Deoxyuridine Inhibition of Nucleoside Phosphorylase in Hela Cells Containing Mycoplasma1

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(CANCER RESEARCH 27 Part 1, 1867-1873, October 1967] 5-Allyl-2'-deoxyuridine Inhibition of Nucleoside Phosphorylase in HeLa Cells Containing Mycoplasma1

JAMES F. HOLLAND, RITA KORN, JUDITH O'MALLEY, HARRY J. MINNEMEYER, AND HOWARD TIECKELMANN Department of Medicine A, Roswell Park Memorial Institute, New York State Department of Health, and the Department of Chemistry, Statt University of New York at Buffalo, Buffalo, New York 1420S

SUMMARY tested (20, 21). 5-Allyl-2'-deoxyuridine was inactive as a growth inhibitor for HeLa cells in concentrations up to 10~4M (19). 5-Allyl-2'-deoxyuridine (AUdR) inhibits nucleoside phosphor- In an effort to test the ability of 5-allyl-2'-deoxyuridine to sub ylase activity associated with mycoplasma-contaminated HeLa stitute for thymidine, the technic of Hakala and Taylor (9) was (HeLa/PPLO) cells. This has been demonstrated in cell culture used. In this system the inhibition of cell growth by the folie acid experiments in which AUdR allowed thymidine to support HeLa/ antagonist methotrexate can be prevented by the simultaneous PPLO growth in a system dependent upon thymidine, in which, incorporation into the medium of glycine, hypoxanthine, and because of phosphorolytic catabolism, thymidine alone had been unable to do so. Inhibition of the exaggerated nucleoside phos- thymidine. When tested at several different concentrations of phorylase activity of HeLa/PPLO by AUdR restored the chemo- thymidine, however, our HeLa cells did not grow. This failure therapeutic activity of 5-fluoro-2'-deoxyuridine on these cells. was demonstrated to be due to cleavage of the thymidine to thymine by mycoplasma contaminating the cultures (8). 5-Ally1- In experiments with isotopically labeled deoxynucleosides, AUdR 2'-deoxyuridine also was unable to support growth of the myco inhibited nucleoside catabolism by HeLa/PPLO, by a mycoplasma-contaminated HeLa cells. When both thymidine and plasma culture, and by purified horse liver thymidine phos- 5-allyl-2'-deoxyuridine were present in the culture medium simul phorylase. taneously, however, abundant growth occurred. The data pre Lesser inhibition of nucleoside phosphorylase activity in one sented herein indicate that this was due to the activity of 5- or more of these systems was obtained with Ar-3-methylthymi- allyl-2'-deoxyuridine as an inhibitor of the enhanced nucleoside dine, A^-S-methyluridine, 5-trifluoromethyl-2'-deoxyuridine, and other 5-substituted 2'-deoxyuridines. phosphorylase activity associated with the mycoplasma infec tion. Preliminary information has been presented (14, 19). AUdR showed no biologic activity other than inhibiting nucleo side phosphorylase, a specificity that recommends its further in vestigation as an adjuvant compound in circumstances where MATERIALS AND METHODS inhibition of phosphorolytic destruction of nucleoside substrates 5-Allyl-2'-deoxyuridine was synthesized as an anomeric mix or drugs is desirable. ture, and the a and ÃŸanomerswere separated (19). Unless other wise indicated, 5-AUdR2 refers to the ÃŸanomer.W-S-Methylthy- INTRODUCTION midine (1) and Ar-3-methyluridine (25) were synthesized. 5-Trifluoromethyl-2'-deoxyuridine was provided by the Cancer The importance of thymidine as a constituent of deoxyribo- Chemotherapy National Service Center. Thymidine-2-I4C, nucleic acid has prompted the synthesis of several analogs for specific activity 25 mc/mmole, was purchased from New England chemotherapeutic purposes. Among the most effective of these Nuclear Corporation. 5-Fluoro-2'-deoxyuridine-2-14C, specific as inhibitors of either the biosynthesis or the utilization of thymidylate have been 5-fluoro-2'-deoxyuridine (10), 5-bromo-2'- activity 10 mc/mmole, was kindly provided by Dr. Robert Duschinsky and by Dr. Charles Heidelberger. The remaining deoxyuridine (16), 5-iodo-2'-deoxyuridine (24), and 5-trifluoro- reagents and cell culture constituents were obtained commer methyl-2'-deoxyuridine (13). The structural similarity of the allyl cially. HeLa/PPLO cells were obtained at various times from group to the methyl group in the 5-position of thymidine three separate laboratories of this Institute and from the Ameri prompted us to synthesize the 5-allyl pyrimidine and its nucleo- can Type Culture Collection and Microbiological Associates, Inc. sides: 5-allyluracil (20), 5-allyluridine (21), and 5-allyl-2'-deoxy- HeLa/PPLO cells were maintained on Eagle's medium with 10% uridine (19). 5-Allyluracil and 5-allyluridine were inactive in all bacterial, mammalian cell culture, and mouse tumor systems 2Abbreviations used: AUdR, 5-allyl-2'-deoxyuridine, and un less otherwise specified, refers to the ÃŸanomer; HeLa/PPLO, 1This investigation was supported in part by Research Grant HeLa cells from which mycoplasma (J. Horoszewicz laboratory No. T-231 from the American Cancer Society and by USPHS Re * 152) were isolated repeatedly. Mycoplasma 152 has not been search Grant Nos. Ã‡A2857 and CA 5834 from the National Cancer discriminately classified and its relation to other uncommon strains Institute. of mycoplasma has not been established; FUdR, 5-fluoro-2'-de- Received October 6, 1906; accepted June 12, 1967. oxyuridine; CF3UdR, 5-trifluoromethyl-2'-deoxyuridine.

OCTOBER 1967 1S()7

horse serum containing 50 Mgof penicillin and 50 Mgof strepto TABLE 1 mycin per ml (4). Pyrex test tubes (16 X 125 mm) were inocu Deoxyribonucleoside-dependent growth of M ycoplasma-contaminated lated with 50,000 cells and incubated at 37Â°C.Twenty-four hours HeLa Cells (HeLa/PPLO) in Deficient Medium Containing Thymidine, S-Allyl-S'-deoxyuridine, or Both" later, tubes were selected for good implants and allocated at ran dom to different treatments. Growth was measured as protein OIM)00.50.70.61.92.32.4100.71.31.84.94.83.1500.62.63.75.96.31.01000.74.55.93.45000.65.96.07.16.33.110000.75.96.37.06.01.2 accumulation after 6 days (23) and expressed as multiples of the Thymidinedot)0510501005005-Allyl-2'-deoxyuridine quantity found in cells that had implanted. Three or more tubes were used for each point. In most experiments the cells were studied in Eagle's medium, as modified by Hakala and Taylor to contain l /UMmethotrexate, 100 MMglycine, and 100 MMhypo- xanthine. In this system, 5 to 6 MMthymidine solutions produced half-maximal growth of uncontaminated HeLa cells and 30-100 MMsupported maximal growth (9). Cultured cells grown in Eagle's medium in pharmacy bottles were used for studies of enzymatic activity. They were harvested " Growth measured as protein content and expressed as multi either mechanically or by trypsinization, centrifuged at slow ples of mean control value (=1) at beginning of experiment. speed, washed once, and suspended in single strength Eagle's Deficient medium (4) is Eagle's single strength medium containing medium containing test compounds, but lacking antibiotics, 10% horse serum and 1 JUMmethotrexate, 100 MMglycine, 100 MM phenol red, and horse serum. Studies were carried out aerobically hypoxanthine, but no thymidine. in a shaking water bath at 37Â°Cusing sterile technic. Aliquots for radiochromatographic separation were removed from the incu at 500 MM.When AUdR and thymidine were present in combi bation mixture and immediately plunged into ice water and pre nation, however, maximal growth occurred; 50 MMconcentrations cipitated with 4% perchloric acid. of each compound produced an approximately 6-fold increase in Horse liver thymidine phosphorylase (6), generously provided growth. This is shown to be dependent on the presence of AUdR, by Dr. Morris Friedkin, was incubated for 30 minutes at 38Â°C because an equivalent addition of thymidine (to 100 MM)did not in 410 M!containing 2.25 mg of liver powder, 20 MinÃ³lesofsodium result in an appreciably greater growth than did 50 MMthymidine. arsenate, 40 Mmoles of tris(hydroxymethyl)aminomethane, and The drug concentrations appear to be logarithmically related 40 Mmoles of thymidine-2-14C at pH 7.2. Varying amounts of one to another, in that an increase in either by tenfold produced thymidine and, where appropriata, inhibitors were added. The the same augmentation of growth. Thus, growth in the presence reaction was terminated with 1 ml of 4% perchloric acid. After of the combination of 5 MMthymidine and 50 MMAUdR was in centrifugation, the supernatant fraction was removed, neutralized creased equally if either the AUdR was increased to 500 MMÂ°r to phenol red with potassium hydroxide, and refrigerated to the thymidine to 50 MM.In the presence of AUdR, thymidine at allow precipitation of potassium perchlorate. The supernatant 500 MMevoked less growth than when concentrations of only 50 solution was chromatographed. MMor 100 MMwere present. Descending Chromatographie separation of fluorouracil-2-14C Because of the apparent protection of thymidine in this system from FUdR-2-14C was accomplished on Whatman # 1 paper in which required thymidine, we studied the effect of AUdR on the isopropanolÂ¡hydrochloric acid:water (4:1:1) overnight at room activity of another deoxyribonucleoside, i.e., inhibition by FUdR temperature. Authentic compounds were run concomitantly. For of HeLa/PPLO cells growing in standard Eagle's medium. In initial identification, radioactive spots were eluted, admixed with Table 2 it can be seen that FUdR, in concentrations up to 1 MM, reference compounds, and rechromatographed in two solvents. had no growth-inhibitory effect. In the presence of 50 MMAUdR, Chromatographie separation of thymine-2-14C from thymidine- however, sensitivity to FUdR was restored, even at 0.01 MM,the 2-14C was accomplished in butanol: ammonia (3:1) run for 46 order of magnitude of the concentration of FUdR that is required hours at room temperature. Chromatographie strips were air- for inhibition in uncontaminated cells (8). dried and divided transversely in 0.5-cm sections and immersed The specificity of the response to AUdR was tested by compari in toluene containing 5 grams of 2,5-diphenyloxazole and 0.3 son in the same experiment with several other related compounds. gram 1,4-bis-2(4-methyl-5-phenyloxazolyl)benzene per liter and 5-Bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-allyluridine, counted in a Packard Tri-Carb liquid scintillation spectrometer 5-allyluracil, and 2-deoxyribose all failed to provide significant sparing of thymidine whereas the activity of AUdR was repeat edly demonstrated. 5-Trifluoromethyl-2'-deoxyuridine exhibits activity as an in RESULTS hibitor of both growth and nucleoside phosphorylase (12). Its in In the first experiment, HeLa/PPLO cells were grown on stand hibitory activity for growth of HeLa/PPLO in standard Eagle's ard Eagle's medium or the modified Eagle's medium containing medium is shown in Table 3. Since a concentration of 100 MM methotrexate, glycine, and hypoxanthine, but lacking thymidine CF3UdR was inhibitory per se, lower concentrations were used to (hereinafter called deficient medium), with additional ATJdR, thy test for protective activity against the destruction of TdR by midine, or combinations of the two. The standard cultures grew HeLa/PPLO in the deficient system. At equimolar concentra 5-fold. It can be seen in Table 1 that AUdR did not support tions, CF3UdR was less active than AUdR (Table 4). growth. Furthermore prevention of growth inhibition by thy The effect of AUdR and CF3UdR on the activity of Friedkin's midine is decidedly less than that reported elsewhere (9), even purified horse liver thymidine phosphorylase was studied. The

1868 CANCER RESEARCH VOL. 27

TABLE 2 TABLE 5 Effect of 5-Allyl-e'-deoxyurdine (AUdR) on B-Fluoro-2'-deoxyuri- Nucleoside Phosphorolysis by HeLa Cells Contaminated with dine (FUdR)-Inhibition of Growth in Standard Eagle's Medium of Mycoplasma (HeLa/PPLO) HeLa Cells Contaminated with Mycoplasma" HeLa/PPLO was incubated for 4 hours at 38Â°Cin the presence of 3.2 Aimoles thymidine. After precipitation of protein with an GIM)06.26.40.016.41.70.16.04.416.22.0 equal volume of 4% HC10Â¿or equal volumes of Ba(OH)2 and AUdROXM)050FUdR ZnSO4, an aliquot of each supernatant was run in the diphenyl amine reaction and read on a Gilford spectrophotometer at 600 m/t.

PrecipitantHC10, found(m/imoles)22.8

" See footnote, Table 1. Ba(OH)2-ZnSO4Deoxyribose 5

TABLE 3 Effect of B-Trifluoromethyl-2'-deoxyuridine (CFtUdR) on studied was substantially less active than had been the case with Growth of HeLa Cells Contaminated with Mycoplasma several other incubations, but nonetheless did demonstrate ap in Standard Eagle's Medium" pearance of thymine from thymidine. When the incubation was terminated by perchlorate (26), deoxyribose was readily demon CF.UdR O,M) strated by the diphenylamine reaction (3). When the reaction was stopped with 0.3 N Ba(OH)2 and 5% ZnSCX,however, the 0 supernatant fraction contained substantially less free or combined 4.91.0 6.110 5.6100 1.5 2-deoxyribose, indicating that it had been precipitated and thus " See footnote, Table 1. had been present as the phosphate (26) (Table 5). Further studies of deoxyribonucleoside cleavage were under TABLE 4 taken with isotopically labeled substrates. HeLa/PPLO cells (3.3 Effect ofS-Trifluoromelhyl-2'-deoxyuridine and B-Allyl-2'-deoxyuri- X IO6)were incubated in 1.1 ml of Eagle's medium deficient in dine on Growth Response to Thymidine of HeLa Cells Contamin horse serum, phenol red, and antibiotics. Recovery of added ated with Mycoplasma in Deficient Medium" FUdR-2-14C as the parent compound plus fluorouracil-2-14C was 90%. There was rapid transformation of FUdR-2-I4C to fluoro- (MM)01.31.21.41.6101.32.95.24.7501.43.92.55.9uracil-2-14Cwith complete disappearance of the deoxyribonucleo ThymidineGull)010501005-Trifluoromethyl-2'-deoxyuridine(/â€¢M)01.31.21.41.6101.31.61.82.2501.31.41.82.25-Allyl-2'-deoxyuridineside within 1 hour. No retardation of the formation of fluorouracil-2-14C was seen when 600 /Â¿MAUdR (ten times the concentration of FUdR-2-I4C) was added directly to the incu bation mixtures. This seemed possibly ascribable to differential rates of transport of substrate and of the necessary concentration of inhibitors to the enzyme site Thus, HeLa/PPLO cells were grown in Eagle's medium containing 50 Â¿IMAUdRfor 4 days. Prior to the incubation, cells were gently washed twice in Eagle's " See footnote, Table 1. medium without AUdR. Then 3.3 X IO6cells were incubated in control medium, in the presence, of either 600 pM AUdR or 600 lyophilized protein powder had a specific enzyme activity for /iM FUdR. The half-disappearance time of FUdR-2-14C was re arsenolysis of approximately 1 Â¿umoleofthymidine per mg per tarded by about one-third by the presence of added AUdR (Chart hour. Its Km was 2.8 X 10~4 (Â±0.6 X IO-").3 In this system AUdR had a A'Â¡of 12 X IQ-4 (Â±3 X IQ-4), while that 1). That this was not due to a nonspecific effect of high concen of CF3UdR was 8.7 X 10~4(Â±3.1 X 10~4).The Kâ€ž/Ktratio for tration of a deoxyribonucleoside is demonstrated by the results obtained when the concentration of FUdR was equimolar to the these two compounds was 0.23 and 0.32, respectively, and the AUdR. The disappearance rate of FUdR was similar to that of ratio of the KÂ¡valueswas 0.72. From these data we conclude that the undiluted radioactive compound, after correction for the dif the two compounds exert essentially similar inhibitory activity ferent specific activities of the two incubation mixtures on the enzyme. Nucleoside phosphorylase activity was not limited to the An experiment was performed to determine whether the deoxy HeLa/PPLO cell. The supernatant culture medium, when incu ribonucleoside cleavage associated with HeLa/PPLO was phos- bated with FUdR-2-14C, caused appearance of fluorouracil-2-14C. phorolytic or hydrolytic. The particular HeLa/PPLO culture This same supernatant fluid also was able to cleave thymidine- 2-14C to thymine-2-14C (Table 6). Thus, the supernatant fluid

*Estimates of K,n and of KÂ¿for the two drugs (assuming com derived from the culture contained either enzyme or mycoplasma, or both. petitive inhibition) were obtained by the maximum neighborhood algorithm for nonlinear least-squares estimation (17) by means of The relationship of the mycoplasma organisms to the nucleo- a modification of IBM Share Program No. 1428. Computations side phosphorylase activity was studied in two strains of myco were performed on an IBM 7044 digital computer through the plasma. Cultures of mycoplasma grown and transferred 4 days courtesy of the Computing Center of the State University of New previously on artificial liquid media in the laboratories of Dr. York at Buffalo. Julius Horoszewicz were collected by centrifugation, resusi>ended,

OCTOBER 1967 1869

60n TABLE 6 Extracellular Nucleoside Phosphorylase Activity HeLa cells contaminated with mycoplasma (HeLa/PPLO) were 50- grown 4 days in Eagle's medium at 37Â°C.The medium was de canted and centrifuged at 3000 rpm for 5 minutes in a clinical centrifuge and 0.1 ml of the supernatant used for 3-hour incuba tions containing 55 rmimoles of 5-fluoro-2'-deoxyuridine-2-"C 40- (FUdR-2-14C) or 40 m/nmoles of thymidine-2-'4C. The reaction was terminated by immersing in ice and the supernatant fluid chromatographed and counted as described.

foundFUdR-2-Â»C5542Fluoroura- ConditionsFresh U- 30- cil-2-Â»C013Thymi-dinc-2-Â»C4029Thy-mine-2-"C011 w 0) onlySupernatantmedium medium from HeLa/PPLOmamÃ³les

20J TABLE 7 Calabolism of Ã´-Fluoro-Ãª'-deoxyuridine-2-llC (FUdR-2-"C) by Mycoplasma Mycoplasma organisms were suspended in a total volume of 1.1 ml Eagle's single strength medium lacking antibiotics, phenol red, and horse serum and were incubated at 37Â°Cina shaking water bath. The medium was made up to GOfjMFUdR-2-"C, and treated flasks contained GOO/IM5-allyl-2'-deoxyuridine (AUdll).

strain#152M.Mycoplasma (min)15301530Control(mamÃ³les (mamÃ³les FUdR)51533023AUdRFUdR)52523131

10 15 30 mergenhagenTime TIME,MIN CHART1. Inhibition of nucleoside phosphorolysis in whole cells. HeLa cells contaminated with mycoplasma cells were grown for 4 days in 50 Â¡t\i5-allyl-2'-deoxyuridine (AUdR). Cells were then Difco Brain Heart infusion broth containing 20% horse serum washed in control medium prior to incubation at 37Â°Cin 1.1 ml. and 0.8% Noble Special Agar) at concentrations of 5, 50, or 500 Control flasks (O D) contained Eagle's medium minus phe /UM.When Mycoplasma 152 and M. mergenhagen were inoculated, nol red, horse serum, and antibiotics. AUdR 600 Â¡ai(O O) or their growth was unaffected. In addition, bacterial sansitivity 5-fluoro-2'-deoxyuridine (FudR) 600 MM(A) was added to the discs 6 mm in diameter were autoclaved, dried, and impregnated same media in experimental flasks. FUdR-2-14C60 n\i was present with 20 n\ of a sterile solution containing 50 tig of AUdR. There in each flask. was no growth inhibition of four strains of coagulase-positive staphylococci, two of Pseudomonas aeruginosa, or single isolates and added to incubation flasks; FUdR-2-14C was added. Recovery of Aerobacter aerogenes, Escherichia coli, Proteus, an enterococcus, of radioactivity as the parent compound plus fluorouracil-2-14C and Bacterium anitratum. was 95-102% in the incubations of strain 152, originally isolated Other thymidine analogs were studied for effects on pyrimidine from our HeLa/PPLO, but only 82-85% in the incubations with nucleoside phosphorylase activity. A*-3-Methylthymidine was a strain Mycoplasma mergenhagen, originally isolated from a human poor substrate for horse liver thymidine phosphorylase and in spleen of a patient with chronic lymphocytic leukemia (7). No hibited the arsenolysis of thymidine only slightly. The thymidine counts were recovered in Chromatographie spots other than phosphorylase of HeLa/PPLO cells was partially inhibited, how FUdR-2-I4C or fluorouracil-2-14C. It can be seen in Table 7 that ever (Table 8). Af-3-Methylthymidine showed little ability to under the conditions of this experiment Mycoplasma 152 did not serve as a substitute for thymidine in HeLa/PPLO cells, but cause significant continuing disappearance of FUdR-2-14C and under circumstances in which thymidine was present in limiting thus no effect of AUdR could be appreciated. There was continu concentrations, the presence of .Y-3-methylthymidine allowed ing decrease in the FTJdR-2-14C recoverable from the system nearly as complete growth as AUdR at equimolar concentrations. incubated with M. mergenhagen. This catabolism was slowed AT-3-Methylthymidine also was capable of slightly increasing the somewhat in the presence of AUdR as can be seen in the 30- activity of FUdR against HeLa/PPLO. When 50 ÃŸMN-3-meÃœiy\- minute specimen. thymidine was present, 1.0 Â¿UMFUdRresulted in 50% growth AUdR was incorporated into solid PPLO media (consisting of inhibition, whereas 10 /UMFUdR was required to reach this level

LS70 CANCER RESEARCH VOL. 27

TABLE 8 AUdR is a new thymidine analog that lacks growth inhibiting Effect of "N-S-Melhyllhymidine on Thymidine Phosphorylase activity for several microorganisms, mycoplasma, or HeLa cells. Activity It is a very weak inhibitor of thymidylate synthetase of Ehrlich ascites tumor cells (13). It cannot replace thymidine in a system thymine in which the concentration of thymidine is limiting. It does, how AdditionExperiment ancy at per mu 300m/i0.1710.0100.0210.0120.2020.0580.0630.0170.0270.0230.0730.040AmÃ³lesenzyme7.780.446.531.260.0110.795ever, have substantial activity in inhibiting pyrimidine nucleoside phosphorylase. This has been demonstrated in several ways. In IThymidineA:-3-MethylthymidineThymidine a system in which the inhibitory effects of methotrexate on the biosynthesis of purine and glycine are circumvented by addition of hypoxanthine and glycine, thymidine concentration becomes critical (9). Mycoplasma-infected HeLa cells are unable to grow-

-3-methylthymi-dineExperiment+ N in this system because of phosphorolytic cleavage of thymidine (8). AUdR is a sufficiently active inhibitor of nucleoside phos phorylase, and a sufficiently inactive compound at other bio IIThymidineA'-3-MethylthymidineThymidine chemical loci, to permit appropriate growth response to thymidine (Table 1). AUdR also can preserve the integrity of 5-fluoro-2'-deoxyuridine, allowing substantially greater growth inhibition than occurs in cell cultures that cleave 5-fluoro-2'- deoxyuridine to 5-fluorouracil (Table 2). None of the other com AT-3-methylthymi-dineConditionIncubateControlIncubateControlIncubateControlIncubateControlIncubateControlIncubateControlAbsorb-+ pounds tested, including several 5-substituted deoxynucleosides that are analogs of thymidine, 5-bromo-2'-deoxyuridine, 5-iodo- 2'-deoxyuridine, 5-trifluoromethyl-2'-deoxyuridine, 5-allyluri- Experiment I: Flasks contained 2.25 mg powder rich in horse dine, 5-allyluracil, and 2-deoxyribose was capable of allow liver thymidine phosphorylase in a total volume of 450 /J. The reaction mixture also contained 20 AmÃ³lesof sodium arsenale, ing growth response to thymidine equivalent to AUdR in the HeLa cell system. 5-Trifluoromethyl-2'-deoxyuridine, a known 40 AmÃ³lesof tns(hydroxymethyl)aminomethane, and 3.2 /Â¿moles of thymidine at a pH of 7.2. A'-S-methylthymidine, 3.2 AmÃ³les,was oncostatic agent (11) and inhibitor of nucleoside phosphorylase added to appropriate flasks. The flasks were incubated for 30 (12), was itself apparently inactivated by the HeLa/PPLO minutes in a water bath at 38Â°C.Thefinal dilution was a factor of system and did not demonstrate anticipated growth-inhibitory 362.5. effects (Table 3). Experiment II: Flasks contained 4.36 X IO6HeLa cells con- The nucleoside cleavage was proved to be phosphorolytic by laminated with mycoplasma in a total volume of 600jul.The reac tion mixture contained single strength Eagle's medium lacking showing precipitability of deoxyribose phosphate with Ba(OH)2 and ZnSO< (Table 5). antibiotics, phenol red, and horse serum. Additions of thymidine and jV-3-methylthymidine were 3.2Aimoleseach. The reaction was The nucleoside phosphorolytic activity of cultured strains of terminated with 1 ml 4% HClCXi.An aliquot was alkalinized with mycoplasma was readily demonstrable with ^Ã.mergenhagen. 0.3 N NaOH. This was partially inhibited by AUdR. The strain 152 isolated from the HeLa cells in this experiment did not cause destruction of growth inhibition without the A"-3-methylthymidine. Thus, of 5-fluoro-2'-deoxyuridine on incubation. This may have been Ar-3-methylthymidine presumptively diminished the catabolism dependent on the number of organisms used, slow drug transport, of FUdll to fluorouracil. or specific characteristics required for phosphorolysis by strain Ar-3-Methyluridine was also capable of promoting growth re 152, which were not satisfied in these conditions independent of sponse to thymidine in the HeLa/PPLO system in deficient HeLa cells, rather than absolute incapability (Table 7). Indeed, medium although less than was Ar-3-methylthymidine. AT- the supernatant solution from the mycoplasma-contaminated 3-Methyluridine was barely active in increasing, the growth- HeLa cell cultures had nucleoside phosphorolytic activity for inhibitory action of FUdR. Neither .Y-3-methylthymidine nor 5-fluoiio-2'-deox}-uridine and thymidine, although it is not pos AT-3-methyluridine was as active as AUdR in potentiation sible to state whether this was due to mycoplasma in the medium of growth dependent upon thymidine nor in increasing inhibition or to soluble nucleoside phosphorylase (Table 6). All HeLa of growth by FUdR. strains, after acquisition and propagation in our laboratory, showed PPLO and had active nucleoside phosphorylase activity. DISCUSSION Attempts deliberately to infect cultures of Henle's intestine with The observations reported herein were recognizable in large 4-day-old cultures of Mycoplasma 152 were apparently unsuc measure because of the nucleoside phosphorolytic activity found cessful, however, since 5-fluoro-2'-deoxyuridine-2-!4C was re in HeLa cells contaminated with mycoplasma (8). Nucleoside covered quantitatively from both control and contaminated cul phosphorolysis has been found to be a characteristic attribute of tures after incubation for 3 hours. mvcoplasma-infected cells. Of 42 cell cultures from which myco The difficult synthesis of AL'dR by the methods we used re plasma were isolated, 38 showed nucleoside phosphorolytic activ stricted the supply of the a anomer for certain biologic ity (15). Effective inhibitors of nucleoside phosphorylase are rare experiments An improved synthesis has recently been described (2, 8). We know of no prior investigation of inhibitors of the (22). A single experiment indicated no difference in activity be nucleoside phosphorylase activity of cells contaminated with tween the a and the ÃŸanomerin jtermitting thymidine to cause mycoplasma. growth responses.

OCTOBER 1967 1S71

Ar-3-Methylthymidine and A'-3-methyluridine were shown to REFERENCES possess appreciable activity in the preservation of thymidine in 1. Beltz, R. E., and Visser, D.W. Growth Inhibition of Escher- the HeLa cell system, although slightly less than that of AUdR. ichia coli by new Thymidine Analogs. J. Am. Chem. Soc., A'-S-Metriylthymidine was more active than AT-3-methyluridine 77: 73G-738,1955. in conserving the activity of o-fluoro-2'deoxyuridine. Only N-3- 2. Birnie, G. D., Kroeger, II., and Heidelberger, C, Studies of methylthymidine was tested in the horse liver thymidine phos- Fluorinated Pyrimidines XVIII. The Degradation of 5-Fluoro- phorylase system, and there it exhibited little activity. 2'-deoxyuridine and Related Compounds by Nucleoside Incubation of mycoplasma-contaminated HeLa cells in vitro Phosphorylase. Biochemistry, a: 566-572, 1963. with FUdR caused a sharp loss of the nucleoside; this was inhib 3. Burton, K. A Study of the Conditions and Mechanism of the ited by AUdR only when cells had been grown in a medium Diphenylamine Reaction for the Colorimetrie Estimation of Deoxyribonucleic Acids. Biochemistry, 62: 315-323, 1956. containing AUdR. This finding suggests that transport of AUdR 4. Eagle, H. Amino Acid Metabolism in Mammalian Cell Cul during the incubation is slow and that subinhibitory concentra tures. Science, ISO:432-437, 1959. tions were present already in the cells grown in AUdR. An alter 5. Edward, D. G. The Pleuropneumonia Group of Organisms; nate possibility is that once inside the cell the nucleoside must A Review, Together with Some New Observations. J. Gen. undergo further chemical reaction. Microbio!., 10: 27-64, 1954. Pyrimidine nucleoside phosphorylase has been observed in 6. Friedkin, M., and Roberts, D. The Enzymatic Synthesis of many mammalian tissues (12, 18). It seems unlikely that in each Nucleosides. I. Thymidine Phosphorylase in Mammalian Tis instance this was due to mycoplasma contamination. This pos sue. J. Biol. Chem., 207: 245-250, 1954. sibility must be considered in each instance, however, because of 7. Grace, J. T., Jr., Horoszewicz, J. S., Stim, T. B., Mirand, E. A., the ubiquity of mycoplasma (5). AUdR inhibited the nucleoside and James, C. Mycoplasmas (PPLO) and Human Leukemia and Lymphoma. Cancer, 18: 1369-1376, 1965. phosphorylase of mycoplasma, of cells infected with mycoplasma 8. Hakala, M. T., Holland, J. F., and Horoszewicz, J. S. Change and of horse liver. Thus, the active sites of these enzymes can be in Pyrimidine Deoxyribonucleoside Metabolism in Cell Cul presumed to share qualitatively similar affinity for substrate and ture caused by Mycoplasma (PPLO) Contamination. Biochem. inhibitor. Biophys. Res. Commun., //.- 466-471, 1963. The activity of AUdR was similar to that of CFjUdR when 9. Hakala, M. T., and Taylor, E. The Ability of Furine and Thy- the effect of the compound on the activity of purified horse liver mine Derivatives and of Glycine to Support the Growth of thymidine phosphorylase was determined. Heidelberger and Mammalian Cells in Culture. J. Biol. Chem., 234: 126-128, Boohar compared the activity of the two compounds on nucleo 1959. side phosphorylase activty in a supernatant fraction of Ehrlich 10. Hartmann, K. U., and Heidelberger, C. Studies on Fluorinated ascites carcinoma cells where both were found to be competitive Pyrimidines. XIII. Inhibition of Thymidylate Synthetase. J. Biol. Chem., 236: 3006-3013, 1961. inhibitors (12). Our data demonstrate that the compounds are 11. Heidelberger, C., and Anderson, S. W. Fluorinated Pyrimi not uncompetitive inhibitors but are not sufficiently extensive to dines. XXI. The Tumor-Inhibitory Activity of 5-Trifluoro- distinguish unequivocally between noncompetitive and competi methyl-2'-deoxyuridine. Cancer Res., Ã•4:1979-1985,1964. tive inhibition. We have accepted Heidelberger and Boohar's 12. Heidelberger, C., and Boohar, J. Fluorinated Pyrimidines. findings (12) and have calculated kinetic constants based upon XXIII. Further Studies on Nucleoside Phosphorylase. Bio- the assumption of competitive inhibition. The inhibitory con chim. Biophys. Acta, 91: 639-641, 1964. stants calculated from these experiments, approximately 10~3M, 13. Heidelberger, C., Boohar, J., and Birnie, G. D. Fluorinated are substantially different from those reported, approximately Pyrimidines. XXII. Effects of Various Compounds on the 10-Â«M(12). Incorporation of (14C)Formate into DNA Thymine in Suspen Since isolated inhibition of a catabolic enzyme without other sions of Ehrlich Ascites Cells. Biochim. Biophys. Acta, 91: 636-638, 1964. apparent effects offers an approach to cellular nutrition or ther 14. Holland, J. F., Minnemeyer, H., Grace, J. T., Jr., Block, R., apy, by protecting a nucleoside substrate or drug, AUdR may O'Malley, J., and Tiecklemann, H. 5-Allyl-2'-deoxyuridine find application in the study of the biochemical disposition of (AUdR) Activity on Metabolism of Pyrimidine Deoxynu- other nucleosides. Because of scarcity of the compound at the cleosides by HeLa cells Infected with Mycoplasma. Proc. time, A^-S-methylthymidine, although of lesser activity, was tried Am. Assoc. Cancer Res., 4: 29, 1963. in one test system as a possible substitute. In spontaneous mouse 15. Horoszewicz, J. S., and Grace, J. T., Jr. PPLO Detection in breast adenocarcinomas refractory to 5-fluoro-2'-deoxyuridine Cell Culture by Thymidine Cleavage. Bacteriol Proc., V89, treatment in doses up to 250 mg/kg/day, no stasis of tumor 1964. growth was obtained in any of 11 other mice when 2V-3-methyl- 16. Kit, S., Beck, C., Graham, O. L., and Gross, A. Effect of 5- thymidine and 5-fluoro-2'-deoxyuridine were given simultane Bromodeoxyuridine on Deoxyribonucleic Acid. Thymine Syn thesis and Cell Metabolism of Lymphatic Tissues and Tumors. ously in daily doses up to 250 mg/kg of each (J. F. Holland and Cancer Res., 18: 598-602, 1958. B. Bryant, unpublished observations.) 17. Marquardt, D. W. An Algorithm for Least-Squares Estimation of Nonlinear Parameters. J. Soc. Ind. Appi. Math., 11:431-441, ACKNOWLEDGMENTS 1963. We are indebted to Dr. William Werkheiser for calculation of 18. Marsh, J. C., and Perry, S. Thymidine Catabolism by Normal the inhibition constants, to Dr. Julius lloroszewicz for the experi and Leiikemic Human Leukocytes. J. Clin. Invest., 43:267-278, ments with mycoplasma, and to both for valuable criticism and 1964. discussions. 19. Minnemeyer, II. J., Clark, P. B., Tieckelmann, II., and Hol-

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land, J. F. The Anomeric 5-Allyl-2'-deoxyuridines. J. Med. Tissue Culture with a Phenol Reagent (Folin-Ciocalteau). Chem., 7: 567-569, 1964. Proc. Soc. Exptl. Biol. Med., 91: 305-307, 1956. 20. Minnemeyer, II. J., Kgger, J. A., Holland, J. F., and Tieckel- 24. Prusoff, W. II. Studies on the Mechanism of Action of 5-Iodo- mann, H. The Synthesis of 5-Allyluracil and the Pyrimidine deoxyuridine, an Analog of Thymidine. Cancer Res., 20: Claisen Rearrangement. J. Org. Chem., 26: 4425-4429, 1961. 92-95, 1960. 21. Minnemeyer, H. J., Tieckelmann, H., and Holland, J. F. 5- 25. Visser. D. W., Barron, G., and Beltz, R. Antimetabolites of Allyluridine. J. Med. Chem., 6: 602-603, 1963. Uridine with Two Structural Alterations. J. Am. Chem. Soc., 22. Montgomery, J. A., and Hewson, K. An Improved Procedure 75: 2017-2019, 1953. for (he Preparation of 5-Allyl-2'-deoxyuridine. J. Heterocy- 26. Wang, T. P. Hydrolytic Nucleosidases. In: S. P. Colowick and clic Chem., g: 313-314, 1965. N. 0. Kaplan (eds.), Methods in Enzymology, Vol. 2, pp. 456- 23. Oyama, V. I., and Eagle, H. Measurement of Cell Growth in 464. New York: Academic Press, 1955.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1967 American Association for Cancer Research. 5-Allyl-2′-deoxyuridine Inhibition of Nucleoside Phosphorylase in HeLa Cells Containing Mycoplasma

James F. Holland, Rita Korn, Judith O'Malley, et al.

Cancer Res 1967;27:1867-1873.

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