 3) showed increased incorporation into acid-insoluble material at all MUdR levels until48 to 54 hr after PHA stimulation. Subsequently, deoxyunidine incorporation decreased at all MUdR levels as well asin the PHA controls. The period from 24 to 48 hr after PHAstimulation has been shown to involve a 3-fold increasedactivity of thymidylate synthetase (24). Inhibition of a netincrease in deoxyunidine incorporation is seen after theincrease in thymidylate synthetase activity ceases.




[3H]unidine incorporation results have been shown to produce an overestimate of actual RNA synthetic rates (8). Theresults of these experiments (Table 1) show that 1 mMMUdR inhibits [3H]adenosine incorporation 26% as compared to that of PHA controls. [3H]Cytidine acid-insolubleincorporation is inhibited 90%, and [3Hjguanosmne acidinsoluble incorporation is stimulated 3-fold by MUdR action. The [3H]adenosine inhibition will be shown to correlateclosely with the net decrease in total cellular RNA inducedby 1 mM MUdR. [3H]Cytidine inhibition magnitude reflectsMUdR effects on cytidine activation by umidine kinase andalso possible increases in cytidine intracellular pool sizedue to potential inhibitory effects of MUdA on cytidineaminohydmolase. The increased [3Hjguanosmne incompomation in the presence of MUdA was subsequently investigated and found to be related to MUdA-induced diminutionof total intracellular guanosine pool size.

Effects of MUdR on Transformation. The inhibitory effects of MUdR on PHA transformation of lymphocytes intolymphoblasts were studied in fixed, Giemsa-stained sampIes removed from the cultures at various times followingaddition of PHA alone on with 1 m@iMUdR. The results mdicated that the percentage of transformation in cultures contaming MUdR was not decreased in comparison to that ofPHA control cultures. Preincubation of PHA and 1 mMMUdR for periods of 2 hr did not increase the inhibitory effects of MUdR. Addition of MUdR as long as 4 hr after PHAincubation with the lymphocytes did not decrease the extentof MUdR inhibition seen in cultures where MUdR had beenpresent simultaneously with added PHA. In order to determine total cell counts in MUdR-tmeated cultures and in PHAstimulated control cultures, a mild trypsinization methodwas used to disaggregate PHA-agglutinated cells. Themethod produced good separation of cells, and viabilitywas not decreased , as determined by trypan blue dye exclusion. Results of the time-course variation in total cell countshowed no increased cell death with 1 mr@iMUdA plus PHA,compared with the PHA controls. Both the PHA and theMUdR plus PHA sets of cultures showed a 35% decrease incell count during the 1st 6 hr of incubation. This is similar tothe percentage loss of lymphocytes we observed duringincubation without PHA. Lucas (24) has reported a 40%decrease in mean DNA content of PHA-stimulated culturesafter 45 hr of incubation.

Effects of MUCIR on DNA, RNA, and Protein Content.Quantitative automadiogmaph studies of cell nuclei byCooper et a!. (6) have indicated that, following the increased activity of DNA-synthesizing enzymes, most lymphocytes contain a DNA complement greaten than diploidbut smaller than tetmaploid. We investigated the effects of 1mM MUdR on total DNA, ANA, and protein content of cul

tunes to determine the magnitude of inhibition.Diphenylammne assay determinations showed a 43% de

crease in DNA at 42 hr after PHA stimulation in the presenceof 1 mM MUdR. Orcinol assay results showed 21% RNAinhibition by the same level of MUdR at 24 hr after stimulation. Lowry protein determinations (23) on 24-hr culturesshowed only at 8% drug-caused decrease in total protein.The inhibition of net RNA synthesis is quite similar to theobserved level of inhibition of adenosine incorporation into

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Chart 3. Effect of MUdR on [3H]deoxyuridine incorporation into PHAstimulated spleen lymphocytes at various times following drug addition. Fordetails, see legend to Chart 2.

Time course studies on [3H]umidine acid-insoluble incomponation (Chart 4) show that almost complete inhibition ofthe net increase of incorporation occurs only at the 1 mMMUdR level. Since PHA control levels of incorporation arerising steadily throughout the time course, the magnitude ofMUdR inhibition increases correspondingly with time at 1mM concentration; however, there is also an increasinginhibition at a lower (0.5 mM) MUdR level. Stimulation ofumidine incorporation by PHA activation has been shown(16) to parallel a marked rise in umidine kinase levels between 12 and 36 hr after addition of PHA.

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6 2 8 24 30 36 42 48 54 60Time After Addition of PHA (hr)

Chart 4. Effect of MUdR on [3H]uridine incorporation into PHA-stimulatedspleen lymphocytes at various times following drug addition. Cells (5 x 10'/ml) were cultured in the presence of PHA and various concentrations of drugfrom 0 hr. Six hr before sampI@ng,cultures were pulse-labeled with uridine (3@.@Ci/culture).Cells were harvested and radioactivity counted as described inâ€œMaterialsand Methods.â€•

Inhibition of Other RibonucleosidesStudies were carried out on MUdR inhibition of acid

insoluble incorporation of other nibonucleosides, since

12 18

Time After Addition of PHA (hr)

. MUdR

/O3MUdR

SEPTEMBER1976 3287



Nucleosidecpm/cultu

meTime

(hr)PHA (A)PHA + MUdR(B)%

incorporation

(B/A)4Adenosine24

48203,187Â±12,937â€•

337,219Â±31,469149,575Â± 7,789

269,781Â± 5,3597480Guanosine24

4874,928Â± 3,071

103,609Â±13,191256,207Â± 4,160

452,890 Â±50,450342437Cytidmne24

48225,962Â± 7,239

361 555 Â± 2,71524,190Â± 880

37,575Â± 94611 10

D. Bogyo et a!.

Table 1Effects of MUdRon acid-insoluble incorporation of ribonucleosides into PHA-stimulated

lymphocytesSpleen lymphocytes(5 x 10/mI) were cultured from 0 hr in the presenceof PHAalone or with

PHAplus 10@M MUdR. Six hr before sampling, the cultures were pulsed with 3 @.tCiof labelednucleoside.Cellswere processed,and acid-insoluble radioactivity wasdetermined asdescribed inâ€œMaterialsand Methods.â€•

cpm PHA+ MUdR@( cpmPHA ia % Incorporation (B/A) = ) x 100.

b Mean Â± SE.

a

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TimeAfter AdditionofLabeledNucleoside(mm)

Chart 6. Effects of MUdR on uptake of [3H]adenosine into acid-solubleand -insoluble intracellular pools. For details, see legend to Chart 2.

MUdR effects on [3H]adenosmne incorporation with increasing time are shown in Chart 6. Acid-soluble incorporation shows an initially rapid uptake, followed by a moregradual phase until near saturation at 4 hr. Acid-insolublelevels of incorporation are linear from 15 mm, on. Again,MUdA does not produce changes in acid-soluble incomporation as has been shown for thymidine inhibition of[3H]umidineuptake (30). The time for acid-soluble saturationis comparable in PHA controls and 1 mM MUdR-tneatedcultures.

Studies with incorporation of tnitiated nucleosides intoacid-insoluble material (Table 2) show that the magnitude ofMUdR inhibition levels off after 1 hr for umidine incomponation but continues to increase oven 4 hr for adenosineincorporation. Stimulation of guanosine incorporation byMUdR isseen after2 hr and increasesthereafter.The importance of choosing a pulse length of sufficient durationfor different tnitiated nucleosides is indicated by these studies.

TimeAfter Additionof LabeledNucleoside(mm)

Chart 5. Effects of MUdR on uptake of [3Hjuridine into acid-soluble andacid-insoluble intracellular pools. Cells were cultured with the addition of thelabeled nucleoside to the PHA-stimulated cultures at 18 hr. Aliquots wereremoved at the times indicated, and both acid-soluble and -insoluble radioactivities determinations were carried out as described in â€œMaterialsandMethods.â€•

acid-insoluble nucleic acids. [3H]Adenosine incorporationinto acid-insoluble material has been shown by other investigators (10, 29) to correlate directly with net ANA synthesis.

Nucleoside Uptake In the Presence of MUdR

The presence of MUdR in mM quantities could inhibitcellular uptake of the tnitiated nucleosides, thereby producing the effects seen. Chart 5 shows MUdR effects on[3H]umidine incorporation with increasing time . Saturationof acid-soluble levels of [3H}unidine occurs in 2 hn, confirming the studies of Cooper (9). At this time, the increase of[3H]urid me incorporation into acid-insoluble material assumes a new mate.MUdR does not prolong the time requiredfor unidine pool saturation to occur, indicating that[3H]umidmneuptake into the cell is not being blocked.

3288 CANCERRESEARCHVOL. 36



Pulseduration

(mm)Effect

of MUdR(cpm)UnidineAdenosineGuanosineThymidinePHAPHA

+MUdR%

incorporation4PHAPHA

+MUdR%

incorporation4PHAPHA

+MUdR%

incorporation4PHA__@PHA

+MUdR%

incorpora

tionâ€•10

15

30

60

120

24023,202

Â± 1,160â€•

40,337Â± 1,210

76,721Â± 6,158

196,400Â± 9,820

319,013Â± 9,5706,945

Â±278

14,416Â± 721

35,823Â± 2,249

95,451Â± 7,636

156,581Â± 6,26330

36

47+

49

4913,556

Â± 678

20,848Â± 1,459

26,021Â± 1,661

65,504Â± 5,240

117,000Â± 5,3508,325

Â± 333

11,575Â± 695

16,627Â± 831

43,997Â± 3,079

97,070Â± 5,82461

56

64

67

8338,312

Â± 766

55,155Â± 4,964

80,444Â± 4,826

118,800Â± 5,940

117,350Â± 7,04113,685

Â± 547

20,207Â± 606

61,369Â± 2,455

145,180Â± 4,355

208,779Â± 12,52136

37

76

120

18013,739

Â±657

69,537Â± 4,867

91,073Â± 5,464

269,410Â± 18,8583,275

Â±197

13,772Â± 551

30,494Â± 1,525

66,209Â± 1,32424

20

30

25


Table 2Effects of MUdR on uptake of labeled nucleoside into acid-insoluble material of PHA-stimulated lymphocytes

Cultures established as described in legend to Table 1 were pulsed at 18 hr after stimulation for all nucleosides except thymidine, inwhich case the addition was at 42 hr poststimulation.

a % incorporation = @c@m PHA + MUdR\cpm PHA ) x 100.

b Mean Â± SE.

Stimulation of [3H]Guanosine Incorporation

The 2- to 3-fold increase in [3H]guanosmneacid-insolubleincorporation seen in the presence of MUdR (Table 1) couldbe due to either increased synthesis of guanosine-nich RNAspecies or increased specific activity of the GTP precursorpool utilized by RNA polymerase. RNA was extracted by thephenol-sodium dodecyl sulfate method from lymphocytespulse-labeled for 6 hr with either [3H]unidine or[3H]guanosine and fractionated into 4, 18, and 28 S specieson 5 to 20% sucrose density gradients. Results of the[3H]unidine experiment (Chart 7) show that all species ofRNA are inhibited by MUdA action, with no major shifts inpeak positions. The sucrose gradient separation of[3H]guanosmne-Iabeled RNA (Chart 8) showed stimulation of

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10 15 20 25

SucroseGradientFraction NumberChart 7. Effects of MUdR on [3H)uridine incorporation into RNA fraction

ated by sucrose density gradient analysis. For details, see â€˜MaterialsandMethods.â€•

incorporation into all 3 ANA species by MUdR action without alteration of the overall qualitative ANA profiles. Todetermine whether the specific activity of any 1 of the 3sucrose density gradient fractions changed in the presenceof MUdR, this experiment was repeated using double labeling with [14C]adenosmneand [3H]guanosmne.The results ofthis experiment showed major differences in the totalpmoles of labeled adenosine incorporated compared tolabeled guanosine incorporated for all species of RNA. Thiscould best be explained if adenosine to guanosine nucleotide interconvemsions were taking place within the lymphocytes.

Lymphocyte RNA was pulse-labeled as before with either[3H]guanosmneor [3H}adenosine, extracted from the cells,and hydrolyzed overnight in 0.5 N KOH; nucleotides wereseparated by descending phase paper chromatography in asatu matedammonium sulfate-isopropyl alcohol-sodium acetate solvent system. [3H]Guanosine-labeled ANA shows noradioactivity in spots other than 3'- and 2' GMP. Table 3

b

EEa

0

00

00a0

Chart 8. Effects of MUdR on [3H]guanosine incorporation into RNA fractionated by sucrose density gradient analysis. For details, see legend toChart 2.

SucroseGradientFractionNumber

SEPTEMBER 1976 3289



PHAPHA+MUdR%oftotal%of

totalradioacradioac

NucleosideRFcpmtivitycpmtivity3'-AMP0.2039021372322'-AMP0.2929116254223'-GMP0.44106257414362'-GMP0.575840

D. Bogyo et a!.

shows the results of chromatography of the [3H]adenosinelabeled ANA. PHA control cultures showed significant conversion of adenosine to guanosine nucleotides as seen inradioactivity appearing in GMP spots. MUdR-tneated cells,on the other hand, showed a decreased conversion of adenosine to guanosine nucleotides.

These data would support the theory that MUdR maydecrease total cellular guanosine nucleotide pools. To testthis theory, experiments were done to determine the vamiation of acid-insoluble incorporation of labeled nucleosidesinduced by pulse-labeling with differing initial specific activity radioactive precursor levels. Using the derivation ofForsdyke (12), V = N(1/x) â€”(P + 1), where V is the induced dilution of labeled isotope, x is the measured acidinsoluble incorporation, P is the total intracellular poolor unknown dilution, and N is the maximal incorporation inthe absence of any dilution, the investigator may derive a

Table 3

Effects of MUdR on [3HJadenosine interconversion andincorporation into RNA

Twenty-five cultures (5 x 106 cells/mI) stimulated with either PHAalone or PHA pluse 10@ M MUdR were pulsed-labeled at 18 hr with[3H]adenosine (3 MCi/culture) for 6 hr. The cells in each group werepooled and processed; the isolated RNA was hydrolyzed, and theradioactivity associated with the paper chromatognaphically separated nucleotides was determined as described in â€œMaterials andMethods.â€•

metabolic activation steps required for the conversion ofMUdR to the niboside.

Inhibition of Thymidine and Uridine Kinases

Lymphocytes cultured with PHA for 24 and 48 hr werecollected, washed, and disrupted by ultrasonic cavitation.The clear lymphocyte extract served as a good source ofthymidine kinase and unidine kinase. With NaF present inthe assay mixture to inhibit phosphatase activity, the prepanation showed linear conversion of substrate with time andshowed a linear increase in substrate conversion with increasing enzyme concentration . Thymidine kinase activity

C0

0

C4,UC0

4,C

â€˜V

0

C

0

C0

0

104106

Chart 9. Estimation of total intracellular uridine pool sizes in the presenceand absence of MUdR by isotope dilution series. To cells cultured as described in legend to Chart 1 was added [3Hjuridine (3 MCi) at 18 hr post-PHAstimulation, for a 6-hr pulse. Graded quantities of cold uridine were added tothe labeled nucleoside before pulsing, resulting in a known series of sampleswith varying specific activities and concentrations. Aliquots were removedand acid-insoluble radioactivity was determined. The method of Forsdyke(12) was used to calculate changes in total intracellular pool size.

C0

0

C4,U

SC.)

0

0

C

0

C0

0

Chart 10. Estimation of total intracellular guanosine pool sizes in thepresence and absence of MUdR by isotope dilution series. For details, seelegend to Chart 9.

cpm/rnl

value of the total intracellular nucleoside-nucleotide concentration (P0. Results of this experiment with [3H]umidmnelabeling shown in Chart 9 indicate that MUdR causes nomajor differences in total intracellular unidine-demivativeconcentration (x intercept values). From the study with[3H]guanosmne(Chart 10) labeling, it can be seen that MUdRproduces a major shift in the x intercept, representing asignificant reduction in total pool size of guanosine demivatives. PHA lymphocytes rapidly convert nucleosides to thenucleotide tniphosphate form so that, at any sampling time,the predominant soluble species measured is the tniphosphate.

Comparison of MUdR and MUR Inhibition

To show that the inhibition of nibonucleoside incorporation is effected by the deoxyniboside form of the drug,studies comparing MUdR and the niboside analog MURwere carried out.

Comparison of their inhibitory effects (Table 4) indicatesthat MUdR produces greaten inhibition of acid-insolubleincorporation for all the labeled nucleosides tested . Thesedata argue against the premise of in vivo MUdR-to-MURconversion, since MUR should be effective without the 2





Table 4

Comparison of effects of MUdR and MUR on the inhibition of incorporation of nucleosides intoacid-insoluble material

Cells were cultured as described in legend to Table 1 with 10@ M MUdR or 10@@M MUR added at0 time. Labeled nucleosides (3 @iCi)were added 6 hr before harvesting of cultures, as described in.. Materials and Methods.â€•

cpm% inhibi

tion

54355236

TimeNucleoside (hr)

Uridine 24

Drug

MUdRMUR

48 MUdRMUR

PHA PHA + drug

243,012 Â±13,981â€•112,231 Â±13,848240,054Â± 4,996155,521 Â±8,389297,220Â±39,699141,818 Â±22,720241,618Â±51,663154,934 Â±37,588

149,575 Â± 8,974148,555 Â± 7,789

2622

Adenosine 24 MUdRMUR

202,187Â±16,174191,289 @:12,937

:@ 2,067

Â± 8,174

Â± 1,878

Â± 13,501

89529022

Cytidine 24 MUdRMUR

48 MUdRMUR

225,962 :t 13,432208,435 Â±12,739361,555 Â±10,412347,661 Â±16,416

24,19099,57537,575

271 870

Thymidine 48 MUdR 953.759 Â±40.537 239,664 Â± 9,237 75MUR 1,005,744Â±76,473 706,520Â±33,545 30

â€œMean Â± SE.

from the 48-hr lymphocyte extract was inhibited 64% by theaddition of MUdR to a final concentration of 1 mM in thesonic extract. Identical inhibition of thymidine kinase wasproduced by 1 mM MUdA in a 24-hr lymphocyte extract. Atthis concentration of MUdR, inhibition of thymidine incorponation into acid-insoluble nucleic acid material of thecultures is 75% (Table 4), a level of inhibition quite compamable to the thymidine kinase inhibition previously noted.

Unidine kinase activity was inhibited 56% by 1 mM MUdR.Thymidine at the same concentration produced no unidinekinase inhibition. The magnitude of umidine kinase inhibition is quite similar to the inhibition of labeled unidine intoacid-insoluble nucleic acids presented earlier (Table 4).

DISCUSSION

The data presented indicate that MUdR is an effectiveinhibitor of DNA synthesis in a mammalian primary spleenlymphocyte culturesystem.The greaterinhibitionby MUdRof deoxyumidine incorporation into acid-insoluble nucleicacid material than of thymidine incorporation reflects thegreaten sensitivity of thymidylate synthetase to MUdR ascompared to thymidine kinase sensitivity. This agrees withstudies of Kalman and Bardos (19) in microbial systems inwhich MUdR was shown to have an apparent K of 1.6 x10@ M fonthymidine kinase and an apparent K ofi x 10@ Mfor thymidylate synthetase. In the latter enzyme system, thephospho rylated derivative (5-mercapto-2'-deoxyu rid ine-5'-phosphate) showed an apparent K of 4 x 108 M. PHAstimulation of lymphocytes produced marked increases inthymidine kinase activity 30 to 42 hr after addition of PHA,and thymidylate synthetase activity shows increases at 24 to48 hr after the addition of the mitogen. The time coursestudies of MUdR inhibition of both deoxyunidine and thymidine incorporation into acid-insoluble material show thatMUdR inhibition increases 48 to 54 hr after PHA stimulation.The progressive inhibition by MUdR follows the period

when these increases in enzyme activity have leveled off.Inhibition by 1 mM MUdR of total cellular DNA, by 43% at

42 hr in comparison to PHA controls, indicates that themajor portion of the S-phase increase in DNA of thesestimulated lymphocytes is blocked. One m@ MUdR does notreduce total cell numbers nor does it block PHA-inducedtransformation. The ability of PHA-stimulated lymphocytesto proceed to the lymphoblast state during major inhibition

of DNA synthesis confirms earlier reports to this effect byKay et al. (20) and Salzman et a!. (26).

The present investigations were also undertaken to elucidate how MUdR inhibits RNA synthesis from radioactivelabeled nucleoside precursors. Initial findings of MUdA inhibition of [3H]umidine incorporation into acid-insoluble nucleic acids were extended to show MUdR inhibition of Iabeled adenosine and cytidine incorporation as well.[3H]Guanosine incorporation, rather than being inhibited,was increased 3-fold in MUdR-treated lymphocytes culturesstimulated with PHA.

MUdR may act by a variety of mechanisms to produce theobserved apparent inhibition of RNA synthesis. Since neithen total cell number nor total percentage of blast cellswere reduced by MUdR, the effects seen do not result from

either cytotoxicity on blockade of PHA stimulation. Companative studies of MUdR and MUR effectiveness argue againstthe type of MUdR breakdown and conversion seen by Hanbemset a!. (14) with 5-fluomo-2'-deoxyunidine.

Time course studies with increasing pulse duration oflabeled nucleoside in the presence of MUdR do not show a

pattern of inhibition resembling the observed blockade ofunidine uptake evidenced with mM levels of thymidine (30).However, the level of acid-soluble nucleoside at saturationlevel is lowered, for umidine and adenosine, in the presenceof 1 mM MUdR. This could be due to any of the following

factors: inhibition of metabolic activation of the labelednucleoside, increased turnover of the acid-soluble pool, orchanges in interconversion of labeled nucleoside and, thus,altered utilization.

3291SEPTEMBER 1976



D. Bogyo et a!.

Evidence from these studies indicates that all 3 of theabove-mentioned parameters may be effected by MUdRaction. Unidine kinase is inhibited by 1 mM MUdR to a levelthat correlates well with the total acid-insoluble unidineincorporation decreases seen with the same level of thedrug. It has been postulated that unidine kinase is the ratelimiting step in incorporation of L3H]unidine into RNA (16).MUdR does interfere with nucleoside activation, at least inthe cases of unidine, cytidine (both initially phosphorylatedby the same kinase), and thymidine.

Effects of MUdR were seen on acid-soluble pool size. Theincreased incorporation of [3H]guanoSine in the presence ofMUdR appears to be due to decreased guanosine poolsresulting from inhibition of adenine to guanine nucleotideconversion within the cell. Adenylate deaminase, whichcatalyzes the 1st step in this conversion, contains an allostenic site that binds pymimidine tniphosphates (2) and showssensitivity to the sulfhydmyl group inhibitor, hydroxymercumibenzoate (31). It is possible that MUdR interacts with thissite by virtue of its 5-mercapto group.

The difficulties known to exist between correlating radioactive precursor incorporation with net synthetic rates havebeen shown again in this study. Any change in specificactivity of the nucleoside tniphosphate cellular pool in drugtreated cultures, compared with controls, will produce incorporation differences that do not reflect changes in RNAsynthesis. In this work, MUdR inhibition of labeled adenosine incorporation into acid-insoluble material correlatedbetter than any other labeled nucleoside with total RNAinhibition produced by the drug. Others have reported that[3H]adenosine incorporation is a good measure of total ANAsynthesis(10,29).

The inhibition of thymidine kinase extracted from PHAstimulated cells at 24 and 48 hr by 1 mM MUdR is identical(65%). By this criterion, qualitative differences in the enzyme before and after major increases in activity triggeredby PHA could not be seen. The inhibition of nibonucleotidesynthesis by the deoxynibonucleoside MUdR was shown foruridine kinase as well. At 1 mM, MUdR inhibits umidinephosphomylation by 56%.

Utilization of this PHA-stimulated primary lymphocyte culture system offers a number of advantages for investigatingpotential chemotherapeutic agents. Drug effects on cellularviability, transformation, uptake, and biosynthetic pathwaysmay all be studied in uniform cell types that are functioningat maximal metabolic capability. These cells have not undengone the irreversible alterations seen in defined celllines. Since PHA triggers the progression of a well-definedtemporal sequence of biochemical events in these cells,drug effects may be studied in relationship to this progression. For example, the MUdR effects on mibonucleotidesynthesis and interconversion seen by 24 hr may effectsubsequent DNA synthesis events occurring at 48 hr. This,in turn, may relate well to the actual in vivo scheduling of adrug dosage regimen. The ease of establishing this primarycell culture system and the potential information it mayaccord argue in favor of more extensive use of the PHAstimulated lymphocyte model in studies of the effects ofantitumon agents.

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3293


SEPTEMBER 1976



1976;36:3284-3293. Cancer Res D. Bogyo, T. J. Bardos and Z. F. Chmielewicz Culture SystemNucleosides into RNA and DNA in a Primary Lymphocyte

-deoxyuridine on the Incorporation of′Effects of 5-Mercapto-2

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