Inhibition of Thymidylate Kinase and DNA Polymerase by the Periodate Oxidation Product of /S-D-Ribosyl-O-Methylthiopurine1

[CANCER RESEARCH 28, 661-685,April 1868]

Inhibition of Thymidylate Kinase and DNA Polymerase by the Periodate Oxidation Product of /s-D-Ribosyl-o-methylthiopurine1

A. P. Kimball,2 M. J. Wilson, J. P. Bell, and G. A. LePage

Life Sciences Research, Stanjord Research Institute, Menlo Park, Calijomia 94036

SUMMARY The Me6MPR-OP has now been found to be a potent inhibitor of the utilization of thymidine for DNA synthesis in the Ehrlich It was demonstrated that the periodate oxidation product ascites tumor. The present study was carried out to determine of /S-D-ribosyl-6-methylthiopurine (Me6MPR-OP) increased the site or sites of blockade of thymidine metabolism by the the survival times of mice bearing the Ehrlich ascites tumor. Me6MPR-OP. Me6MPR-OP at 75 mg/kg inhibited the incorporation of thymidine-3H into DNA thymine of the Ehrlich ascites tumor by MATERIALS AND METHODS 90-95%. Enzyme studies showed that Me6MPR-OP inhibited thymidine-5'-monophosphate kinase (TMP kinase) and DNA Survival Studies polymerase to produce a sequential blockade in the incorpora Female Swiss mice (Simonsen Laboratories, Gilroy, Cali tion of TMP into DNA. At molarities of Me6MPR-OP that fornia) were each implanted with 7 X IO5 to 2 X IO6 Ehrlich were inhibitory, the /3-D-ribosyl-6-methylthiopurine did not in ascites cells. Drug therapy was started 24 hours later. hibit the thymidine or TMP kinases, or significantly inhibit Me6MPR-OP was dissolved in 95% ethanol and saline was DNA polymerase. added so that the final concentration with respect to ethanol was 4%. Paper chromatography in ethanol:saturated sodium INTRODUCTION tetraborate:ammonium acetate (5 M, pH 9.5) :0.5 M versene (220:80:20:0.5) (9) showed that Me6MPR-OP (RP = 0.70) The nucleoside analog, Me6MPR3 has been shown to be a was free of MeGMPR (RF = 0.53). carcinostatic agent effective against the murine L1210 ascites In Vitro Studies with Cell Suspensions tumor resistant to 6-mercaptopurine (1). Me6MPR is con verted to nucleotide by adenosine kinase to yield a potent Incubations of Ehrlich ascites cell suspensions were carried feedback inhibitor of de novo purine biosynthesis (l, 2, 3, 5). out in 50 ml Erlenmeyer flasks, on a metabolic shaker at 37 Â°C,with an air atmosphere. Each flask contained 16 ml of Also, the mechanism of action of Me6MPR has been ascribed Krebs original Ringer phosphate medium, pH 7.4, without to feedback inhibition of the de novo purine biosynthetic path CaCl2, glucose, 5.5 X 10~3 M, thymidine-3H, 5 X IO-4 M, 4 way ( 1). Periodate oxidation of nucleosides can yield products MC/Mmole(base-labeled, Schwarz BioResearch, Inc.), and ascites that have carcinostatic properties (4). Periodate oxidation of Me6MPR gave a new compound (Me6MPR-OP) that also cells, 25 mg wet weight per ml of medium. Me6MPR-OP was had antileukemic activity.4 Me6MPR-OP, synthesized first for dissolved in the medium and used at 10~3 M. The cells were preincubated in the medium for 15 minutes at 37Â°C with testing as an immune suppressant, was found to be somewhat superior to MeGMPR as an immunosuppressant drug4 (7). Me6MPR-OP before adding the thymidine-3H. At the desig nated times, 2.0 ml aliquots were withdrawn and added to chilled tubes containing 0.22 ml of ice-cold 50% TCA. The cells 1 This work was supported by Contract PH 43-65-575 with the were extracted twice more with 4 ml portions of ice-cold 5% Cancer Chemotherapy National Service Center, National Cancer Institute, National Institutes of Health. TCA (80 volumes). The acid was removed with washes (2-4 2 Present address : Department of Biophysical Sciences, Uni ml each) of ice-cold 95% ethanol, and the tissue residue was versity of Houston, Houston, Texas. dissolved in 1.5 ml of hyamine hydroxide. Aliquots, 0.5 ml, 3 The abbreviations used are : Me6MPR, /8-D-ribosyl-6-methyl- were taken for counting in a toluene scintillation solution. thiopurine; Me6MPR-OP, periodate oxidation product of ÃŸ-o- Counting efficiency was 6%. ribosyl-6-methylthiopurine; 6-MP, 6-mereaptopurine ; TMP, TDP, and TTP, thymidine-5'-mono-, di-, triphosphate ; dUMP, deoxy- In Vivo Studies uridylate; TCA, trichloroacetic acid; Tris buffer, tris-(hydroxy- methyDaminomethane buffer; dATP, 2'-deoxyadenosine-5*triphos- Female Swiss mice bearing 6-day implants of the Ehrlich phate; dCTP, 2'-deoxycytidine-5'-triphosphate; dGTP, 2"-deoxy- ascites tumor each received Me6MPR-OP, 75 mg/kg, in saline guanosine-5'-triphosphate. containing 4% ethanol. Controls received saline containing 4% 4 J. P. Bell, G. A. LePage, and A. P. Kimball, unpublished ethanol. One hour later, the mice each received thymidine-sH, experiments. 3 /imoles, 4 MC/wnole, and metabolic utilization was allowed to Received September 18, 1967; accepted December 23, 1967. occur for 1 hour. The mice were sacrificed and the ascites cells

APRIL 1968 661

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1968 American Association for Cancer Research. A. P. Kimball, M. J. Wilson, J. P. Bell, and G. A. LePage were harvested. Cells and ascitic fluid were separated by cen- RESULTS AND DISCUSSION trifugation at 1470 X g for 2 minutes and the ascitic fluids Survival Studies were discarded. The ascites cells were extracted three times with 7-volume portions of ice-cold 5% TCA, and these washes The Me6MPR-OP effectively inhibited the growth of the were discarded. Acid was removed by washing the cells twice Ehrlich ascites tumor in Swiss mice when given once daily for with 10-ml portions of ice-cold 95% ethanol. The extraction 6 days at 20 mg/kg. The survival times of mice treated with of nucleic acids and the preparation of DNA thymine has been this dose were approximately doubled compared with controls described (6). (Table 1). An experiment was conducted to provide a more direct measure of the inhibition of tumor growth. Swiss mice Enzyme Studies Thymidinc and Thymidine-5'-monophosphate Kinases. The Table 1 preparation of enzyme extracts from the Ehrlich tumor has Me6MPR-OP survival been described (6). The incubation tubes (in duplicate) each dosageSaline20 of mice55 time(days)14.6 survivors0100 contained thymidine-2-14C, 25 mMmoles, 14.1 X IO6 cpm/Vmole (New England Nuclear), or TMP-2-14C, 10 mwnoles, 4.4 to Â±0.433.4 mg/kg/day X 6 Â± 75 36.1 X IO6 cpm/Mmole (New England Nuclear) ; Tris buffer, Saline20 910Average 14.6Â±1.931 pH 7.9, 50 Mmoles; MgCl2, 6 AmÃ³les; creatine phosphate, 5 mg/kg/day X 6Number .6Â±10550-day AmÃ³les; creatine phosphokinase, 75 Mg; ATP, 1 pinole ; and Survival study with Swiss mice bearing the Ehrlich ascites enzyme extract from Ehrlich cells equivalent to 0.07-0.10 ml of tumor subline resistant to 6-mercaptopurine. Female Swiss mice, cells in a final volume of 0.70 ml. Drugs or H20 were prein- 27-31 gm, each received i.p. implants of 7 X IO5 Ehrlich ascites cubated with the enzyme and all reagents except thymidine- cells. Treatment, which was started 1 day later, was once daily 2-I4C or TMP-2-14C for 15 minutes at 37 Â°C,then the sub for 6 consecutive treatments by i.p. injection. The survival times strates were added and incubations were continued. The re are reported as average survivals Â± average mean deviations. A actions were stopped by heating at 100Â°Cfor 2 minutes. Protein mouse surviving 50 days was computed as surviving only 50 days. was spun down and 0.025-ml aliquots of the supernatant Me6MPR-OP, periodate oxidation product of jS-D-ribosyl-6- fluids were chromatographed on Whatman No. 3 MM paper methylthiopurine. along the 50 Mgeach of TMP, TDP, and TTP as carriers. The papers were developed in isobutyric acid: water: concentra ted Table 2 NH4OH (66:33:1) for 15-16 hours (TTP, RP = 0.14; TDP, Dosage (mg/kg once cell volume inhibition RF = 0.27; TMP, RF = 0.51; thymidine, RF = 0.77). The car daily X2)Saline18S37575.0Packed permouse0.60 (Daya)99398.7973 rier spots were removed from the developed chromatograms Â±0.090.0040.0080.016% and counted at 50% efficiency in a toluene scintillation solution.

DNA Polymerase The incubation tubes (in duplicate) each contained TMP- 2-14C, 10 mMmoles, 1.7 X IO6 to 14.5 X IO6 cpm/Mmole, or Inhibition of Ehrlich ascites cell growth in female Swiss mice (30-31 gm) by various doses of the periodate oxidation product TTP-2-14C, 10 mamÃ³les, 3.3 X IO6 to 6.6 X IO6 cpm/Mmole; of /3-D-ribosyl-6-methylthiopurine. Each mouse received i.p. im Tris buffer, pH 7.9, 50 jumÃ³les;MgCl2, 50 /umoles; creatine plants of 2 X IO6 Ehrlich ascites cells. Drug therapy was started phosphate, 5 Mmoles; creatine phosphokinase, 75 Mg; ATP, 24 hours later, once daily for 2 doses. Cells were quantitatively 1 Â«noie; denatured DNA, 100 Mg; dGTP, dATP, and dCTP, removed on Day 5. Cells from drug-treated mice (5 mice/group) 15 mMmoleseach; and enzyme extract from Ehrlich cells equiv were pooled and centrifuged in accurately calibrated tubes for 2 alent to 0.07 to 0.100 ml cells in a final volume of 0.70 ml. minutes at 1470 X g. Drug or H20 was preincubated with enzyme and all reagents except TMP-2-14C or TTP-2-Â«C, dGTP, dATP, and dCTP Table 3 for 15 minutes at 37Â°C.Then the substrates were added and incubation continued. The reaction mixtures were inactivated by the addition of 2.0 ml of ice-cold 5% TCA. The tubes were DrugSalineMe6MPR-OP, (cpm/mlcells)49,3002,37029,0602,992DNAthymine(cpm/^imole)8,6004407,703661 placed in an ice-bath for 10 minutes, then centrifuged at mg/kgSalineMe6MPR-OP,75 1470 X g for 3 minutes. The precipitates were each resuspended in 1.5 ml of ice-cold 5% TCA and left at 0Â°Cfor 10 minutes; 75 mg/kgDNA then they were centrifuged for 3 minutes. This washing pro In Vivo inhibition of thymidine-3H incorporation into DNA cedure was repeated once more. The precipitates were each dissolved in 0.40 ml of concentrated formic acid; 0.10-ml ali thymine by the periodate oxidation product of ÃŸ-D-ribosyl-6- methylthiopurine (Me6MPR-OP). Female Swiss mice bearing quots were plated and counted in an end-window gas flow coun 6-day implants of the Ehrlich ascites tumor each received saline ter at 20% efficiency. This method has been described more or Me6MPR-OP, 75 mg/kg. Thymidine-3H, 3 Mmoles, 4 MC/Mmole, fully elsewhere (10). Protein was determined by the method was given 1 hour later and metabolic utilization allowed to occur of Lowry et cd. (8). No mercaptoethanol was added to the for 1 hour. Each value is the average of separate determination incubation mixtures. on 3 mice.

662 CANCER RESEARCH VOL. 28

Table 4 were each implanted with 2 X IO8 Ehrlich ascites cells and Me6MPR-OP treatment was started 24 hours later. Me6MPR- incorporation OP was given daily i.p. for 2 doses at 18.8, 37.5, or 75.0 mg/kg. Incubation cells)Controls04,6409,44012,64012,06015,120+(cpm/gm time(minutes)01020304050Thymidine-SH 10-3HMe6MPR-OP02,0001,7603,9202,5603,600%inhibition6683087Â»76The tumor cells were quantitatively removed from each mouse on Day 5 and the packed cell volumes were measured. There was 97-99% inhibition of tumor cell growth by these dosages of Me6MPR-OP (Table 2) as measured by packed cell volumes. Studies I'MVivo

The Me6MPR-OP at 75 mg/kg gave a 90-95% inhibition of the incorporation of thymidine-3H into the DNA thymine of In Vitro inhibition of thymidine-3H incorporation into Ehrlich the Ehrlich ascites tumor in vivo (Table 3). This blockade ascites cell suspensions by the periodate oxidation product of could result from the inhibition of thymidine kinase, of thymid- /Ã®-D-ribosyl-6-methylthiopurine (Me6MPR-OP). The incubation mixture contained in Kreb's original Ringer phosphate medium ylate kinase, or of DNA polymerase. (with no CaCl2), pH 7.4, the following: glucose, 5.5 X 10-* M; thymidine-3H, 5 X 10â€”4M, 4 /ac//amole; 25 mg wet weight of cells Studies in Vitro per ml of medium; in a final volume of 16.0 ml in 50-ml Ehrlen- Ehrlich Ascites Cell Suspensions. The Me6MPR-OP at KH meyer flasks. Incubations were carried out in air in a metabolic M inhibited the in vitro incorporation of thymidine-3H into shaker at 37Â°C.Me6MPR-OP (10-3 M), dissolved in medium, was a cold acid-insoluble product of whole Ehrlich cell preparations preincubated with cells in medium for 15 minutes at 37Â°Cbefore (presumably DNA), by about 60% (Table 4), during the linear adding the thymidine-3H. At the designated times, 2.0 ml aliquote portion of the incorporation. This correlated with the in vivo were removed for analysis. inhibition of incorporation of thymidine-3H into the DNA thy mine of this ascites tumor. Thymidine Kinase. The thymidine kinase of the Ehrlich Table 5 ascites cell-free extract gave a linear conversion of thymidine to TMP for at least 15 minutes. In addition, the conversion Incubation /Â¿moltControl04.799.8012.10sformed+TMP formed mamÃ³lesTTP + TDP time 1.4 X 10-Â«M 1.4 X 10-* M of TMP (formed from thymidine) to TTP + TDP was linear (minutes)051015m Me6MPR-OP04.588.8913.00Control00.400.811.67+Me6MPR-OP00.120.500.90for 15 minutes. Table 5 gives the results of a study of the effects of Me6MPR-OP at 1.4 X 1(H M on the conversion of thy midine to TMP + TDP + TTP. There was no effect on the formation of TMP from thymidine. However, there was in hibition of the conversion of TMP to TDP + TTP. The effects Effect of Me6MPR-OP on thymidine kinase. Each tube con of MeGMPR and Me6MPR-OP on thymidine kinase were com tained thymidine-2-14C, 25 mamÃ³les, 14.1 X IO6 cpm//umole; pared. The results showed (Table 6) that, at 2.8 X 10~4 M, Tris buffer, pH 7.9, 50 AmÃ³les; MgCl2, 6 AmÃ³les; ATP, l /Â¿mole; neither drug inhibited thymidine kinase. The conversion of creatine phosphate, 5 jumÃ³les; creatine phosphokinase, 75 /ag; TMP (formed from thymidine) to TDP + TTP was inhibited Ehrlich cell free extract, 2.00 mg protein, in a final volume of 70% by Me6MPR-OP, while Me6MPR was without effect. 0.70 ml. Me6MPR-OP (1.4 X 10-* M) or H2O was preincubated Thymidine-5'-monophosphate Kinase: Ehrlich Ascites Tu with the enzyme and all reagents except thymidine-2-i4C for 15 minutes at 37Â°C then the thymidine-2-14C was added and mor. The effects of Me6MPR and Me6MPR-OP on thymidine- incubations continued. TMP, TDP, TTP, thymidine-5'-mono-, 5'-monophosphate kinase at 1.4 X 10~4 M and 2.8 X 10~4 M di-, triphosphate ; Me6MPR-OP, the periodate oxidation product are shown by data presented in Table 7. At 1.4 X IO"4 M, of jS-D-ribosyl-6-methylthiopurine. Me6MPR had no effect on TMP kinase but Me6MPR-OP de-

TableÃ³Drug formed 4- TDPformed(m/imoles)150 M)Control(2.8 X 10-* (m/imoles)11.85

Me6MPR 1250 2.06 Me6MPR-OPTMP 11.70inhibitionNone 2TTP 057inhibitionstimulation77 Effect of Me6MPR and Me6MPR-OP on thymidine kinase. Each tube contained thymidine- 2-14C, 25 mamÃ³les, 14.1 X 10Â«cpm//imole; Tris buffer, pH 7.9, 50 Â¿uÃ±Ã³les;MgCl2,6 /Â¿moles; ATP, 1 /amolÃ©,creatine phosphate, 5 /amÃ³les; creatine phosphokinase, 75 /ag; Ehrlich cell free extract, 1.44 mg protein, in a final volume of 0.70 ml. Me6MPR, Me6MPR-OP, or H2O was preincubated with enzyme and all reagents except thymidine-2-14C for 15 minutes at 37Â°C, then the thymidine-2-14C was added and incubations continued for 15 minutes. Me6MPR, /3-D-ribosyl-6-methylthiopurine; Me6MPR-OP, the periodate oxidation product of Me6MPR; TMP, TDP, TTP, thymidine-5'-mono-, di-, triphosphate.

APRIL 1968 663

Table? 9DrugControlMe6MPR-OPMe6MPR-OPMe6MPR-OPControlMe6MPR-OPMe6MPRMolarity1.4Table m/umoles TTP-2-MC TMP converted incorporated0.630.480.41OJ260.580540.51%inhibition2435595912 Drug Molarity to TTP + TDP inhibition Me6MPRMe6MPRMe6MPRMe6MPR-OPMe6MPR-OPMe6MPR-OPNone1.4X X10-*2.1 10-*M2.8 X10-*2.8 X 10-*MNone1.4X X10-*2.8

10-*M2.8 Â»2.8X 10â€” X 10-* M1.652.001.791.580.%0.48stimulationstimulation3969 X 10-*m/tmoles Effects of Me6MPR and Me6MPR-OP on thymidine-5'-mono- Effects of various molarities of Me6MPR-OP on DNA poly- phosphate kinase. Each tube contained TMP-2-14C, 10 mamÃ³les, merase using TTP-2-14C as precursor. Each tube contained TTP- 36.1 X IO6 cpm/Aimole; Tris buffer, pH 7.9, 50 /Â¿moles; MgCl2, 2-14C, 10 mamÃ³les; 3.3 X IO8 cpm/^mole; Tris buffer, pH 7.9, 50 6 AmÃ³les; creatine phosphate, 5 /limÃ³les; creatine phosphokinase, /umoles: MgCl.â€ž6 jumÃ³les; creatine phosphate, 5 /Â¿moles;creatine 75 Mg; ATP, l Mmole; Ehrlich cell-free extract, 2.00 mg protein, in phosphokinase, 75 Mg; ATP, 1 /amolÃ©;denatured DNA, 100 Mg; a final volume of 0.70 ml. Drugs or H2O were preincubated with dGTP, dATP, dCTP, 20 m/tmoles each ; and Ehrlich enzyme ex enzyme and all reagents except TMP-2-14C for 15 minutes at tract, 1.44 mg protein, in a final volume of 0.70 ml. Me6MPR-OP, 37Â°C.Then the TMP-2-14C was added and incubations continued MeoMPR, or H.,O was preincubated with enzyme and all re for 15 minutes. For abbreviations see Table 6. agents except TTP-2-Â»4C, dGTP, dATP, and dCTP for 15 minutes at 37Â°C.Then these were added and incubations continued for Table 8 15 minutes. For abbreviations see Tables 6 and 8.

TMP-2-Â»C incorporated+ (presumably DNA), catalyzed by enzyme extracts of the Ehr Radioactive time(minutes)01530450153045mumoles1.4 X 10-Â«M ControlMe6MPR-OP0 lich ascites tumor, proceeded linearly for at least 45 minutes. precursorTMP-2-i*CTMP-2-Â«CTMP-2-WCTMP-2-"CTTP-2-i*CTTP-2-i*CTTP-2-i*CTTP-2-i*CIncubationMe6MPR-OP at 1.4 X 10~4 M depressed the incorporation of 00.66 TMP-2-14C into DNA about 60% (Table 8). However, the 023130 incorporation of TTP-2-14C into DNA was only inhibited 0.471.95 about 15% by Me6MPR-OP at 1.4 X IO-4 M in this study. 058miimoles These data are consistent with the interpretation that there is TTP-2-HCincorporated+ a block in the formation of TTP from TMP. The effect of higher molarities of Me6MPR-OP on the "DNA polymerase" 1.4 X 10-Â«M ControlMe6MPR-OP0 using TTP-2-14C as the radioactive precursor is shown in 00.74 Table 9. Also, the Me6MPR-OP and Me6MPR were compared 0.621.08 at the highest molarity of Me6MPR-OP used (2.8 X 10-* M). 0.931.18 At 1.4 X 10~4 M, Me6MPR-OP gave minimal inhibitory effects 0.98%inhibition656470161412on the "DNA polymerase"; but with increasing concentration, Effects of Me6MPR-OP on the incorporation of TMP-2-14C the inhibition increased. The "DNA polymerase" activity was and TTP-2-14C into an acid-insoluble product ("DNA poly depressed about 60% at 2.8 X 10~4 M Me6MPR-OP. Inhibi merase") of an Ehrlich cell-free system. Each tube contained tion produced by MeoMPR at 2.8 X 10~4 M was not significant TMP-2-i*C, 10 mamÃ³les, 1.7 X IO8 cpm/Vmole; or TTP-2-i4C, (12%). 10 mamÃ³les, 6.6 X IO6 cpm//umole; Tris buffer, pH 7.9, 50 Â¡amÃ³les; Periodate oxidation of MeoMPR yielded a derivative with MgCl2, 6 /umoles; creatine phosphate, 5 /amÃ³les; creatine phospho activity not found with the parent compound. Me6MPR-OP kinase, 75 Mg; ATP, 1 Â¿uÃ±Ã³le;denatured DNA, 100 Mg; dGTP, inhibited thymidylate kinase and DNA polymerase. Thus a dATP, and dCTP, each 15 Ã±amÃ³les; and Ehrlich cell free ex tract, 228 mg protein for TMP-2-14C incorporation or 2.72 mg sequential block was produced in the metabolism of thymine protein for TTP-2-14C incorporation; in a final volume of 0.70 ml. for DNA synthesis, the conversion of TMP to TTP and the Me6MPR-OP, 1.4 X 10~4 M, or H.,O was preincubated with en incorporation of TTP into DNA. Me6MPR is converted to zyme and all reagents except TMP"-2-i*C or TTP-2-14C, dATP, the active antitumor agent, Me6MPR-5'-phosphate (1); how dCTP, and dGTP for 15 minutes at 37Â°C.Then the TMP-2-l4C ever, the active form of the Me6MPR-OP has, as yet, not been or TTP-2-14, dATP, dCTP, and dGTP were added and incuba determined. The Me6MPR-OP is easily prepared from inosine tions continued. dATP, 2'-deoxyadenosine-5'-triphosphate; dCTP, in good yield. In light of the inhibitions produced on TMP 2'-deoxycytidine triphosphate ; dGTP, ^-deoxyguanosine triphos- kinase and DNA polymerase, and the activity in experimental phate; for other abbreviations see Table 6. tumors, MeoMPR-OP appears worthy of further study as a chemotherapeutic agent. pressed this phosphorylation about 40%. At 2.8 X 10~* M Me6MPR was still without effect on TMP kinase but a 70% inhibition was obtained with the Me6MPR-OP at 2.8 X ACKNOWLEDGMENTS IO-4 M. We wish to thank Dr. Leon Goodman and Mr. 0. Crews for "DNA Polymerase" of the Ehrlich Ascites Tumor. The in supplies of the /3-D-ribosyl-6-methylthiopurine and its periodate corporation of TMP-2-14C into a cold acid-insoluble product oxidation product.

664 CANCER RESEARCH VOL. 28

REFERENCES 5. Henderson, J. F. Feedback Inhibition of Purine Biosynthesis in Ascites Tumor Cells by Purine Analogs. Biochem. Phar- 1. Bennett, L. L., Jr., Brockman, R. W., Schnebli, H. P., Chumley, macol., le: 551-556, 1963. S., Dixon, G. J., Schabe!, F. M., Dulmadge, E. A., Skipper, 6. Kimball, A. P., Bowman, B., Bush, P. S., Herriot, J., and H. E., Montgomery, J. A., and Thomas, J. H. Activity and LePage, G. A. Inhibitory Effects of the Arabinosides of 6- Mechanism of Action of 6-Methylthiopurine Ribonucleoside Mercaptopurine and Cytosine on Purine and Pyrimidine in Cancer Cells Resistant to 6-Mercaptopurine. Nature, SOS: Metabolism. Cancer Res., 36: 1337-1343, 1966. 1276-1279, 1965. 7. Kimball, A. P., Herriot, S. J., and Allinson, P. S. Studies on 2. Bennett, L. L., Jr., Schnebli, H. P., Vail, M. H., Allan, P. W, Immune Suppressive Drugs. Proc. Soc. Exptl. Biol. Med., and Montgomery, J. A. Furine Ribonucleoside Kinase Activity 126: 181-184, 1967. and Resistance to some Analogs of Adenosine. Mol. Phar- 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, macol., 2: 432-443, 1966. R. J. Protein Measurement with the Folin Phenol Reagent. 3. Caldwell, I. C., Henderson, J. F., and Paterson, A. R. P. Re J. Biol. Chem., 19S: 265-275, 1951. sistance to Purine Ribonucleoside Analogs in an Ascites Tumor. 9. Reichard, P. Chromatographie Separations of Cytosine Con Can. J. Biochem., 45: 735-744, 1967. taining Compounds. Acta Chem. Scand., 12: 2048, 1958. 4. Dvonch, W., Fletcher, H., Gregory, F. J., Healy, E. H., War 10. York, J. L., and LePage, G. A. A Proposed Mechanism for the ren, G. H., and Alburn, H. E. Antitumor Activity of Periodate- Action of 9-/8-D-Arabinofuranosyladenine as an Inhibitor of oxidation Products of Carbohydrates and Their Derivatives. the Growth of some Ascites Cells. Can. J. Biochem., 44-' 19- Cancer Res., 26: 2386-2389, 1966. 26, 1966.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1968 American Association for Cancer Research. Inhibition of Thymidylate Kinase and DNA Polymerase by the Periodate Oxidation Product of β-d-Ribosyl-6-methylthiopurine

A. P. Kimball, M. J. Wilson, J. P. Bell, et al.

Cancer Res 1968;28:661-665.

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