Effects of 5-Mercapto-2'-Deoxyuridine on the Incorporation of Nucleosides Into RNA and DNA in a Primary Lymphocyte Culture System1

Home , Deoxyuridine

[CANCER RESEARCH 36, 3284-3293, September 19761 Effects of 5-Mercapto-2'-deoxyuridine on the Incorporation of Nucleosides into RNA and DNA in a Primary Lymphocyte Culture 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 Exogenous guanosine incorporation into lymphocyte acid-insoluble material is increased by MUdR. This in The effects of 5-mercapto-2'-deoxyuridine (MUdR) on creased utilization of exogenous nucleoside is apparently DNA synthesis in a primary munine spleen lymphocyte cul the result of MUdR inhibition of conversion of adenosine to tune system stimulated by phytohemagglutinin (PHA) were guanine nucleotides within the lymphocytes and a conse studied. Inhibition of thymidine incorporation into acid quent diminution of the total intracellular guanine nucleo insoluble nucleic acid material was 50% at 0.5 mM MUdR tide pool size. I concentration, while inhibition of deoxyuridine incorpora The active inhibitory compound is the deoxymibonucleo tion into acid-insoluble nucleic acids was 50% at 0.01 mM side or deoxynibonucleotide. Comparison with the niboside MUdR. Time course studies, at 0.5 and 0.05 mM MUdR, analog 5-mercaptoumidine showed that MUdR was a more showed that the magnitude of inhibition of incorporation for efficient inhibitor of nucleoside incorporation. thymidine and deoxyuridine, respectively, increased from a time point after PHA stimulation when increased synthesis of thymidine kinase and thymidylate synthetase had leveled INTRODUCTION off. At 1 mM MUdR, total cellular DNA in cultures was de The isostemic substitution of the mercapto group for the creased 43% at 42 hr after PHA stimulation. Neither the total methyl group at the pymimidine 5-position was the initial number of cells nor the percentage of PHA-transformed rationale of Baranski et al. (3) for the synthesis and testing cells was decreased in comparison to that of controls. of MUdR4 as a thymidine analog and inhibitor of DNA syn MUdR therefore blocks the increase in DNA content of thesis. It was found that MUdR effectively blocks DNA syn lymphocytes that is initiated during the S phase of the cell thesis in bacterial cells (3, 19). The mechanism of this cycle. inhibitory effect of MUdR involves competition at the deoxy Millimolar levels of MUdR inhibited incorporation of un nibonucleoside level for thymidine kinase and activation of dine, adenosine, and cytidine into acid-insoluble material in the analog to the corresponding monophosphate, which in PHA-stimulated primary munine lymphocyte cultures. Total turn is a potent inhibitor of thymidylate synthetase. Recent cellular RNA synthesis was inhibited at these levels of results in this laboratory show that MUdR is an effective MUdR, with no differential effects on 4, 18, or 28 S RNA chemotherapeutic agent against both transplanted and car species observed. Uptake of these nucleosides into the total cinogen-induced animal tumors (Z. F. Chmielewicz, A. cellularacid-solublematerialwas not blocked. Carter, and T. J. Bardos, unpublished observation) and Uptake of different labeled nucleosides into cellular, acid that MUdR has significant inhibitory effects on human skin solublepools occurs at differentmates.Thus, choice of a neoplasms (28). suitable minimum pulse time to achieve saturation for dif PHA-stimulated primary lymphocyte culture has been ferent labeled nucleosides must relate to this consideration. proposed as an effective screening system for antimetabo Thymidine kinase from whole-cell sonic extracts of PHA lites (11). In particular, DNA biosynthetic enzymes such as stimulated lymphocytes was inhibited 65% by 1 mM MUdR thymidine kinase, thymidylate synthetase, and DNA polym at 24 and 48 hr after stimulation. Uridine kinase extracted erase show elevated activities following mitogenic stimula from the PHA-stimulated cells was also significantly in tion that are similar to the changes seen in vimus-trans hibited by 1 mM MUdR at 24 hr (56%). formed cells (21) on regenerating tissue (4). We undertook these studies to determine the effectiveness of MUdR in blocking DNA biosynthesis in this mammalian system. In , Thisworkwas partlysupported by NationalCancerInstituteGrantCA particular, the inhibition of thymidine kinase and thymidyl 06695 and American Cancer Society Grant CH-20C. a Trainee under USPHS Training Grant PHS-5T01GM00555, which partly ate synthetase activities were focused upon, since the pre supported this work. Part of this work is from a dissertation submitted to vious work cited indicates that these are primary loci of Faculty of State University of New York at Buffalo. in partial fulfillment of the MUdR action. requirements for the Ph.D. degree. Present address: Department of Experi mental Therapeutics and Grace Cancer Drug Center, Roswell Park Memorial

Institute, Buffalo. N. Y. 4 The abbreviations used are: MUdR, 5-mercapto-2'-deoxyuridine; PHA, 3 To whom requests for reprints should be addressed. phytohemagglutinmn; RPMI, Roswell Park Memorial Institute; MUR. 5-mer Received September 16, 1975; accepted May 25, 1976. captouridine.

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Our preliminary studies using the PHA-stimulated munine were centrifuged for 10 mm at 600 x g. Tothe cell pellet was lymphocyte system indicated that MUdR, at higher concen added 1 ml of the fully Supplemented medium with gentle trations, also inhibited RNA synthesis. Further work was mixing. A quantity of cell suspension (0.1 ml) was mixed carried out to investigate the effects of MUdR on mibonu with 0.5 ml of trypan blue dye (0.4%), and cell counts were cleoside on nibonucleotide synthesis and interconversion. done on a Neubauer grid hemacytometer at x 10 magnifica tion. Determination of Percentage of Blasts. Cultures were MATERIALS AND METHODS incubated with 0.01 ml of 106M colchicine for 2 hr before harvesting to produce metaphase arrest. Cells were fixed by Materials the method of Hastingseta!. (15), and drops were placed on glass covenslips to aim-dry. Covenslips were stained for 10 Unlabeled nucleosides and nucleotides were obtained mm with Giemsa stain (pH 6.8), washed, aim-dried, and from Sigma Chemical Co. , St. Louis, Mo. (unidine, guano mounted on glass slides with Canadian balsam. The criteria sine, 3'-UMP, UMP, 3'-CMP, CMP, 3'-GMP, GMP, 3'-AMP, of Cooper et al. (6) were used for grading cells, and both and AMP). The following radioactively labeled precursors type 2 and type 3 cells were considered blasts. were purchased from ICN Chemical Aadioisotopes Division DNA determinations were carried out on washed cell pel (Irvine, Calif.): [methy/-3H]thymidine (16.7 Ci/mmole), [6- lets of 5 x 106cells by the diphenylamine method of Burton 3H]deoxyunidine (21.2 Ci/mmole), [5-3H]umidine (21.7 Ci/ (5). mmole), [8-3H]guanosine (16.3 Ci/mmole, and [5- RNA content was determined by a modified oncinol 3H]cytidine (22 Ci/mmole). [2-3H]Adenosine (10.3 Ci/ method (22), and cellular protein was assayed by the mmole) was obtained from New England Nuclear, Boston, method of Lowry et a!. (23). Mass. , as was Aquasol. Calf thymus DNA was obtained from Nucleoside Kinase Assay. Twenty cultures containing 5 Sigma Chemical Co. Purified PHA was acquired from Bun x 106 cells/mI were grown with PHA for the appropriate roughs-Wellcome, Research Triangle Park, N. C. NCS solu time and washed , and cells were collected by centnifugation bilizer was purchased from Amensham-Seanle, Arlington for 10 mm at 600 x g in 3 ml of 0.9% NaCI solution per Heights, III. The RPMI 1640 medium and all supplements culture. To each culture pellet was added 1 ml of 0.1 M Tnis, were obtained from Grand Island Biological Co. , Grand pH 8.0, at 0Â°.Thecells were pooled, collected by centnifuga Island, N. Y., as was the trypsin-EDTA solution. Giemsa tionat 1500 x g for 10 mm, and suspended in3 ml of the stain type B was obtained from Hanleco, Philadelphia, Pa. Tnis buffer. The cells were sonically extracted on ice with a Branson sonicatom, microtip, using four 15-sec bursts at a Methods power setting of 0.4 maximum alternating with 15-sec rest intervals. The sonic extract was centrifuged at 3500 x g for Lymphocytes were isolated from the spleens of 6-week 10 mm, and the supemnatant was used for the kinase assay. old male BALB/c mice bythe method ofAdlemetal. (1). They The nucleoside kinase assay was carried out as described were suspended at a concentration of 5 x 106 cells/mI in by Ives et al. (18). Incubation time was 90 mm for the assay. RPMI 1640 med ium with N â€˜-2-hydmoxyethylpipemazine-N'- DEAE discs were washed with 6 x 10 ml of 1 mM ammonium ethanesulfonic acid supplemented with 10% fetal calf se fommateand , similarly, with distilled water. The reaction rate mum, penicillin (100 units/mI), streptomycin (100 j@g/ml), was linear over the 90-mm incubation period, and reaction and L-glutamine (1%). PHA was added (1 @g)toeach ml of velocity increased linearly with addition of increasing en the cell suspension. Aliquots of 1 ml of the cell suspension zyme. in 15- x 45-mm glass screw-top vials were incubated in a humidified aimatmosphere containing 10% CO2.Amounts of Isolation of Spleen Lymphocyte RNA MUdR listed in the test refer to final concentrations in the 1- ml lymphocyte cultures. Two sets of 24 cultures (120 x 106cells) were established, 1 with PHA and the other with PHA and 10@@MMUdR from Incorporation of Labeled Nucleosides zero time. Each culture received a 6-hr pulse of tnitiated nucleoside (3 MCi) starting at 18 hr after initiation of incuba Radioactive nucleosides were added (3 @Ci/cultume)6hr tion. All cultures were then centrifuged at 600 x g for 10 before the harvesting of cells, except in the uptake studies mm. The cell pellets were washed with 3 ml of 0.14 M NaCI where they were added for the time periods indicated. and then were suspended in 1 ml of ice-cold acetate buffer The cells were processed for liquid scintillation counting (10 mM), pH 5.1 , pen vial, with 0.1 M NaCI and 5% bentonite. as described previously (25). NCS solubilizer was used in RNA was extracted from the pooled cell cultures by the stead of Hyamine hydroxide. Aliquots (0.2 ml) of the 1st method of Cooper (7). Bentonite was purified by the method tnichlomoacetic acid supemnatant were dissolved in 3 ml of of Frankel-Conmat et al. (13). Rat liver RNA was used as a Aquasol and used for acid-soluble incorporation studies. marker and coprecipitating agent for labeled RNA was pre Determination of Cell Number. PHA-agglutinated cell pared by the method of Hiatt (17). cultures were centrifuged at 600 x g for 10 mm at room temperature. To the cell pellet was added 0.5 ml of a 0.05% Sucrose Density Centrifugation trypsin-0.02% EDTA solution with gentle mixing. After 10 mm of incubation at 37Â°,2.5 ml of RPMI 1640 medium with The total labeled RNA extract from both the PHA and the 10% fetal calf serum were added to each culture. The vials PHA plus MUdA cultures was dissolved in 2 ml of the ace

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tate buffer (10 mM), pH 5.1 , and layered oven the tops of 2 l00 linear 5 and 20% sucrose gradients (33 ml) prepared with a ! :@Deoxyuridine Buchler gradient marker. Over a 3rd 5 to 20% gradient was 80 @ layered 1 ml of the rat liven RNA marker (600 @g)plus1 ml of A...â€¢@

the acetate buffer. The gradients were centrifuged at 25,000 . 0 @.,Thymidine @ rpm (113,000 x g) for 14 hr with the use of a Spinco SW-27 60 @ head. The sucrose density gradients were placed in a Plex Uridine iglas piercing apparatus, and the cellulose nitrate tubes 5e 40 were tapped by gravity flow from the bottom. One-mI frac tions were collected . The tubes were read for sucrose con 20 centration on a Bausch and Lomb refractometer. The marker gradient fractions were read at 260 nm to determine

the position of 4, 18, and 28 S RNA peaks. Aliquots of 0.2 ml l0@ 5@l0@ 0' 5xl0@ 10' 5,i0@ I0@ 5x103 of the labeled RNA fractions were removed, mixed with 3 ml Concentration,MUdR(N) of Aquasol in glass screw-cap vials (15 x 45 mm), and counted in 10-mI plastic scintillation vials in a Packard Tn Chart 1. Effects of MUdR on [3H]uridine, [3H]deoxyuridine, and [3H]thymidine incorporation into PHA-stimulated spleen lymphocytes. Cells Cambliquid scintillation counter. (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 (thymi dine or deoxyuridine), at the level of 3 MCi/culture. Six hr later, the cells were Separation of Nucleotides harvested and radioactivity was counted as described in Materials and Methods. â€˜Inhibitionis expressed as the percentage of uridine incorporated into cultures containing PHA and no drug. Total labeled RNA, extracted from PHA and PHA plus 10@ M MUdR cultures, as described for the sucrose density poration time course (Chart 2) indicated that, until 48 hr centnifugation, was hydrolyzed at a final concentration of after PHA stimulation, there was increased thymidine incom 0.5 M KOH for 15 hr at 37Â°.Thisdigest was neutralized with poration at all levels of MUdR tested. From that time point perchlonic acid and centrifuged for 10 mm at 1500 x g. on, no increase in incorporation was seen at the 2 highest Aliquots of the supemnatant were spotted on Whatman No. 2 levels of MUdA, and the absolute magnitude of inhibition chromatography strips along with the 3' and 2' standards of thus increased. Determination of acid-soluble thymidine AMP, CMP, UMP, and GMP. Descending phase paper chro levels within the lymphocytes indicated that, at all levels of matogmaphy was carried out with the solvent system, 80% MUdA tested, thymidine levels in total soluble pools in satu matedammonium sulfate-2% isopropyl alcohol-18% so creased over the whole time course. The deoxyunidine in dium acetate, 1 M (v/v/v). The chromatogmam was devel corporation time course (Chart 3) showed increased incor oped for 16 hr. Spots were identified with a Mineralight UV poration into acid-insoluble material at all MUdR levels until 12 scanner, measured for AF values, and cut out from the 48 to 54 hr after PHA stimulation. Subsequently, deoxyuni chromatognam. Ten ml of Aquasol were added to each dine incorporation decreased at all MUdR levels as well as sample in glass scintillation counting vials, and radioactivity in the PHA controls. The period from 24 to 48 hr after PHA was determined. stimulation has been shown to involve a 3-fold increased activity of thymidylate synthetase (24). Inhibition of a net increase in deoxyunidine incorporation is seen after the RESULTS increase in thymidylate synthetase activity ceases. Effects of Different Concentrations of MUdR on Nucleo side Incorporation. When munine spleen lymphocytes are cultured in the presence of PHA, a marked increase in the mateof unidine incorporation is seen starting at approxi E mately 12 hr, followed by an increased rate of thymidine @. @6 C 32 incorporation starting at approximately 24 hr after stimula 0 @ tion. 28 0 Incorporation of thymidine remains high after 48 hn, but a. 0 U Sasaki and Norman (27) have shown that, after this time, an .5 20 increasing proportion of PHA-stimulated cells show labeled 4, mitoses indicative of a 2nd cell division. Our initial studies C on MUdR inhibition of labeled nucleoside incorporation @0 E were carried out only up to 48 hr after PHA stimulation to .C eliminate this component of net cellular increase. I- The dose-response curves for MUdR inhibition of thymi 66 72 dine, deoxyunidine, and uridine incorporation (Chart 1) mdi Time After Addition of PHA (hr) cate 50% inhibitory levels of MUdR to be 0.5 mM for thymi Chart 2. Effect of MUdR on [3H]thymidine incorporation into PHA-stimu dine, 0.01 mM for deoxyuridine, and 1 m@ifor umidine. lated spleen lymphocytes at various times following drug addition. Cells (5 x Effects of MUdR with Increasing Time. Time course stud 10'/ml) were cultured in the presence of PHA and various concentrations of drug from 0 hr. Six hr before sampling, cultures were pulse-labeled with ies of MUdR effects were carried out at several concentra thymidine (3 MCi/culture). Cells were harvested and radioactivity counted as tions in the 10 to 90% inhibitory mange.The thymidine incon described in Materials and Methods.

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[3H]unidine incorporation results have been shown to pro duce an overestimate of actual RNA synthetic rates (8). The results of these experiments (Table 1) show that 1 mM MUdR inhibits [3H]adenosine incorporation 26% as com 0 pared to that of PHA controls. [3H]Cytidine acid-insoluble incorporation is inhibited 90%, and [3Hjguanosmne acid E insoluble incorporation is stimulated 3-fold by MUdR ac E a U tion. The [3H]adenosine inhibition will be shown to correlate C closely with the net decrease in total cellular RNA induced 0 0 by 1 mM MUdR. [3H]Cytidine inhibition magnitude reflects a MUdR effects on cytidine activation by umidine kinase and 0 U also possible increases in cytidine intracellular pool size C due to potential inhibitory effects of MUdA on cytidine aminohydmolase. The increased [3Hjguanosmne incompoma 4) tion in the presence of MUdA was subsequently investi C â€˜V gated and found to be related to MUdA-induced diminution of total intracellular guanosine pool size. 0 0) Effects of MUdR on Transformation. The inhibitory ef

I fects of MUdR on PHA transformation of lymphocytes into lymphoblasts were studied in fixed, Giemsa-stained sam 12 18 pIes removed from the cultures at various times following Time After Addition of PHA (hr) addition of PHA alone on with 1 m@iMUdR. The results mdi Chart 3. Effect of MUdR on [3H]deoxyuridine incorporation into PHA cated that the percentage of transformation in cultures con stimulated spleen lymphocytes at various times following drug addition. For taming MUdR was not decreased in comparison to that of details, see legend to Chart 2. PHA control cultures. Preincubation of PHA and 1 mM Time course studies on [3H]umidine acid-insoluble incom MUdR for periods of 2 hr did not increase the inhibitory ef ponation (Chart 4) show that almost complete inhibition of fects of MUdR. Addition of MUdR as long as 4 hr after PHA the net increase of incorporation occurs only at the 1 mM incubation with the lymphocytes did not decrease the extent MUdR level. Since PHA control levels of incorporation are of MUdR inhibition seen in cultures where MUdR had been rising steadily throughout the time course, the magnitude of present simultaneously with added PHA. In order to deter MUdR inhibition increases correspondingly with time at 1 mine total cell counts in MUdR-tmeated cultures and in PHA mM concentration; however, there is also an increasing stimulated control cultures, a mild trypsinization method inhibition at a lower (0.5 mM) MUdR level. Stimulation of was used to disaggregate PHA-agglutinated cells. The umidine incorporation by PHA activation has been shown method produced good separation of cells, and viability (16) to parallel a marked rise in umidine kinase levels be was not decreased , as determined by trypan blue dye exclu tween 12 and 36 hr after addition of PHA. sion. Results of the time-course variation in total cell count showed no increased cell death with 1 mr@iMUdAplus PHA, compared with the PHA controls. Both the PHA and the En200 MUdR plus PHA sets of cultures showed a 35% decrease in

E cell count during the 1st 6 hr of incubation. This is similar to . MUdR E 60 the percentage loss of lymphocytes we observed during a incubation without PHA. Lucas (24) has reported a 40% 0 decrease in mean DNA content of PHA-stimulated cultures @ 20 0 after 45 hr of incubation. a 0 @ 80 Effects of MUCIR on DNA, RNA, and Protein Content. 4) Quantitative automadiogmaph studies of cell nuclei by 0 /O3MUdR Cooper et a!. (6) have indicated that, following the in :@ 40 creased activity of DNA-synthesizing enzymes, most lym phocytes contain a DNA complement greaten than diploid 6 2 8 24 30 36 42 48 54 60 but smaller than tetmaploid. We investigated the effects of 1 Time After Addition of PHA (hr) mM MUdR on total DNA, ANA, and protein content of cul Chart 4. Effect of MUdR on [3H]uridine incorporation into PHA-stimulated tunes to determine the magnitude of inhibition. spleen lymphocytes at various times following drug addition. Cells (5 x 10'/ Diphenylammne assay determinations showed a 43% de ml) were cultured in the presence of PHA and various concentrations of drug from 0 hr. Six hr before sampI@ng,cultures were pulse-labeled with uridine (3 crease in DNA at 42 hr after PHA stimulation in the presence @.@Ci/culture).Cellswere harvested and radioactivity counted as described in of 1 mM MUdR. Orcinol assay results showed 21% RNA â€œMaterialsandMethods.â€• inhibition by the same level of MUdR at 24 hr after stimula tion. Lowry protein determinations (23) on 24-hr cultures Inhibition of Other Ribonucleosides showed only at 8% drug-caused decrease in total protein. Studies were carried out on MUdR inhibition of acid The inhibition of net RNA synthesis is quite similar to the insoluble incorporation of other nibonucleosides, since observed level of inhibition of adenosine incorporation into

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. D. Bogyo et a!. Table 1 Effects of MUdRon acid-insoluble incorporation of ribonucleosides into PHA-stimulated lymphocytes Spleen lymphocytes(5 x 10/mI) were cultured from 0 hr in the presenceof PHAalone or with PHA plus 10@M MUdR. Six hr before sampling, the cultures were pulsed with 3 @.tCioflabeled nucleoside. Cellswere processed,and acid-insoluble radioactivity wasdetermined asdescribed in â€œMaterialsandMethods.â€• meTime

incorpo ration (B/A)4Adenosine24Nucleosidecpm/cultu (hr)PHA (A)PHA + MUdR(B)% Â±12,937â€• Â± 7,789 80Guanosine24 48203,187337,219Â±31,469149,575269,781Â± 5,35974

Â± 3,071 Â± 4,160 437Cytidmne24 4874,928 103,609Â±13,191256,207452,890 Â±50,450342

Â± 7,239 Â± 880 48225,962 361 555 Â± 2,71524,190 37,575Â± 94611 10 cpm PHA+ MUdR@ a % Incorporation (B/A) = ( cpmPHA i) x 100. b Mean Â± SE.

a a 0

@ is 0 E C E a C.' 4, 0 V â€˜V 0 0

0 C 0 0 0 a a 0 CD C Va .0 3 0 (I, â€˜0 0 20 80 TimeAfter Additionof LabeledNucleoside(mm) TimeAfter Additionof LabeledNucleoside(mm) Chart 5. Effects of MUdR on uptake of [3Hjuridine into acid-soluble and acid-insoluble intracellular pools. Cells were cultured with the addition of the Chart 6. Effects of MUdR on uptake of [3H]adenosine into acid-soluble labeled nucleoside to the PHA-stimulated cultures at 18 hr. Aliquots were and -insoluble intracellular pools. For details, see legend to Chart 2. removed at the times indicated, and both acid-soluble and -insoluble ra dioactivities determinations were carried out as described in â€œMaterialsand MUdR effects on [3H]adenosmne incorporation with in Methods.â€• creasing time are shown in Chart 6. Acid-soluble incorpora tion shows an initially rapid uptake, followed by a more acid-insoluble nucleic acids. [3H]Adenosine incorporation gradual phase until near saturation at 4 hr. Acid-insoluble into acid-insoluble material has been shown by other inves levels of incorporation are linear from 15 mm, on. Again, tigators (10, 29) to correlate directly with net ANA synthesis. MUdA does not produce changes in acid-soluble incompora tion as has been shown for thymidine inhibition of Nucleoside Uptake In the Presence of MUdR [3H]umidineuptake (30). The time for acid-soluble saturation is comparable in PHA controls and 1 mM MUdR-tneated The presence of MUdR in mM quantities could inhibit cultures. cellular uptake of the tnitiated nucleosides, thereby produc Studies with incorporation of tnitiated nucleosides into ing the effects seen. Chart 5 shows MUdR effects on acid-insoluble material (Table 2) show that the magnitude of [3H]umidine incorporation with increasing time . Saturation MUdR inhibition levels off after 1 hr for umidine incompona of acid-soluble levels of [3H}unidine occurs in 2 hn, confirm tion but continues to increase oven 4 hr for adenosine ing the studies of Cooper (9). At this time, the increase of incorporation. Stimulation of guanosine incorporation by [3H]urid me incorporation into acid-insoluble material as MUdR isseen after2 hr and increasesthereafter.Theim sumes a new mate.MUdR does not prolong the time required portance of choosing a pulse length of sufficient duration for unidine pool saturation to occur, indicating that for different tnitiated nucleosides is indicated by these stud [3H]umidmneuptake into the cell is not being blocked. ies.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. Effect of MUdR on Lymphocyte RNA and DNA Synthesis Table 2 Effects 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, in which case the addition was at 42 hr poststimulation. (cpm)UnidineAdenosineGuanosineThymidinePHAPHA of MUdR

Pulse incor incor incor incor duration + pora + pora + pora + pora (mm)Effecttionâ€•10 MUdR% tion4PHAPHA MUdR% tion4PHAPHA MUdR% tion4PHA__@PHA MUdR%

Â± 1,160â€• Â±278 Â± 678 Â± 333 Â± 766 Â± 547 15 Â±657 Â±197 30 40,337 14,416 36 20,848 11,575 56 55,155 20,207 37 Â± 1,210 Â± 721 Â± 1,459 Â± 695 Â± 4,964 Â± 606 60 76,721 35,823 47 26,021 16,627 64 80,444 61,369 76 69,537 13,772 20 Â± 6,158 Â± 2,249 + Â± 1,661 Â± 831 Â± 4,826 Â± 2,455 Â± 4,867 Â± 551 120 196,400 95,451 49 65,504 43,997 67 118,800 145,180 120 91,073 30,494 30 Â± 9,820 Â± 7,636 Â± 5,240 Â± 3,079 Â± 5,940 Â± 4,355 Â± 5,464 Â± 1,525 24023,202319,013 156,581 4913,556117,000 97,070 8338,312117,350 208,779 18013,739269,410 66,209 25 Â± 9,5706,945 Â± 6,26330 Â± 5,3508,325 Â± 5,82461 Â± 7,04113,685 Â± 12,52136 Â± 18,8583,275 Â± 1,32424

a % incorporation = @c@m PHA + MUdR\ cpm PHA ) x 100. b Mean Â± SE.

Stimulation of [3H]Guanosine Incorporation incorporation into all 3 ANA species by MUdR action with out alteration of the overall qualitative ANA profiles. To The 2- to 3-fold increase in [3H]guanosmneacid-insoluble determine whether the specific activity of any 1 of the 3 incorporation seen in the presence of MUdR (Table 1) could sucrose density gradient fractions changed in the presence be due to either increased synthesis of guanosine-nich RNA of MUdR, this experiment was repeated using double label species or increased specific activity of the GTP precursor ing with [14C]adenosmneand [3H]guanosmne.The results of pool utilized by RNA polymerase. RNA was extracted by the this experiment showed major differences in the total phenol-sodium dodecyl sulfate method from lymphocytes pmoles of labeled adenosine incorporated compared to pulse-labeled for 6 hr with either [3H]unidine or labeled guanosine incorporated for all species of RNA. This [3H]guanosine and fractionated into 4, 18, and 28 S species could best be explained if adenosine to guanosine nucleo on 5 to 20% sucrose density gradients. Results of the tide interconvemsions were taking place within the lympho [3H]unidine experiment (Chart 7) show that all species of cytes. RNA are inhibited by MUdA action, with no major shifts in Lymphocyte RNA was pulse-labeled as before with either peak positions. The sucrose gradient separation of [3H]guanosmneor [3H}adenosine, extracted from the cells, [3H]guanosmne-Iabeled RNA (Chart 8) showed stimulation of and hydrolyzed overnight in 0.5 N KOH; nucleotides were separated by descending phase paper chromatography in a satu matedammonium sulfate-isopropyl alcohol-sodium ace tate solvent system. [3H]Guanosine-labeled ANA shows no radioactivity in spots other than 3'- and 2' GMP. Table 3

0 b E E E a E a

0 0

0 0 0 0 a 0 0 0 0 C a 0

10 15 20 25 SucroseGradientFraction Number SucroseGradientFractionNumber Chart 7. Effects of MUdR on [3H)uridine incorporation into RNA fraction Chart 8. Effects of MUdR on [3H]guanosine incorporation into RNA frac ated by sucrose density gradient analysis. For details, see â€˜Materialsand tionated by sucrose density gradient analysis. For details, see legend to Methods.â€• Chart 2.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. D. Bogyo et a!. shows the results of chromatography of the [3H]adenosine metabolic activation steps required for the conversion of labeled ANA. PHA control cultures showed significant con MUdR to the niboside. version of adenosine to guanosine nucleotides as seen in radioactivity appearing in GMP spots. MUdR-tneated cells, Inhibition of Thymidine and Uridine Kinases on the other hand, showed a decreased conversion of aden Lymphocytes cultured with PHA for 24 and 48 hr were osine to guanosine nucleotides. collected, washed, and disrupted by ultrasonic cavitation. These data would support the theory that MUdR may The clear lymphocyte extract served as a good source of decrease total cellular guanosine nucleotide pools. To test thymidine kinase and unidine kinase. With NaF present in this theory, experiments were done to determine the vamia the assay mixture to inhibit phosphatase activity, the prepa tion of acid-insoluble incorporation of labeled nucleosides nation showed linear conversion of substrate with time and induced by pulse-labeling with differing initial specific ac showed a linear increase in substrate conversion with in tivity radioactive precursor levels. Using the derivation of creasing enzyme concentration . Thymidine kinase activity Forsdyke (12), V = N(1/x) â€”(P + 1), where V is the in duced dilution of labeled isotope, x is the measured acid C insoluble incorporation, P is the total intracellular pool 0 or unknown dilution, and N is the maximal incorporation in 0 C 4, the absence of any dilution, the investigator may derive a U C 0 Table 3 4, C Effects of MUdR on [3HJadenosine interconversion and â€˜V incorporation into RNA Twenty-five cultures (5 x 106 cells/mI) stimulated with either PHA alone or PHA pluse 10@ M MUdR were pulsed-labeled at 18 hr with 0 [3H]adenosine (3 MCi/culture) for 6 hr. The cells in each group were pooled and processed; the isolated RNA was hydrolyzed, and the C radioactivity associated with the paper chromatognaphically sepa 0 C rated nucleotides was determined as described in â€œMaterialsand 0 Methods.â€• PHAPHA+MUdR%oftotal%of 0 104106 totalradioac radioac NucleosideRFcpmtivitycpmtivity3'-AMP0.2039021372322'-AMP0.2929116254223'-GMP0.44106257414362'-GMP0.575840cpm/rnl Chart 9. Estimation of total intracellular uridine pool sizes in the presence and absence of MUdR by isotope dilution series. To cells cultured as de scribed in legend to Chart 1 was added [3Hjuridine (3 MCi) at 18 hr post-PHA stimulation, for a 6-hr pulse. Graded quantities of cold uridine were added to the labeled nucleoside before pulsing, resulting in a known series of samples with varying specific activities and concentrations. Aliquots were removed and acid-insoluble radioactivity was determined. The method of Forsdyke value of the total intracellular nucleoside-nucleotide con (12) was used to calculate changes in total intracellular pool size. centration (P0. Results of this experiment with [3H]umidmne labeling shown in Chart 9 indicate that MUdR causes no C major differences in total intracellular unidine-demivative 0 concentration (x intercept values). From the study with 0 C 4, [3H]guanosmne(Chart 10) labeling, it can be seen that MUdR U S produces a major shift in the x intercept, representing a C.) significant reduction in total pool size of guanosine demiva tives. PHA lymphocytes rapidly convert nucleosides to the nucleotide tniphosphate form so that, at any sampling time, 0 the predominant soluble species measured is the tniphos phate. 0

C Comparison of MUdR and MUR Inhibition 0 C 0 To show that the inhibition of nibonucleoside incorpora tion is effected by the deoxyniboside form of the drug, 0 studies comparing MUdR and the niboside analog MUR were carried out. Comparison of their inhibitory effects (Table 4) indicates that MUdR produces greaten inhibition of acid-insoluble incorporation for all the labeled nucleosides tested . These Chart 10. Estimation of total intracellular guanosine pool sizes in the data argue against the premise of in vivo MUdR-to-MUR presence and absence of MUdR by isotope dilution series. For details, see conversion, since MUR should be effective without the 2 legend to Chart 9.

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Table 4 Comparison of effects of MUdR and MUR on the inhibition of incorporation of nucleosides into acid-insoluble material @ Cells were cultured as described in legend to Table 1 with 10 M MUdR or 10@@MMUR added at 0 time. Labeled nucleosides (3 @iCi)were added 6 hr before harvesting of cultures, as described in

.. Materials and Methods.â€• cpm Time % inhibi Nucleoside (hr) Drug PHA PHA + drug tion Uridine 24 MUdR 13,848240,054243,012 Â±13,981â€•112,231 Â± 54 MUR 8,389297,220Â± 4,996155,521 Â± 35 48 MUdR 22,720241,618Â±39,699141,818 Â± 52 MUR Â±51,663154,934 Â±37,588 36

Adenosine 24 MUdR 202,187Â±16,174 149,575 Â± 8,974 26 MUR 191,289 @:12,937 148,555 Â± 7,789 22

Cytidine 24 MUdR 225,962 :t 13,432 24,190 :@ 2,067 89 MUR 208,435 Â±12,739 99,575 Â± 8,174 52 48 MUdR 361,555 Â±10,412 37,575 Â± 1,878 90 MUR 347,661 Â±16,416 271 870 Â± 13,501 22

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

â€œMean Â± SE.

from the 48-hr lymphocyte extract was inhibited 64% by the when these increases in enzyme activity have leveled off. addition of MUdR to a final concentration of 1 mM in the Inhibition by 1 mM MUdR of total cellular DNA, by 43% at sonic extract. Identical inhibition of thymidine kinase was 42 hr in comparison to PHA controls, indicates that the produced by 1 mM MUdA in a 24-hr lymphocyte extract. At major portion of the S-phase increase in DNA of these this concentration of MUdR, inhibition of thymidine incor stimulated lymphocytes is blocked. One m@ MUdR does not ponation into acid-insoluble nucleic acid material of the reduce total cell numbers nor does it block PHA-induced cultures is 75% (Table 4), a level of inhibition quite compa transformation. The ability of PHA-stimulated lymphocytes mable to the thymidine kinase inhibition previously noted. to proceed to the lymphoblast state during major inhibition Unidine kinase activity was inhibited 56% by 1 mM MUdR. of DNA synthesis confirms earlier reports to this effect by Thymidine at the same concentration produced no unidine Kay et al. (20) and Salzman et a!. (26). kinase inhibition. The magnitude of umidine kinase inhibi The present investigations were also undertaken to eluci tion is quite similar to the inhibition of labeled unidine into date how MUdR inhibits RNA synthesis from radioactive acid-insoluble nucleic acids presented earlier (Table 4). labeled nucleoside precursors. Initial findings of MUdA in hibition of [3H]umidine incorporation into acid-insoluble nu cleic acids were extended to show MUdR inhibition of Ia DISCUSSION beled adenosine and cytidine incorporation as well. [3H]Guanosine incorporation, rather than being inhibited, The data presented indicate that MUdR is an effective was increased 3-fold in MUdR-treated lymphocytes cultures inhibitor of DNA synthesis in a mammalian primary spleen stimulated with PHA. lymphocyte culturesystem.The greaterinhibitionbyMUdR MUdR may act by a variety of mechanisms to produce the of deoxyumidine incorporation into acid-insoluble nucleic observed apparent inhibition of RNA synthesis. Since nei acid material than of thymidine incorporation reflects the then total cell number nor total percentage of blast cells greaten sensitivity of thymidylate synthetase to MUdR as were reduced by MUdR, the effects seen do not result from compared to thymidine kinase sensitivity. This agrees with either cytotoxicity on blockade of PHA stimulation. Compan studies of Kalman and Bardos (19) in microbial systems in ative studies of MUdR and MUR effectiveness argue against which MUdR was shown to have an apparent K of 1.6 x the type of MUdR breakdown and conversion seen by Han @ 10@ M fonthymidine kinase and an apparent K ofi x 10 M bemset a!. (14) with 5-fluomo-2'-deoxyunidine. for thymidylate synthetase. In the latter enzyme system, the Time course studies with increasing pulse duration of phospho rylated derivative (5-mercapto-2'-deoxyu rid ine-5'- labeled nucleoside in the presence of MUdR do not show a phosphate) showed an apparent K of 4 x 108 M. PHA pattern of inhibition resembling the observed blockade of stimulation of lymphocytes produced marked increases in unidine uptake evidenced with mM levels of thymidine (30). thymidine kinase activity 30 to 42 hr after addition of PHA, However, the level of acid-soluble nucleoside at saturation and thymidylate synthetase activity shows increases at 24 to level is lowered, for umidine and adenosine, in the presence 48 hr after the addition of the mitogen. The time course of 1 mM MUdR. This could be due to any of the following studies of MUdR inhibition of both deoxyunidine and thymi factors: inhibition of metabolic activation of the labeled dine incorporation into acid-insoluble material show that nucleoside, increased turnover of the acid-soluble pool, or MUdR inhibition increases 48 to 54 hr after PHA stimulation. changes in interconversion of labeled nucleoside and, thus, The progressive inhibition by MUdR follows the period altered utilization.

SEPTEMBER 1976 3291

Evidence from these studies indicates that all 3 of the REFERENCES above-mentioned parameters may be effected by MUdR action. Unidine kinase is inhibited by 1 mM MUdR to a level that correlates well with the total acid-insoluble unidine 1. Adler, W. H., Takiguchi, T., March, B. , and Smith, R. T. Cellular Recog nition by Mouse Lymphocytes in Vitro. J. Exptl. Med., 131: 1049-1078, incorporation decreases seen with the same level of the 1970. drug. It has been postulated that unidine kinase is the rate 2. Askari, A. , and Franklin, T. Effects of Monovalent Cations and ATP on limiting step in incorporation of L3H]unidine into RNA (16). Erythrocyte AMP Deaminase. Biochim. Biophys. Acta, 110: 163-173, 1965. MUdR does interfere with nucleoside activation, at least in 3. Baranski, K., Bardos, T. J. , Bloch, A. , and Kalman, T. I. 5-Mercaptodeox the cases of unidine, cytidine (both initially phosphorylated yuridine: Its Enzymatic Synthesis and Mode of Action in Microbiological Systems. 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Ribonucleic Acid Metabolism in Lymphocytes Stimulated stenic site that binds pymimidine tniphosphates (2) and shows by Phytohemagglutmnmn. J. Biol. Chem., 243: 34-43, 1968. 8. Cooper, H. L. Studies on RNA Metabolism during Lymphocyte Activa sensitivity to the sulfhydmyl group inhibitor, hydroxymercu tion. Transplant. Rev., 11: 3-38, 1972. mibenzoate (31). It is possible that MUdR interacts with this 9. Cooper, H. L. Effects of Mitogens on the Mitotic Cycle: A Biochemical site by virtue of its 5-mercapto group. Evaluation of Lymphocyte Activation. In: A. M. Zimmerman, G. Padilla, and I. Cameron (eds.), Drugs and the Cell Cycle. pp. 137-193. New York: The difficulties known to exist between correlating radio Academic Press, Inc., 1974. active precursor incorporation with net synthetic rates have 10. Emerson, D.. and Humphreys, T. A Simple and Sensitive Method for Quantitative Measurement of Cellular RNA Synthesis. Anal. Biochem., been shown again in this study. Any change in specific 40: 254-266, 1971. activity of the nucleoside tniphosphate cellular pool in drug 11. Farrow, M. G. , and Van Dyke, K. A Micro-system for Screening Antileu treated cultures, compared with controls, will produce in kemic Drugs Utilizing Human Whole Blood. Chemotherapy, 16: 76-84, 1971. corporation differences that do not reflect changes in RNA 12. Forsdyke, D. Studies ofthe Incorporation of[5-3H]-Uridine during Activa synthesis. In this work, MUdR inhibition of labeled adeno tion and Transformation of Lymphocytes Induced by Phytohemaggluti sine incorporation into acid-insoluble material correlated nm. Biochem. J., 107: 197-205, 1968. 13. Frankel-Conrat, H., Singer, B., and Tsugita, A. Purification of Viral RNA better than any other labeled nucleoside with total RNA by Means of Bentonite. Virology, 14: 54-65, 1961. inhibition produced by the drug. Others have reported that 14. Harbers, E., Chandhuri, N. K., and Heidelberger, C. Studies on Fluori nated Pyrimidines. VIII. Further Biochemical and Metabolic lnvestiga [3H]adenosine incorporation is a good measure of total ANA tions. J. Biol. Chem., 234: 1255-1262, 1958. synthesis(10,29). 15. Hastings. J., Freedman, S., Rendon, 0., Cooper, H. L. , and Hirschhorn, The inhibition of thymidine kinase extracted from PHA K. Culture of White Cells Using Differential Leukocyte Separation. Na ture, 192: 1214â€”1215,1961. stimulated cells at 24 and 48 hr by 1 mM MUdR is identical 16. Hausen, P., and Stein, H. On the Synthesis of RNA in Lymphocytes (65%). By this criterion, qualitative differences in the en Stimulated by Phytohemagglutinin. European J. Biochem., 4: 401-406, zyme before and after major increases in activity triggered 1968. 17. Hiatt, H. A Rapidly Labeled RNA in Rat Liver Nuclei. J. Mol. Biol., 5: 217- by PHA could not be seen. The inhibition of nibonucleotide 229, 1962. synthesis by the deoxynibonucleoside MUdR was shown for 18. Ives, D. H., Durham, J. P., and Tucker, V. S. 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Cancer Res., 26: 638- at maximal metabolic capability. These cells have not un 646, 1966. 22. Lin, R. I., and Schjeide, D. A. Micro Estimation of RNA by the Cupric Ion dengone the irreversible alterations seen in defined cell Catalyzed Orcinol Reaction. Anal. Biochem., 27: 473-483, 1969. lines. Since PHA triggers the progression of a well-defined 23. Lowry, 0. H., Rosebrough, N. J. , Farr, A. L. , and Randall, R. J. Protein temporal sequence of biochemical events in these cells, Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193: 265- 275,1951. drug effects may be studied in relationship to this progres 24. Lucas, Z. J. Regulatory Control of Nucleic Acid Synthesis during Blasto sion. For example, the MUdR effects on mibonucleotide genesis of Lymphocytes in Culture. In: E. Mihich (ed), Drugs and Cell synthesis and interconversion seen by 24 hr may effect Regulation, pp. 159-180. New York: Academic Press, Inc., 1971. 25. Oppenheim, J. , Wolstercraft, R., and Gell, P. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. Effects of 5-Mercapto-2′-deoxyuridine on the Incorporation of Nucleosides into RNA and DNA in a Primary Lymphocyte Culture System

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

Cancer Res 1976;36:3284-3293.

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