Association of Decreased Uridine and Deoxycytidine Kinase With

Home , Uridine kinase

(CANCER RESEARCH 30, 2180-2186, August 1970] Association of Decreased Uridine and Deoxycytidine Kinase with Enhanced RNA and DNA Polymerase in Mouse Leukemic Cells Resistant to 5-Azacytidine and 5-Aza-2'-deoxycytidine

V Y J. Vesely, A. CihÃ¡k, and F. Sorm Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia

and the liver proteosynthesis presumably by abolishing the SUMMARY heavy polyribosomes (6, 20). 5-Aza-2'-deoxycytidine inhibits Resistance of mouse leukemic cells to 5-azacytidine and the incorporation of 2'-deoxycytidine into mouse leukemia 5-aza-2'-deoxycytidine is associated with the partial loss of cells in vivo and in vitro (35). 5-Azacytidine, but not its deoxycytidine kinase; at the same time, in 5-azacytidine- 2'-deoxy derivative, is an effective inhibitor of the hormonal resistant leukemic cells uridine kinase is decreased. Further and dietary induction of different liver enzymes both in more, the 5-azacytidine-resistant subline exhibits increased normal and regenerating rat liver (6, 7). DNA-dependent RNA and DNA polymerase activities, while Previously, we have observed that the development of its 5-aza-2-deoxycytidine-resistant counterpart shows an resistance of mouse leukemic cells to 5-azacytidine is enhanced RNA polymerase only. The deletion of both associated with the stepwise decline of uridine kinase activity kinases could explain cross-resistance of 5-azacytidine-resis (33). In analogy, resistance of leukemic cells to 5-aza-2'- tant leukemic cells to 5-aza-2-deoxycytidine. 5-Aza-2- deoxycytidine results in a diminished activity of deoxy deoxycytidine-resistant leukemic cells with unchanged cytidine kinase (35). These findings suggested the specific uridine kinase retain their sensitivity toward 5-azacytidine. decrease of uridine or deoxycytidine kinase activity in The significance of enhanced RNA and DNA polymerase 5-azacytidine- and 5-aza-2'-deoxycytidine-resistant sublines, activities in resistant cells is discussed. respectively. However, the results of chemotherapeutic trials on cross-resistance and sensitivity of mouse leukemic cells INTRODUCTION resistant toward both analogs (34) were not entirely compatible with such an assumption, and for this reason we In the studies on resistance to pyrirnidine (13, 25) and have undertaken to study in the mutant sublines the purine (2, 4) analogs carried out on resistant mammalian activities of DNA-dependent RNA and DNA polymerases cells, support was obtained for the hypothesis that which take part at later stages of cell nucleic acid synthesis. nucleotide formation was essential for biological activity of these compounds; resistance to such analogs was associated MATERIALS AND METHODS with a complete or partial deletion of the enzymes Reagents. 5-Azacytidine and 5-aza-2'-deoxycytidine were responsible for their anabolic conversion to corresponding 5'-phosphates that could be anabolized to 5'-triphosphates synthesized by Dr. A. Pi'skala and Dr. J. Piimi of this Institute. Ribonucleoside and deoxyribonucleoside 5'-triphos- and incorporated into nucleic acids (10, 14). These alterations, induced by exogenous substances such as nucleic phates, pyruvate kinase, 2-phosphoenolpyruvate, ribonuclease acid analogs, lead to different biochemical modifications by 5 times crystallized (bovine pancreas), deoxyribonuclease I means of which the cell circumvents the primary lesion (bovine pancreas), and cytosine l-ÃŸ-D-arabinofuranoside were situated at a locus of its metabolic pathway. Similar obtained from Calbiochem (Luzerne, Switzerland). Actino- modifications would be interesting as model systems, and mycin D was kindly supplied by Merck, Sharp and they would also be of practical value since they could be Dohme (Rahway, N. J.). TTP-2-'4C (25 mCi/mmole) was made use of as targets for chemotherapy in malignant cells. purchased from New England Nuclear Corp. (Boston, Mass.). 5-Azacytidine and 5-aza-2'-deoxycytidine (27, 28) are UTP-U-14C1(175 mCi/mmole), ATP-U-14C (75 mCi/mmole), 2'-deoxycytidine-2-14C (25 mCi/mmole, and uridine-U-I4C highly active inhibitors of the growth of leukemic cells in AKR mice (31, 33). In mammalian tissue, 5-azacytidine is (200 mCi/mmole) were prepared at the Institute for phosphorylated and incorporated into different types of Research, Production and Use of Radioisotopes in Prague. nucleic acids (15); it depresses the synthesis of ribonucleic Animals and the Development of Resistance. For the acids de novo by blocking orotidylic acid decarboxylase (34)

'UTP-U-14C, ATP-U-14C, and uridine-U-14C refer to the uniformly Received February 2, 1970; accepted April 16, 1970. labeled compounds.

2180 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1970 American Association for Cancer Research. Mouse Leukemic Cells Resistant to 5-Azapyrimidines experiments, inbred AKR mice were used. Resistance adding 3 ml of ice-cold 10% trichloroacetic acid. After centrifugation (4Â°), the sediments were washed 3 times with development has been described elsewhere (33). Control leukemic mice AKR/s were in transplant generations 240 to cold 5% trichloroacetic acid. After resuspension, the samples 250, leukemic mice resistant to 5-azacytidine (AKR/r-AzCR) were filtered onto Millipore filters and washed with 5 were in transplant generations 56 to 69, and leukemic mice additional portions of ice-cold acid. The radioactivity was resistant to 5-aza-2'-deoxycytidine (AKR/r-AzCdR) were in counted in a Frieseke-Hoepfner gas flow counter. The transplant generations 132 to 144. enzyme activity is expressed in /j/amoles of incorporated Incorporation of 2'-Deoxycytidine into Leukemic Cells in nucleoside 5'-phosphate/100 /ug of DNA. Vitro. Cell suspensions from mouse leukemic livers were DNA-dependent DNA Polymerase. Livers were excised from prepared by gently homogenizing the tissue with a hand- leukemic mice and, after mincing, homogenization was operated Teflon pestle in Eagle's minimal medium enriched carried out with a hand-operated glass pestle in a glass with tryptose phosphate (10%), calf serum (8%), and homogenizer in 5 volumes of ice-cold 0.01 M Tris-HCl (pH embryonal bovine serum (2%). Cell suspension was filtered 7.5) with 0.15 M KC1. Homogenate was spun at 105,000 X g, 2 hr at 2Â°.Supernatant fraction was used as a source of through 2 layers of gauze, and cells were counted in a hemocytometer. Incubation was carried out as 37Â° in a DNA polymerase activity, which was determined in the assay Dubnoff shaking incubator in a total volume of 1 ml in the system as given by Keir (17). The reaction mixture (total presence of 0.1 mM 2'-deoxycytidine-2-14C. At given time volume of 0.3 ml) was incubated at 37Â°for 30 min and intervals, samples were immersed in ice-cold water and contained Tris-HCl (pH 7.5), 20 mM; potassium chloride, centrifuged, and the sediments were extracted with ice-cold 10.0 mM; Mg2+ ions, 4.0 mM; EDTA; 0.30 mM; mercapto ethanol, 6.0 mM; unlabeled deoxyribonucleoside 5'-triphos- 5% trichloroacetic acid. Supernatant fractions were dis carded, and the incorporation of the precursor into nucleic phate, 0.2 mM; TTP-2-14C, 0.02 mM; 5 fig of denatured acids was determined as described (33). Escherichia coli DNA; and supernatant fraction correspond Uridine and Deoxycytidine Ritinse Assay. Incubation was ing generally to 250 tig of protein. Further steps were carried out at 37Â°in the following medium in a total volume similar to those as described for RNA polymerase. Protein of 1 ml: uridine-U-14C or 2-deoxycytidine-2-14C, 0.015 mM; was determined according to Lowry et al. (22). The enzyme Tris-HCl (pH 8.1), 60 mM; ATP with equimolar Mg2+ ions, activity was expressed in Â¿/Â¿umolesofincorporated TMP/250 1.5 mM; and partially purified uridine kinase (24) corre /ug of protein in a 30-min period of incubation. sponding to 50 Mg of protein or supernatant fraction from leukemic cells (35). The reaction was terminated by heating RESULTS the reaction mixture at 100Â°for 1 min. Aliquots (0.1 ml) were chromatographed with Whatman No. 1 paper in a Metabolic Alterations of Pyrimidine Pathway in Leukemic solvent system composed of isobutyric acid:ammonium Mouse Cells. Uridine kinase in the 5-azacytidine-resistant line hydroxide:water (66:1.5:33). Chromatographie spots were is decreased about 74%, and in 5-aza-2'-deoxycytidine- cut out, and their radioactivity was counted in a Packard resistant cells it is decreased only 10%; however, deoxy Tri-Carb scintillation spectrometer. Uridine or deoxycytidine cytidine kinase activity in both resistant sublines is depressed kinase activities are expressed as AmÃ³les of UMP or dCMP nearly equally, i.e., 71 to 75% (Table 1). It is generally formed per 100/ug of protein in a 10-min period. supposed that the kinase depression in resistant cells is DNA-dependent RNA Polymerase. The assay of DNA- specific for a given nucleoside or deoxynucleoside anti- dependent RNA polymerase was carried out according to the metabolite (10). Comparable deletion of deoxycytidine modified procedure of Cunningham and Steiner (9). kinase activity in 5-aza-2'-deoxycytidine- and 5-azacytidine- Leukemic spleens were homogenized in a glass homogenizer resistant cells was rather unexpected. Moreover, we have observed that, whereas the uptake of 2'-deoxycytidine into with a Teflon pestle driven by a motor at 800 rpm in 6 volumes of ice-cold 0.25 M sucrose with 1 mM Mg2* ions. 5-aza-2'-deoxycytidine-resistant mutants is decreased, its The sediment was washed twice in the cold in the same incorporation into 5-azacytidine-resistant leukemic cells is medium and resuspended in 0.25 M sucrose. The nuclei were within normal limits (Chart 1). In the cell-free system, counted in a Biirker hemocytometer and their deoxy- deoxycytidine kinase from mouse leukemia cells has a lower ribonucleic acid content was determined according to Burton affinity for 5-aza-2'-deoxycytidine than for 2'-deoxycytidine; (3). After centrifugation, the sediment was treated with 15 the analog fails to inhibit the enzyme activity even at 10 mM Tris-HCl (pH 8.1) containing 0.5 mM EDTA and 1 mM times the concentration of the natural substrate (Chart 2). mercaptoethanol. A 0.1-ml portion of the nuclear enzyme, The same goes for the uridine kinase activity in the presence corresponding usually to IO7 nuclei or to 65 jug of DNA, of 5-azacytidine. was added to the reaction mixture (total volume of 0.5 ml) DNA-dependent RNA Polymerase. RNA polymerase in containing Tris-HCl (pH 8.1), 100 mM; Mn2+ ions, 5 mM; mouse leukemic spleens infected with Friend murine mercaptoethanol, 60 mM; 2-phosphoenolpyruvate, 2 mM; leukemia virus has been studied by Lin and Rich (21). The pyruvate kinase, 5 jug; sodium fluoride, 10 mM; ammonium enzyme used in the present experiments has been obtained sulfate, 200 mM; unlabeled ribonucleoside 5'-triphosphates, from leukemic spleens of AKR mice. For the isolation of the 0.6 mM; and ATP-U-I4C or UTP-U-14C, 0.06 mM. nuclear enzyme, the modification of the method reported by Incubation was carried out in a Dubnoff shaking incubator Cunningham and Steiner (9) for liver enzyme was used. at 37Â° for 10 min, and the reaction was terminated by During the course of our study, a similar procedure was

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Table 1 90 Uridine and deoxycytidine kinase of mouse leukemia cells resistant to 5-azacytidine and 5-aza-2'-deoxycyfidine Incubation was for 10 min at 37Â°in a total volume of 0.2 ml with partially purified undine kinase or deoxycytidine kinase, respectively. The experiment was repeated 3 times with enzyme preparations from 60 6 I different transplant generations with essentially the same results. Enzyme activity is expressed in ju/umoles of nucleoside 5 -phos phate/ 100 MS of protein. i 30 kinaseLeukemic Uridine kinaseMMHioles36.4 with 5T&ZO-2-deoxycytidine cellsAKR/s

I AKR/r-AzCR 120.2 26 10.7 2925 3 6 10 20 30 AKR/r-AzCdRM/nmoles468.4 421.3%100 90Deoxycytidine9.2%100 Time .min Time imin

Chart 2. Uridine and deoxycytidine kinase in the presence of 5-azacytidine and 5-aza-2'-deoxycytidine. Incubation at 37 in a total volume of I ml in 60 mM Tris-HCl (pH 8.1). Uridine-U-14C or

O AKR/s 2'-deoxycytidine-2-14C, 0.015 mM (0.1 /uCi); ATP with equimolar 0 AKR/r-AzCR Mg2+ ions, 1.5 mM; and 0.2 ml of supernatant fraction from mouse Â® AKR/r-AzCdR leukemic cells (2 mg of protein). The enzyme activity is expressed as the percentage of 5'-phosphates formed of the total radioactivity at given time intervals. 5-Azacytidine or 5-aza-2^deoxycytidint (0.15 mM).

O AKR/s â€¢AKR/r-AzCR 40 Â® AKR/r-AzCdR

Time,min Chart 1. Uptake of 2"-deoxycytidine by mouse leukemic cells 20 AKR/s, AKR/r-AzCR, and AKR/r-AzCdR in vitro. Incorporation of 0.1 mM 2-deoxycytidine-2-14C was carried out at 37 in a total volume of 1 ml with 2 X IO7 cells suspended in Eagle's medium.

published for lymphoid tissue RNA polymerase (16). The 12 enzyme activity required all 4 ribonucleoside 5'-triphosphates Time,min and Mn2+ ions and was RNase and actinomycin D sensitive. Chart 3. Time course of DNA-dependent RNA polymerase from The activities of RNA polymerase from leukemic mice sensitive and resistant to 5-azacytidine and 5-aza-2'-deoxy- mouse leukemic cells AKR/s, AKR/r-AzCR, and AKR/r-AzCdR. Incubation at 37 in a total volume of 0.5 ml in 100 mM Tris-HCl cytidine are given in Table 2. In both resistant sublines, (pH 8.1) with 0.1 ml of the nuclear enzyme corresponding to 100 RNA polymerase is enhanced 56 to 64%. This increase is of DNA. Enzyme activity is expressed in MMmoles of UMP- C statistically significant and is shown also in Chart 3, where incorporated during the incubation. the time course of the reaction in different types of cell lines is presented. The incorporation of UMP is maximal during the first 7 min of the incubation period. The in Chart 4 with essentially same results. The activity of DNA decreased activity after this time is presumably due to the polymerase in 5-azacytidine-resistant cells has further been degradation of newly formed RNA. tested in a double reciprocal plot of reaction rate against DNA-dependent DNA Polymerase. As a source of DNA DNA primer (17). These plots, expressed according to the polymerase, high-speed supernatant fraction from leukemic method of Lineweaver-Burk, do not indicate any change in livers was used. DNA polymerase activity is significantly KM constants of the respective enzyme activities, while increased in the 5-azacytidine-resistant leukemic cell, while demonstrating an increased velocity of DNA polymerase the enhancement in the mutant subline resistant to 5-aza-2'- activity from 5-azacytidine-resistant subline (Chart 5). Sensitivity of 5-Aza-2'-deoxytidine-resistant Leukemic Cells deoxycytidine has not been found to be statistically significant (Table 3). The time course of the reaction is given to 5-Azacytidine and Cross-resistance of Both Mutant Suh-

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Table 2 Changes of DNA-dependent RNA polymerase in spleens ofleukemic AKR mice Incubation was for 10 min at 37Â°ina total volume of 0.5 ml. Enzyme activity is expressed in MMmolesof AMP-14C incorporated/100 Mgof DNA (AKR/s = 100%).

RNA polymerase (MMmoles/100MgDNA)

Experiment No. AKR/s AKR/r-AzCR AKR/r-AzCdR 1222.2 Â±1.2 Â±2.1" 24 .4 Â±1.3(100)(100)34.8 38.3 Â±2.3"(157) (156)34.5+4.1"39.9 Â±4.5"(156)(164) ap<0.05.

Table 3 Changes of DNA-dependent DNA polymerase in livers ofleukemic AKR mice Incubation was for 30 min at 37 in a total volume of 0.3 ml. Enzyme activity is expressed in MamÃ³lesof TMP-14C incorporated/250 Mgof protein in a reaction mixture (AKR/s = 100%).

DNA polymerase (MamÃ³les/250Mgprotein)

Experiment No. AKR/s AKR/r-AzCR AKR/r-AzCdR 7.72 + 1.13 (100) 14.70 Â±1.27Â° (190) 9.77 Â±1.60* (127) 7.90+0.14 (100) 13.45 Â±0.32" (170) 9.20 + 1.40* (117)

Â«p<0.02. ftp>0.05.

O AKR/s 20 0.8 â€¢AKR/r-AzCR

10 0.4 O AKR/S â€¢AKR/r-AzCR Â® AKR/r-AlCdR

1 20 Â¿0 60 O 6 12 18 Time.min I/s2Â« IO2 Chart 4. Time course of DNA-dependent DNA polymerase from Chart 5. DNA-dependent DNA polymerase of mouse leukemic cells mouse leukemic cells AKR/s, AKR/r-AzCR, and AKR/r-AzCdR. AKR/s and AKR/r-AzCR in a double reciprocal plot of reaction rate Incubation at 37Â°in a total volume of 0.3 ml in 20 mM Tris-HCl (pH against DNA primer. For experimental conditions, see Chart 4, 7.5) with 5 Mg of denatured E. coli DNA and soluble fraction of incubation period 30 min. enzyme corresponding to 250 Mg of protein. Enzyme activity is expressed in MMmolesof TMP incorporated during the incubation.

40% but, as expected, does not possess any antileukemic lines to Cytosine Arabinoside. It has been shown that action against both mutant sublines. Furthermore, Table 4 cytosine arabinoside (8) inhibits the growth of murine indicates that 5-azacytidine is effective in suppressing the growth of 5-aza-2'-deoxycytidine-resistant leukemic cells, leukemias (11). A requisite for the action of this drug is prior conversion to cytosine arabinoside 5'-phosphate (5), for since their uridine kinase functions normally (Table 1). which deoxycytidine kinase is responsible. The partial loss of this enzyme in 5-azacytidine- and 5-aza-2'-deoxycytidine- DISCUSSION resistant leukemic cells led us to examine the effect of cytosine arabinoside on both resistant cell types (Table 4). Previously, we have demonstrated that 5-azacytidine- The analog prolongs the life-span of AKR leukemic mice resistant leukemic cells are cross-resistant to 5-aza-2'-

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Table 4 Difference in the sensitivity ofleukemic mice resistance to 5-azacytidine and 5-aza-2'-deoxycytidine toward 5-azacytidine and cy tosine l-ÃŸ-D-arabinofuranoside Administration i.p. of 5-azacytidine (3 mg/kg) and cytosine l-ÃŸ-D-arabinofuranoside(50 mg/kg) (single dose) on 4 consecutive days was started 24 hr after s.c. inoculation of leukemic cells: 10-15 mice (22 g Â±2.3) in each group. (Survival of control AKR/s = 100%.)

survivalAdministrationControlAKR/s survivalDays survivalDays Â±S.E.7.0 Â±S.E.7.510.5 Â±S.E.7. Â±0 2 Â±0.1 5-Azacytidine 13.5 Â±1.5" 193 7.4 Â±0.6 99 12.2 Â±0.6" 170 Cytosine arabinosideDays 9.8 Â±0.7"%100140AKR/r-AzCR7.2 Â±0%100 96AKR/r-AzCdR7.3 Â±0.1%100102 "p<0.01. deoxycytidine (34). This phenomenon was difficult to Resistance to 5-Azocytidine : explain in biochemical terms, as the incorporation of 2'-deoxycytidine into the cells was not altered (Chart 1), and poipnerase consequently it was assumed that their deoxycytidine kinase Uridine UMP UTP â€¢â€¢} RNA activity was not changed. In the present experiments, however, we have been able to show that deoxycytidine 21-Deoxycytidine kinase in 5-azacytidine-resistant cells is impaired to the same dCMP dCTP DMA extent as in their 5-aza-2'-deoxycytidine-resistant counter parts (Table 1). It has been found, furthermore, that 5-aza- Chart 6. Metabolic alterations in 5-azacytidine-resistant leukemic 2-deoxycytidine at 10 times the concentration of 2-deoxy- mice cells. cytidine does not inhibit the phosphorylation of the natural substrate by the cell-free extract prepared from leukemic apparently due to a mechanism similar to that observed in cells (Chart 2). The low affinity of deoxycytidine kinase for the case of DNA polymerase; its enhancement, associated 5-aza-2Kleoxycytidine may explain the inconsistency between with the decreased activity of uridine kinase (Table 1), may the lack of the antileukemic action of 5-aza-2-deoxycytidine compensate for a deficient UMP formation (Chart 6). and the normal uptake of 2^deoxycytidine in 5-azacytidine- In the 5-aza-2'-deoxycytidine-resistant subline, the enhance resistant leukemic cells, provided that the phosphorylation of ment of DNA-dependent RNA polymerase (Chart 3, Table 2) 5-aza-2-deoxycytidine is indispensable for its biological cannot be regarded as a result of the decreased activity of effect. uridine kinase (Table 1). DNA-dependent DNA polymerase The unaltered incorporation of 2'-deoxycytidine into the activity is in these cells not significantly increased in com 5-azacytidine-resistant subline associated with the decreased parison with the controls (Chart 4, Table 3), although a activity of deoxycytidine kinase suggested the existence of a considerable depression of deoxycytidine kinase activity has compensatory mechanism located at higher levels of been observed (Table 1). Nevertheless, a possible action of metabolic pathway. Deoxycytidine kinase is rate-limiting and 5-aza-2-deoxycytidine on DNA polymerase, in analogy to specific in distinction to mono- and diphosphokinases (32) cytosine arabinoside (12), should be excluded. and is largely responsible for further anabolic trans The results of chemotherapeutic trials (Table 4) are in formations of the natural substrate and corresponding agreement with the biochemical findings. 5-Azacytidine is analogs. Although its activity is decreased in 5-azacytidine- effective in suppressing 5-aza-2-deoxycytidine-resistant leuke resistant cells, it is presumably sufficient to account for the mic cells in which uridine kinase functions normally, and the phosphorylation of the natural precursor to such a degree as activity of RNA polymerase is increased so that the phos to satisfy the demands of DNA polymerase, which is able, in phorylation and the incorporation of the analog is possible this instance, to utilize the substrate more efficiently than its (Chart 7). The cross-resistance of 5-azacytidine-resistant cells counterpart from 5-azacytidine-sensitive cells (Chart 5); the toward 5-aza-2-deoxycytidine may be explained by the overall result would be a normal uptake of 2'-deoxycytidine deletion of deoxycytidine kinase. The lack of response in (Chart 6). The phosphorylation of 5-aza-2'-deoxycytidine by 5-azacytidine- and 5-aza-2-deoxycytidine-resistant leukemic deoxycytidine kinase is less extensive than that of 2'- cells to cytosine arabinoside (Table 4) is in accordance with deoxycytidine, so that the enhanced DNA polymerase the partial loss of deoxycytidine kinase in the sublines, since activity is probably not able to counterbalance the primary in cytosine arabinoside-resistant cells the activity of this deficiency of the phosphorylation. Consequently, the enzyme is considerably depressed (5, 19, 23, 30). anabolic transformation of 5-aza-2'-deoxycytidine would be The metabolic alterations of 5-azacytidine- and 5-aza-2- decreased and its antileukemic effect would be abolished. deoxycytidine-resistant leukemic cells are due to uridine and The increased activity of DNA-dependent RNA polymerase deoxycytidine kinase deletions which, in the case of resis in 5-azacytidine-resistant leukemic cells (Chart 3, Table 2) is tance to 5-azacytidine, occur simultaneously although both

2184 CANCER RESEARCH VOL. 30

Resistanceto 5-Azo-2:deoxycytidine: 12. Furth, J. J., and Cohen, S. S. Inhibition of Mammalian DNA Polymerase by the 5'-Triphosphates of 1-ÃŸ-D-Arabinofuranosyl- kinast potymtrast cytosinÃ¨ and the 5' -Triphosphate of 9-(3-D-Arabinofuranosyl- UrÂ¡dine UMP UTP RNA adenine. Cancer Res., 28: 2061-2067, 1968. 13. Handschumacher, R. E., Calabresi, P., Welch, A. D., Bono, V., Fallow, H., and Frei, E., III. Summary of Current Information 2-Deoxycytidine dCMP DNA on 6-Azauridine. Cancer Chemotherapy Rept., 21: 1-18, 1962. Chart 7. Metabolic alterations in 5-aza-2'-deoxycytidine-resistant 14. Hutchison, D. J. Studies on Cross-Resistance and Collateral Sensitivity. Cancer Res., 25: 1581-1595, 1965. leukemic mice cells. 15. Juroviik, M., Ra'ska, K., Sorm, F., and SormovÃ¡, Z. Anabolic Transformations of the New Antimetabolite 5-Azacytidine and enzymes are distinctly different (18, 24). The enhancement Its Incorporation into Ribonucleic Acid. Collection Czech. Chem. of DNA-dependent RNA and DNA polymerases may be Commun., 30: 3370-3376, 1965. 16. Kehoe, J. M., Lust, G., and Beisel, W. R. Lymphoid Tissue- regarded as secondary, and in 5-azacytidine-resistant cells it Corticosteroid Interaction: An Early Effect on Both Mg2+- and is probably of compensatory character. The finding of Mn2+-activated RNA Polymerase Activities. Biochim. Biophys. increased polymerase activities in resistant cells with deleted Acta, 174: 761-763, 1969. nucleoside and deoxynucleoside kinases (25, 29) could offer 17. Keir, H. M. DNA Polymerase from Mammalian Cells. Progr. a new opportunity for chemotherapeutic attack. At present, Nucleic Acid Res., 4: 81-128, 1965. it is not known whether cells resistant to purine analogs with 18. Kessel, D. Properties of Deoxycytidine Kinase Partially Purified deleted phosphoribosyltransferases (1, 4, 26) respond from L 1210 Cells. J. Biol. Chem., 243: 4739-4744, 1968. similarly. 19. Kessel, D., Hall, T. C., and Rosenthal, D. Uptake and Phos- phorylation of Cytosine Arabinoside by Normal and Leukemia Human Blood Cells in Vitro. Cancer Res., 29: 459-463, 1969. ACKNOWLEDGMENTS 20. Levitan, I. B., and Webb, T. E. Effects of 5-Azacytidine on Polyribosomes and on the Control of Tyrosine Transaminase We extend our appreciation to Mrs. Jana Miillerova for skilled Activity in Rat Liver. Biochim. Biophys. Acta, 182: 491-500, technical assistance. 1969. 21. Lin, F. H., and Rich, M. A. RNA Polymerase Activity following REFERENCES Infection with Murine Leukemia Virus. Biochim. Biophys. Acta, 757: 310-321, 1968. 1. Bennet, L. L., Jr., Schnebli, H. P., Vail, M. H., Allan, P. W., and 22. Lowry, O. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J. Montgomery, J. A. Purine Ribonucleoside Kinase Activity and Protein Measurement with the Folin Phenol Reagent. J. Biol. Resistance to Some Analogs of Adenosine. Mol. Pharmacoi., 2: Chem., 193: 265-275, 1951. 432-443, 1966. 23. Momparler, R. L., Chu, M. I., and Fischer, G. A. Studies on a 2. Brockman, R. W. Resistance to Purine Antagonists in Experi New Mechanism of Resistance of L 5178 Y Murine Leukemia mental Leukemia System. Cancer Res., 25: 1596-1605, 1965. Cells to Cytosine Arabinoside. Biochim. Biophys. Acta, 767: 3. Burton, K. A Study of the Conditions and Mechanism of the 481-493, 1968. Diphenylamine Reaction for the Colorimetrie Estimation of 24. Orengo, A. Regulation of Enzymic Activity by Metabolites. I. Deoxyribonucleic Acid. Biochem. J., 62: 315-323, 1956. Uridine-Cytidine Kinase of Novikoff Ascites Rat Tumor. J. Biol. 4. Caldwell, I. C., Henderson, J. F., and Paterson, A. R. P. Chem., 244: 2204-2209, 1969. Resistance to Purine Ribonucleoside Analogues in an Ascites 25. Pasternak, C. A., Fischer, G. A., and Handschumacher, R. E. Tumor. Can. J. Biochem. Physiol., 45: 735-744, 1967. Alteration in Pyrimidine Metabolism in L 5178 Leukemia Cells 5. Chu, M. Y., and Fischer, G. A. Comparative Studies of Leukemic Resistant to 6-Azauridine. Cancer Res., 21: 110-117, 1961. Cells Sensitive and Resistant to Cytosine Arabinoside. Biochem. 26. Paterson, A. R. P., and Hori, A. Resistance to 6-Mercaptopurine. Pharmacol., 14: 333-341, 1965. I. Biochemical Differences between the Ehrlich Ascites 6. CihÃ¡k, A., VeselÃ¡, H., and Sorm, F. Thymidine Kinase and Carcinoma and a 6-Mercaptopurine-resistant Subline. Can. J. Polyribosome Distribution in Regenerating Rat Liver following Biochem., 40: 181-194, 1962. 5-Azacytidine. Biochim. Biophys. Acta, 767: 277-279, 1968. 27. Piskala, A., and Sorm, F. Nucleic Acid Components and Their 7. CihÃ¡k, A., Vesely, J., and Sorm, F. Difference in the Inhibition Analogues. LI. Synthesis of 1-Glycosyl Derivatives of 5-Azauracil of Hormone and Substrate Induction of Rat Liver Tryptophan and 5-Azacytosine. Collection Czech. Chem. Commun., 30: Pyrrolase by 5-Azacytidine. Collection Czech. Chem. Commun., 2801-2811, 1965. 32: 3427-3436, 1967. 28. Piimi, J., and Â§orm,F. Synthesis of a 2'-Deoxy-D-ribofuranosyl- 8. Cohen, S. S. Biochemistry of D-Arabinosyl Nucleosides and 5-Azacy tosine. Collection Czech. Chem. Commun., 29: Nucleotides. Progr. Nucleic Acid Res., 5: 1-35, 1966. 2576-2577, 1964. 9. Cunningham, D. D., and Steiner, D. F. Extraction of RNA 29. Reichard, P., SkÃ¶ld,O., Klein, G., RÃ©vÃ©sz,L.,and Magnusson, Polymerase from Rat Liver Nuclei in a Soluble Form. Biochim. P.-H. Studies on Resistance against 5-Fluorouracil. I. Enzymes of Biophys. Acta, 145: 834-836, 1967. the Uracil Pathway during Development of Resistance. Cancer 10. Elion, G. B., and Hitchings, G. H. Metabolic Basis for the Res., 21: 110-117, 1961. Actions of Analogs of Purines and Pyrimidines. Advan. 30. Schrecker, A. W. Nucleoside Kinases in Leukemia L 1210. Proc. Chemotherapy, 2: 91, 1965. Am. Assoc. Cancer Res., 10: 77, 1969. 11. Evans, J. S., Musser, E. A., Bostwick, L., and Mengel, G. D. The 31. Sorm, F., and Vesely, J. 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J. Veselý, A. Cihák and F. Sorm

Cancer Res 1970;30:2180-2186.

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