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

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Effects of 5-Mercapto-2'-Deoxyuridine on the Incorporation of Nucleosides Into RNA and DNA in a Primary Lymphocyte Culture System1 [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. 3284 CANCERRESEARCHVOL. 36 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1976 American Association for Cancer Research. Effect of MUdR on Lymphocyte RNA and DNA Synthesis 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 0°.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 ‘-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.
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