Some Factors Affecting the Phosphorylation of Thymidine by Transplantable Rat Hepatomas'

Home , Thymidine, Thymidine diphosphate, Thymidine triphosphate

JOSEF BUKOVSKY2 AND JAY S. Ro'rii@

(Institute of Celluk@r Biology, University of Connecticut, Storr8, Connecticut)

SUMMARY The phosphorylation of thymidine and thymidylate and some factors affecting the in vitro reactions have been investigated in a series of transplantable hepatomas of different growth rates. The tumors (Novikoff, McCoy MDAB, Dunning, Novikoff ascites, and Morris 5123, in order of decreasing growth rate) all were capable of phosphorylating thymidine, and a correlation between growth rate and rate of thy midine utilization was observed. All hepatomas were responsive to feedback in hibition by TTP4 and this effect was potentiated by low levels of Mg@+. Some properties of the kinase systems in the different hepatomas were examined. Thymidine kinase required only endogenous levels of Mg@ while thymidylate kinase required added Mg@. Both activities were abolished by excess ethylenediamine tetraacetate. Kinase activity was moderately stable to storage in the frozen state. TMP kinase was generally undetectable in the Dunning hepatoma. TMP phos phatase was inhibited by adenosine triphosphate and activated by Mg@, but the action of this phosphatase does not explain the lack of TTP formation by Dunning hepatoma supernatant fraction. The activity of the kinases varied with the growth stage of the tumor and was generally low at early and late times in the tumor growth cycle. These changes were interpreted in terms of the number of actively proliferating tumor cells present.

A number of recent publications have established the liver. In addition, there appears to exist a complicated importance of phosphorylation of thymidine and thymidyl and as yet incompletely understood series of interacting ate as a major pathway in mammals, plants, and some feedback controls for the phosphorylation of thymidine bacteria for the production of TTP needed for DNA and for other related reactions (19). synthesis (1, 2, 4, 5, 9, 15, 17, 19, 20, 22, 23, 27, 29, 34). Since it is often stated that cancer tissues lack normal The importance of this pathway is manifested by the control of metabolism, and since the phosphorylation of existence of high levels of thymidine phosphorylating en thymidine appears to be one of the more important zymes in rapidly proliferating tissues such as certain regulatory points leading to DNA synthesis, it seemed to tumors (4, 5, 8, 19, 22), regenerating liver (1, 2, 4, 5, 9, 27), be of particular interest to examine factors affecting and embryonic tissues (26; J. S. Roth and J. Askew, un phosphorylation of thymidine in tumors. The availability published experiments), and the absence or low activity of a series of hepatomas with different biochemical char of these enzymes in nongrowing tissues such as adult rat acteristics and growth rates (28) is particularly fortunate in that it may be possible to correlate enzyme activity or

i This project has been aided by grants from the National other biochemical parameters with growth rate or the Cancer Institute, National Institutes of Health, CA-07824; The ability of the tumor to regulate particular metabolic American Cancer Society, P-303, and the U.S. Atomic Energy sequences. Commission, AT(30-1)2737. A portion of this material was presented at the Federated Societies meeting in Chicago, Illinois, It is important to keep in mind that when unpurified April,1964. preparations are used, the phosphorylation of thymidine S Abstracted from a thesis presented in partial fulfillment of and thymidylate in vivo and in vitro occurs amidst several the requirements for the Ph.D. degree, June, 1964. related competing reactions, including the action of phos 3 Supported by Research Career Award RC-31-63 from the National Cancer Institute, National Institutes of Health. phatases, nucleosidases, thymine reductases, etc. Some

4 The abbreviations used were: TTP, thymidine triphosphate; attention has been given to the significance of these re TMP, thymidylate; DNA, deoxyribonucleic acid; TDP, thymidine actions (8, 12, 16). diphosphate; dCMP, deoxycytidylate; dUMP, deoxyuridylate; CTP, cytidine triphosphate; UTP, uridine triphosphate; GTP, MATERIALS AND METHODS guanosine triphosphate; RNA, ribonucleic acid; dCDP, deoxy cytidine diphosphate; dADP, deoxyadenosine diphosphate. Propagation and transplantation of the hepatoma@.â€”For Received for publication October 19, 1964. the most part, 5 different hepatomas carried in our 358

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. BUKOVSKY AND RoTHâ€”Factors Affecting the Phosphorylation of Thymidine 359 laboratory were used. The tumors were grown intra TABLE 2 peritoneally by technics already described (33). For the EFFECT OF STORAGEAT â€”17Â°C.ON THE PHOSPHORYLATION McCoy MDAB, Novikoff, and Dunning hepatomas, OF THYMIDINE only material from the main tumor mass attached to the gastrohepatic and gastrosplenic omentum was used for AS:Thymi OP ACTIVITYKEcOVERED HEPATOMAPERcENTAGE â€” experimentation and transplants. The Morris 5123 D @px@pbTDPTn'McCoyMDAB develops as red-brown granules about 5 mm in diameter with color and consistency similar to liver. The Novikoff ascites tumor was transplanted by withdrawing 2 ml of the ascitic fluid and immediately injecting it into another 36 2 37 6 55 Holtzman strain rat. 103 8 45 5 42 The solid tumors normally grow to a large size (about Dunning 0 2 55 3 40 10 gm) before the animal dies. Experiments described 27 3 55 4 38 here were usually carried out with tissues taken at the 92 6 88 1 5 time of transplantation, some time before maximum tumor growth. Table 1 lists the growth rates of the different Morris 5123 D0 0 50 20 4 26 hepatomas used. 4 75 11 2 12 Preparation of tissues.â€”Animals were killed by ex 65 8437 107 158 5 sanguination via heart puncture under ether anesthesia. The tumors were excised, immersed in a beaker containing a Thymidine-2-C14 was the substrate. b The abbreviations used are : TMP, thymidylate; TDP, thymi ice-cold 0.25 M sucrose solution, washed free of blood, and dine diphosphate; TTP, thymidine triphosphate. freed of other tissues. After weighing, the required amount of tissue was homogenized in a 7 ml all-glass Ten Broeck homogenizer in ice-cold sucrose medium containing 0.03 ml of water to give a total volume of 0.155 ml. 0.23 M sucrose, 0.005 M KC1 and 0.01 M Tris buffer (pH Incubations were carried out at 37Â°C.for 20 min. and 8.0). As a standard procedure, 2 gm of tumor were sub terminated by immersing the tubes in boiling water for jected to 12 strokes of the homogenizer and made up to 3 min. The tubes were then cooled in ice water and 8.0 ml with the sucrose solution. The homogenate was centrifuged in the International refrigerated centrifuge at centrifuged at 105,000 X g for 1 hr. and the supernatant 1600 X g for 15 mm. Aliquots of 75 @lwere withdrawn fraction carefully decanted. Normally, it was used im for chromatography. mediately, but occasionally aliquots were stored at â€”17Â°C. Thymidytate kinase.â€”This enzyme was assayed by es in test tubes so that comparable experiments could be sentially the same procedure using 17.5 m@moles of performed on supernatant fractions frozen and thawed thymidylate-2-C'4. only once. Full enzymatic activities were sometimes re Chromatography and counting procedure.â€”Strips of tained for several months in the frozen state, depending Whatman AE 81 filter paper 2@ cm wide and 45 cm long on the tumor (Table 2). The activity decreased rapidly were soaked in 0.35 M ammonium formate solution (pH if the supernatant fraction was refrozen and thawed once 5.5) for 24 hr. and then in 2 changes of distilled water for more. Bojarski and Hiatt (3) reported rapid loss of 24 hr. each and air dried. Seventy-five microliters of TMP kinase activity on storage in solution at 0Â°C. or supernatant fraction from the final centrifugation were 37Â°C. applied as a narrow band 5.5 cm from one end of the strip; Enzyme a@says.â€”The technic, modified from that of the strip was then attached to a trough with stainless Delamore and Prusoff (10), depends on a paper chromato steel clips and developed by descending chromatography graphic separation of thymidine, TMP, TDP, and TTP. with 0.65 M ammonium formate (pH 5.5â€”6.0)for approxi Thymidine kinase.â€”The assay mixtures in centrifuge mately 2@ hr. The solvent front travels about 30 cm in tubes contained 0.05 ml of supernatant fraction; 0.05 ml this period of time. The strips were removed, dried in of thymidine-2-C'4 (5 m@mo1es) prepared in 80 m@& Tris the air, and then placed in a Scanogram 411 chromatogram buffer (pH 8.0) containing 0.75 Mmoles Mg@; 0.025 ml of scanner (Atomic Accessories, Valley Stream, Long Island) ATP, containing 0.8 @@moles,adjustedto pH 8.0; plus and counted. The radioactivity in each peak is expressed as a percentage of the total count on the individual strips. TABLE 1 In the 5 hepatomas studied, recovery of radioactivity in the 4 peaks was close to 100%. In normal liver, however, GROWTH RATES OF DIFFERENT HEPATOMAS there was a considerable loss of radioactivity due to active ba:TransplantationDeathDAYS AlTER nim*i@ INJECTION catabolic enzyme systems. HEPATOKAAPPROXIMATE Protein was determined by the method of Lowry et at. animalMcCoy of (24). Thymidine-2-C'4 was obtained from the New England Nuclear Corporation and had a specific activity MDABG of 30 mc/mmole. Thymidylate-2-C'4 was obtained from Novikoff 4â€”S 5-7 Schwarz Bioresearch and had an activity of 9 mc/rnmole. Dunning 7-9 9-12 Novikoff ascites 7 14â€”21 N ,N-dimethyl-p-(m-tolylazo)-aniline, p-dimethylamino Morris 5123 D4.5 8â€”10weeks4.7 12â€”14weeks azobenzene and 2-fluorenamine were obtained from East man Kodak Company. 1 , 1 ,3-Tricyano-2-amino-1-pro a MDAB, N,N-dimethyl-p-(m-tolylazo) aniline. pene was generously supplied by Mrs. Mary S. Duggan,

Biological Screening Office, The UpJohn Company. A extremely slow or, most often, negligible. Occasionally, solution of 10 mg per 0.1 ml of 2-methoxyethanol was significant phosphorylation of thymidine to TTP was ob prepared. 3-Phosphoglyceric acid CTP, UTP, GTP, served (Table 2), but in a large series of experiments, phospho(enol) pyruvic acid, pyruvate kinase creatine when TMP was the substrate, no appreciable phosphoryl phosphate, and creatine phosphokinase were obtained ation was detected (Table 4). It is possible that the few from Sigma Chemical Company. examples of significant thymidine phosphorylation to RESULTS TTP were artifacts. The reason for this interesting behavior is unknown, but Generalpropertiesof the phosphorylationof thymidine in is being explored. One suggestion is that TMP kinase in different hepatoma.@.â€”The formation of phosphorylated the Dunning hepatoma is less stable than in the other derivatives from thymidine by the McCoy MDAB, tumors. It is also possible that TTP in this tumor is Novikoff, Novikoff ascites, Dunning, and Morris 5123 D manufactured by an alternate pathway. The extremely hepatomas is illustrated in Chart 1, A through E, re low activity of dCMP deaminase (33) in the Dunning spectively, which presents typical experiments. The hepatoma (which would normally produce dUMP and McCoy MDAB, under our experimental conditions, then TMP), and consequent low endogenous levels of consistently showed the most rapid and complete pro TMP, would be in keeping with the probability of greater duction of TTP. Qualitatively, the Novikoff solid hepa enzyme instability (2, 3). toma was similar to the McCoy MDAB, except for a The Morris 5123 D hepatoma showed a slow but sig more rapid and greater production of TMP and a slower nificant rate of phosphorylation of thymidine; on the decline in the concentration of this latter nucleotide. The other hand, the 5123 D is one of the most active tumors Novikoff ascites hepatoma showed a little less activity studied in the phosphorylation of TMP (8). than the solid form, but was otherwise quite similar to it. The investigations of Ives et at. (19) have shown that The Dunning hepatoma presented a considerably differ thymidine and thymidylate kinases have different affinities ent picture from the 3 tumors already discussed. TMP for their substrates. Four of the 5 transplantable hepa production was rapid, but formation of TTP was usually tomas of varying growth rates studied herein demon strated similar properties. With the exception of the A â€¢Mccoy MDAB B = Novikouf Dunning hepatoma, the other 4 tumors showed con @@@ ILAJ , I I II sistently greater formation of TTP from TMP than from 80 \ T thymidine, perhaps because of full saturation of TMP \ kinase immediately from the beginning of incubation. 60.\ ,â€œ S The response of the tumor enzymes to magnesium was, in general, also similar to that reported by Ives et at. (19) and some data are presented in Table 3. Thymidine kinase showed no need for added magnesium, while : __ thymidylate kinase required it. When magnesium was

C â€¢Novikoff alcitea D Dunning omitted from the incubation mixture and EDTA added, IIII the activity of thymidine kinase was also completely in hibited (compare Experiments 1 and 4, Table 3). This C T \I result suggests that thymidine kinase also requires a metal .@ ion but that endogenous levels are sufficient for this 0 VS enzyme. Prolonged dialysis did not affect the activity of thymidine kinase, suggesting that the metal is tightly a,C U A bound. 0. I \_@@-_TTP None of the tumors studied could utilize CTP, UTP, or â€”Ã¨--.--4-â€”I.iJ2L@ 0 10 20 30 40 50 60 GTP as phosphatedonorsto an appreciableextent. E = Morris 5123 D minutes Thymidylate kinase showed no activity at all with these donors, while with thymidine kinase 10â€”20% of the ac 80 tivity obtained with ATP was observed. Various ATP 60 â€”1 generating systems were also investigated, as follows: TIP (a) ATP, creatine phosphate and creatine kinase; (b) ATP, 40 â€”â€”IMP phospho(enol) pyruvic acid and pyruvate kinase; (c) ATP â€”â€”-TDP and 3-phosphoglyceric acid. When different amounts of 20 . TTP __@ @-@- @MP these 3 combinations were used, the activity was not, in @+â€”.--.--.-,-- -r any case, higher than with the optimum concentration of 0 ) 10 20 30 40 50 60 minutes ATP and Mg@. Stability of the kinases.â€”After storage in a frozen state CHART 1.â€”Formation of phosphorylated derivatives from thy midine by various rat hepatomas. T, thymidine. A series of (â€”17Â°C.) for several months, thymidine kinase and tubes were assayed in duplicate using standard conditions at the thymidylate kinase in the tumors maintained, to some illustrated time intervals. The amounts of protein were essen extent, their ability to phosphorylate their substrates, tially the same in all the hepatoma high speed supernatant fractions utilized except for the Morris 5123 D, which had approxi thymidine and TMP, respectively. The observation of mately 35â€”40%moreprotein. (See Table 5 for a typical example.) Bojarski and Hiatt (3), that thymidylate kinase rapidly

TABLE 3 Bresnick also recently reported that all of a series of EFFECT OF EDTAa ON THE PHOSPHORYLATION OF transplantable hepatomas examined were inhibited by THYMIDINE TTP (7). There was a difference in response of the tumors to dCTP, however, as not all hepatomas were OP RADIO ACTIVITY AS: inhibited by this triphosphate. In general, the slow SAMPLE AND CONDITIONS@'PERCENTAGE growing minimal-deviation hepatomas were sensitive to TrPExperiment Th@ymi- TMÃ˜ TDP dCTP, while some of the more rapidly proliferating hepatomas were not affected. 1 When TDP was tested as a feedback inhibitor (not McCoy MDAB illustrated), it gave results similar to those obtained with McCoy MDAB + Mg@ 6 68 5 21 TTP. It seems likely that the diphosphate is rapidly McCoy MDAB + EDTA 99 1 0 0 converted to the triphosphate, as Ives has shown recently Experiment 2 that TDP kinase has an extremely high turnover number Morris 5123 D 38 62 0 0 (18). When increasing amounts of TMP were added to Morris 512.3 D + EDTA 94 6 0 0 the reaction mixture, no inhibition could be demonstrated. Experiment 3 It was observed, however, that thymidine kinase in the Novikoff 8 92 0 0 Novikoff, Novikoff ascites, MDAB, and 5123 hepatomas Novikoff + EDTA 90 10 0 0 Experiment 4 converted thymidine to TMP in the constant ratio of McCoy MDAB + Mg1@ 6 11 20 63 approximately 1 :30 over a wide range of concentrations McCoy MDAB + Mg@ + EDTA6 894 890 30 0 of both substrates. Thymidine and Thymidylate kinase activities during a The abbreviations used are : EDTA, ethylenediaminetetra tumor growth.â€”In a number of studies on thymidine and acetate; TMP, thymidylate; TDP, thymidine diphosphate; TTP, thymidylate kinases, it was observed that the activities of thymidine triphosphate. the enzymes appeared to increase up to a certain point b Standard incubation conditions : 0.75 pmoles Mg@ and 1.0 and then decrease as the tumor grew large and old. It pmoles EDTA when indicated; thymidine-2-C14 substrate. was therefore of interest to determine the pattern of activity of the kinase with respect to tumor development. loses its activity if stored in solution at 0Â°C.or at higher These experiments were performed by utilizing lesser temperatures, but is stabilized for a considerable period in omentum adipose tissue which becomes converted into the presence of thymidine or TMP, was confirmed. How hepatoma as the tumor grows. ever, the stability of TMP kinase in the frozen state Groups of rats were inoculated with hepatoma to give appeared to vary from one hepatoma to the next. Al at least 2 rats per day for investigation. The kinases and though the stability depends to some extent on tissue also phosphatase activity were assayed each day. concentration of thymidine and TMP, nevertheless, a The results with several hepatomas are shown in Table TMP kinase preparation of high specific activity generally 4. In the McCoy MDAB, Novikoff, and Dunning maintained its activity better on storage in the frozen hepatomas, thymidine kinase activity increased from very state than one of low initial activity. low levels to a maximum at 3-6 days. In the Novikoff Feedback inhibition of thymidine kina.se in tumors.â€” Several investigators (6, 20, 26) have demonstrated that TTP is a potent feedback inhibitor of thymidine kinase ac tivity in a number of different systems. In some tumors that have been investigated, for example Novikoff hep a, atoma in tissue culture and leukemic human leukocytes, > ID this control is intact. The 5 tumors examined herein > also demonstrated feedback inhibition with TTP, and the a, a, results of a typical experiment are shown in Chart 2. Al ID though thymidine kinase activity in all of the hepatomas 0 .C was completely inhibited by 30 m@zmolesofTTP in the re 0. action mixture, they demonstrated varying degrees of in 0 0. hibition by 7.5 m@moles of TTP. There is, essentially, no 0 correlation between the extent of the inhibition by low levels a,C of TTP and the growth rate of the tumor, although the U a, slow-growing Morris 5123 D was inhibited to the greatest 0. extent at all concentrations. In the absence of magnesium, much lower concen trations of TTP inhibit, and the results under these experimental conditions are shown in Chart 3. Once m@imoIeslIP again, the 5123 hepatoma exhibited the greatest inhibition at the lowest concentrations, but all the hepatomas were CHART 2.â€”Inhibition of the phosphorylation of thymidine by TTP in several rat hepatomas. Standard assay conditions were strongly inhibited by remarkably low concentrations of used. Total amount of TTP per assay tube is indicated on the TTP. abscissa.

particulate fractions of various hepatomas all contain con siderable TMP phosphatase activity, and in some hepa tomas, for example the Dunning, the supernatant fraction @ may also contain a sizeable amount of activity. :@ These earlier investigations employed high substrate

.@ concentrations and were carried out in the absence of @ ATP and Mg@. For this reason, a quantitative study @ was undertaken to determine phosphatase activity under @. varying conditions which included those used in the .@ standard assay of this report.

.@ Chart 4 shows the effect of Mg@ concentration on the @ hydrolysis of TMP by phosphatase in several tumors. @ In these experiments, ATP concentration was kept con

0. stant at 0.8 Mmoles, the amount normally used. With Dunning hepatoma, little hydrolysis of TMP was observed when the standard quantity of Mg+@was present (0.75 4.5 6.0 7.5 @moles). Novikoff and McCoy MDAB hepatomas cx m@imolesTIP hibited a slight hydrolysis of TMP ; Novikoff ascites CHART 3.â€”Inhibition of the phosphorylation of thymidine by exhibited considerable hydrolysis under the same con TTP in the absence of magnesium, in several rat hepatomas. ditions. Morris 5123 D hydrolyzed about 50 % of the Conditions as in Chart 2 except no magnesium added. substrate in the presence of 0.75 Mmoles of Mg@ and increasing amounts at higher concentrations of Mg+4. ascites, activity was constant and maximal from the first ATP chelates Mg@+, thus reducing phosphatase activity time of sampling (4 days) to the 9th day and then declined which appears to be stimulated by Mg++. For this to zero by the 14th day. Activity in the Dunning also reason, phosphatase activity was also investigated in the declined precipitously on the 9th day. The changes in absence of ATP and in the presence of standard amounts thymidylate kinase were similar except that this enzyme (0.75 @@moles)ofMg@+. Table 5 shows the results of this appeared later than thymidine kinase and, in general, study. The trend of phosphatase activity shown in activities were more erratic. The absence or low activity Chart 4 is maintained by the McCoy MDAB, Novikoff, of thymidylate kinase activity in the Dunning hepatoma and Morris 5123 D hepatomas, but not by Dunning is again indicated in Table 4. Phosphatase activity was hepatoma. This latter tumor, which showed no hydrolysis high in all the tumors and remained relatively constant of TMP in the presence of ATP, exhibited rather high throughout the experimental period, except for the Dun- activity in the absence of ATP. The Novikoff ascites ning, in which it tended to increase at about the 6th day hepatoma, which had high phosphatase activity in the to very high levels. These high levels of phosphatase activity, which have also been observed by other methods TABLE 4 of assay (8), may be another reason for the low thymidylate THYMIDINEANDTHYMIDYLATEKINASEACTIVITIESDURING kinase activity in the Dunning hepatoma. It should be TUMORGROWTHINVARIOUSHEPATOMAS noted, however, that the presence of ATP in the incubation TEYMIDINE RINASE TEYMIDYLATE RINASZ PEOSPRATASE mixture usually markedly reduces the rate of thymidylate ACTIVITY ACTIVITY ACTIVITY DAYS (% (% (% dephosphorylation. The phosphatase assays in Table 4 EYDROLYZED)b INOCULA ,[email protected]@rxi) [email protected]@m) were conducted in the absence of ATP. TION It is believed that the best interpretation of the results Mb N D NA M N D NA M N D NA in Table 4 is that thymidine and thymidylate kinase activities are maximal at all times, (at about the highest 0 1 1 0 0 0 0 30 32 46 levels indicated in the table) in actively growing tumor 1 7 5 3 0 0 0 42 42 42 19 44 32 cells. At early stages, when there are relatively few 2 13 0 0 0 tumor cells and relatively large numbers of connective 3 42 23 3 0 0 0 39 30 23 4 95 94 64 1 24 0 58 50 25 3' tissue cells and adipose tissue cells which have little or no 5 98 95 44 64 90 3' 0 56 36 30 40 22 enzyme activity, the measured activity is low. At late 6 98 92 59 88 3 41 33 72 â€˜7 stages, when tumor growth has largely slowed down due 7 94 66 20 2 57 75 22 to exhaustion of nutrients or for other reasons, and a 8 95 67 0 62 56 17 large proportion of the hepatoma cells are no longer 9 19 64 0 61 74 21 actively proliferating, activity rapidly falls off since these 10 40 8 20 enzymes are unstable, have a high turnover rate, and if 13 4 0 10 synthesis is not maintained, their activity would tend to 14 0 0 8 decline rapidly (32). a The abbreviations used are TMP, thymidylate; M, McCoy Thymidylate phosphatase.â€”Differences in the activities MDAB, N, Novikoff, D, Dunning, NA, Novikoff ascites. of thymidine and thymidylate kinase in various hepatomas Conditions of assay as described under â€œMaterialsand Methods.â€• may be a reflection of differences in phosphatase activity. B Five-minute incubation only, no adenosine triphosphate Previous studies (8) have demonstrated that the 3 major present.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. BUKOVSKY AND ROTHâ€”Factors Affecting the Phosphorylation of Thymidine 363 presence of ATP, demonstrated only modest hydrolysis in TABLE 5 the absence of ATP. The reason for these differences is HYDROLYSIS OF TMP@ BY PHOSPHATASE IN THE ABSENCE not clear. Fluoride has been reported to be inhibitory to OF ADENOSINE TRIPHOSPHATE deoxynucleotide phosphatase activity at pH 8.0 (25). In several experiments, the minimal concentration of fluoride HepatomaProtein/O.05 ml hydrolyzed Activitr (mg)TMP (m@@moIcs)Specific(mpmoles/mg required for maximal inhibition of phosphatase activity protein/hr.)McCoy with TMP as substrate was found to be 2 m@smoles. A fraction (10 %) of the phosphatase activity remained un MDAB affected, even at higher concentrations of fluoride. In Novikoff 0.6.5 22.3 103 several experiments, not illustrated, fluoride at a concen Dunning 0 .63 37 .6 179 tration of 2 m@moles did not alter the activity of thymidine Novikoff ascites 0.75 15.4 61 kinase and had no consistent effect on thymidylate kinase Morris 5123 D0.65 1.0528.0 67 .0129 192 activity. The changes were not in any case of major a The abbreviations used are : TMP, thymidylate. significance. Attempt@s to iiviuce thymidine and thymidylate kinase ac tivity in normal rat liver.â€”It is well known that partial RNA synthesis. When the drug was injected as described, hepatectomy leads to a rapid increase in thymidine and however, no change was noted in kinase activity in liver. thymidylate kin.ase activity in the regenerating liver (1, 2, Injection of cortisone for 7 days also was ineffective. 4, 5, 9, 27). In spite of extensive research, the mechanism Evidence has been presented that thymidylate kinase for this increase is unknown. Gebert and Potter (14) activity in resting tissue of mouse liver is actually high, have reported that force-feeding protein hydrolysate to possibly higher than in growing tissue or hepatoma (21). 160â€”200gm rats will lead to the induction of thymidine It has also been demonstrated that thymidylate kmnase is kinase activity in liver. particle-bound and may be released from mouse liver by We attempted to induce thymidine or thymidylate alternate freezing and thawing in the presence of TMP kinase activity in adult rats by : (a) extensive starvation (21). We have confirmed this, but when this procedure (6 days) followed by refeeding (animals were assayed 3, was applied to adult rat liver there was no increase in the 6, 9, 12, and 24 hr. after refeeding) ; (b) injection of DNA phosphorylation of thymidine, and this has been confirmed hydrolysate daily for 1 week (14) ; (c) injection of 3-methyl by V. R. Potter, (personal communication). Unlike cholanthrene, N ,N-dimethyl-p-(m-tolylazo) aniline, 2- Eseherichia coli thymidine kinase (30), dCDP, and dADP fluorenamine, and p-dimethylaminoazobenzene daily for 1 had no stimulating effect on the unpurified enzyme in the week; and (d) injection of diethylstilbestrol. None of tumors, and higher concentrations of dADP were inhibi these treatments had any significant effect on the phos tory to thymidine kinase. phorylation of thymidine by adult rat liver. It has been reported (11) that 1 , 1 ,3-tricyano-2-amino-1-propene has DISCUSSION some mitotic and inductive activity and also increases In the present experiments, high thymidine kinase ac tivity was found in 4 transplantable hepatomas, the McCoy MDAB, Novikoff, Novikoff ascites, and Dun ning and somewhat lower activity in the Morris 5123 D. Thymidylate kinase activity was high in all the tumors studied, with the exception of the Dunning hepatoma in which this enzyme was nearly always undetectable by the methods employed. All of the hepatomas showed good

.@ feedback control by TTP acting on the thymidine kinase reaction, suggesting that if a loss of control of DNA HI- 0 anabolism exists, it lies elsewhere in this series of hepa a, C tomas. E There are many other possible focal points for loss of >@ control and it seems likely that metabolic pathways other a)C than thymidine phosphorylation may be affected in some U of the tumors. Some earlier reactions that may be of 4) 0. significance are illustrated below.

UDP -+ dUDP -@dUMP -@TMP I dCMP 0 0.375 0.75 1.5 3.0 Exploration of the metabolic control of these pathways pmolesMg@'@'@ in a series of hepatomas is under way. It is quite possible, of course, that some of the tumors CHART 4.â€”Hydrolysis of TMP by several hepatomas in the have biochemical lesions in control, not at the enzyme presence of increasing concentrations of magnesium. Standard assay conditions. Total amount of magnesium per assay tube is level, but at the gene level, in repression or derepression indicated on the abscissa. mechanisms involved in synthesis of messenger RNA.

There is evidence that templates for thymidine kinase 12. FIALA, S.; FIALA, A.; TOBAR,G.; ANDMCQUILLA,H. Deoxy (31), thymidylate kinase and deoxycytidylate deaminase nucleotidase Activity in Rat Liver and Certain Tumors. J. NatI. Cancer Inst., 28:1269â€”89,1962. (32) are more stable in some hepatomas than in normal 13. FIRSHEIN,W. The Induction of Deoxycytidylic Acid Kinase liver. This increased template stability in combination Activity by a Dinucleotide Fraction Derived from an Easy with accelerated protein synthesis could account for higher matic DNA Digest in Pneumococci. Biochem. and Biophys. levels of these enzymes observed in some hepatomas Res. Communs.,11:187-94,1963. 14. GEBERT,R. A., AND POTTER, V. R. The Effect of Dietary without, however, necessitating any changes in the control Protein Content on Liver Thymidine Kinase. Federation of enzyme synthesis at the gene level or in the control of Proc. 23:268, 1964. enzyme activity at the enzyme level, as by allosterism or 15. Ga&v, H. J., ANDSa,@Luz, R. M. S. The Mechanism of Forma other mechanisms. tion of Thymidine Triphosphate by Enzymes from Land A considerable degree of caution has to be exercised in schutz Ascites-Tumour Cells. Biochem. J., 89:486â€”9,1963. 16. GRAY, E. D.; WEISSMAN,S. M.; RICHARDS,J.; BELL, D.; relating the in tntro activity of thymidine and thymidylate KEIR, H. M.; SMELLIE, R. M. S.; AND DAVIDSON, J. N. Studies kinases to their relative functional activities in vivo. As On the Biosynthesis of Deoxyribonucleic Acid by Extracts of has been demonstrated, there are many factors that affect Mammalian Cells. V. Factors Interfering with Biosynthesis. enzyme activity in ritro, such as ATP and Mg@ concen Biochim. et Biophys. Acta, 45:111â€”20,1960. 17. HOTTA, Y., AND STERN, H. Transient Phosphorylation of trations, the level of various endogenous nucleotides which Deoxyribosides and Regulation of Deoxyribonucleic Acid stabilize the enzyme e.g., thymidine and TMP, and TDP Synthesis. J. Biophys. Biochem. Cytol., 11311â€”19,1961. and TTP which exert feedback control; little is known 18. Ivzs, D. H. Thyinidine Diphosphate as an Intermediate in about the importance of these factors in vivo. In view of Thymidine Triphosphate Synthesis in Mammalian Tumor. Federation Proc., 23:169, 1964. the significant feedback effect of low concentrations of 19. IVES, D. H. ; MORSE,P. A., JR. ; ANDPOTFER,V. R. Feedback TTP, it would probably be preferable to assay for TTP Inhibition of Thymidine Kinase by Thymidine Triphosphate. synthesis in a system in which the TTP is removed as J. Biol. Chem., 238:1467â€”74,1963. rapidly as it is formed, e.g., in the presence of DNA 20. KIELLEY, R. K. Some Characteristics of Deoxynucleotide polymerase and the 3 other deoxynucleoside triphosphates. Kinase Activities in Mouse-Liver Homogenates. Biochim. et Biophys. Acta, 53:150-58, 1961. Experiments in vivo to determine whether differences in 21. . Particle-Bound Thymidylate Kinase in Mouse Liver, enzyme activity are paralleled by the capacity of the tumor A Possible Factor in the Control of DNA Synthesis. Biochem. tissue to incorporate radioactive thymidylate residue into and Biophys. Res. Communs., 10249-53, 1963. DNA would also be useful. 22. . Patterns of Synthesis of Thymidine Triphosphate and other Deoxyribonucleoside Triphosphates in Mouse Liver and in Mouse Ascites Hepatoma. Cancer Res., 23801â€”10,1963. REFERENCES 23. KIT, S.; PIEKARSKI, L. J.; AND DUBBS, D. R. Induction of 1. BIANCHI,P. A. ; CRATHORN,A.R. ; ANDSHOOTER,K.V. Thymi Thymidine Kinase by Vaccinia-Infected Mouse Fibroblasts. dine Kinases and Deoxyribonucleic Acid Synthesis in Normal J. Molecular Biol., 6:22â€”33,1963. and Regenerating Rat Liver. Biochim. et Biophys. Acta, 24. LOWRY,0. H. ; ROSEBROUGH,N. J. ; FAmt, A. L. ; ANDRANDALL, 61:728â€”35,1962. R. J. Protein Measurement with the Folin Phenol Reagent. 2. BlANCH!, P. A.; BUTLER, J. A. V.; CRATHORN,A. R.; AND J. Biol. Chem., 19&265â€”75,1951. SHOOTER, K. V. The Thymidine Phosphorylating Kinases. 25. MALEY, F., ANDMALEY, G. F. Nucleotide Interconversion& Ibid., 53:123-31, 1961. II. Elevation of Deoxycytidylate Deaminase and Thymidylate 3. BOJARSKI,T. B., ANDHIATT, H. H. Stabilization of Thymi Synthetase in Regenerating Rat Liver. Ibid., 236:2965-70,1960. dylate Kinase Activity by Thymidylate and by Thymidine. 26. . On the Nature of a Sparing Effect by Thymidine on Nature, 188:1112â€”14,1960. the Utilization of Deoxycytidine. Biochemistry, 1:847â€”51, 4. BOLLUM,F.J., ANDPOTFER,V. R. Incorporation of Thymidine 1962. into Deoxyribonucleic Acid by Enzymes from Rat Tissues. J. 27. MANTSAVINOS,R., AND CANELLAKIS, E. S. Studies on the Biol. Chem., 233:478-82, 1958. Biosynthesis of Deoxyribonucleic Acid by Soluble Mammalian 5. . Nucleic Acid Metabolism in Regenerating Rat Liver. Enzymes.J. Biol. Chem., 234.628.-35,1959. VI. Soluble Enzymes which Convert Thymidine to Thymidine 28. MORRIS, H. P. Some Growth, Morphological and Biochemical Phosphates and DNA. Cancer Res., 19-.561-65,1959. Characteristics of Hepatoma 5123 and Other New Trans 6. BREITMAN,T. R. The Feedback Inhibition of Thymidine plantable Hepatomas. Progr. Exptl. Tumor Res., 3371â€”411, Kinase. Biochim. et Biophys. Acta, 67:153-55, 1963. 1962. 7. BRE5NICK,E. ; THOMPSON,U. B. ; MORRIS, H. P. ; ANDLIEBELT, 29. Ox@tzAx@,R., ANDKORNBERG,A. Deoxythymidine Kinase of A. G. Inhibition of Thymidine Kinase Activity in Liver and Escherichia coli. I. Purification and Some Properties of the Hepatomas by TTP and d-CTP. Biochem. and Biophys. Res., Enzyme. J. Biol. Chem., 239:269â€”74,1964. Communs., 16:278-84, 1964. 30. . Deoxythymidine Kinase of Eacherichia coti. II. Kine 8. BUKOV5KY, J., AND ROTH, J. S. Studies on Thymidylate tics and Feedback Control. Ibid., 239:275-84, 1964. Phosphatase, Thymidine Kinase and Thymidylate Kinase 31. PITOT, H. C.; PERAINO,C.; Bi.&xE, R.; ANDKENNAN, A. L. Activities in Some Transplantable Rat Hepatomas. In: G. Altered Template Stability in Rat Hepatomas. Proc. Am. WEBER (ed.) Control of Enzyme Activity in Liver, 2371â€”82, Assoc. Cancer Res., 552, 1964. New York, The Macmillan Co., 1964. 32. RoTn, J. S. Increased Stability of Templates for Deoxycyti 9. CANELLAKJS,E.S., ANDMANTSAVINOS,R.The Conversion of dylate Deaminase, Thymidine Kinase and Thymidylate [email protected]'monophosphates to the Corresponding Kinase in Some Rapidly Proliferating Tissues. Life Sciences di- and Triphosphates by Soluble Mammalian Enzymes. 3:1145â€”49,1964. Biochim. et Biophys. Acta, 27643â€”45,1958. 33. ROTH, J. S. ; SHEID, B. ; ANDMoRRIS, H. P. Some observations 10. DELAMORE,I.W., ANDPRUSOFF,W. H. Separation of Thymi on the Deamination of Deoxynucleotides and Deoxynucleo dine Mono-, Di- and Triphosphate by Paper Chromatography. sides by Normal Rat Liver and Hepatomas. Cancer Res., Biochem. Pharmacol., 8:336â€”37,1961. 23:454â€”61,1963. 11. EGYHAZI,E., ANDHYDEN,H. Experimentally Induced Changes 34. WEISSMAN,S. M.; SMELLIE,R. M. S.; ANDPAUL, J. Studies on in the Base Composition of the Ribonucleic Acids of Isolated the Biosynthesis of Deoxyribonucleic Acid by Extracts of Nerve Cells and Their Oligodendroglial Cells. J. Biophys. Mammalian Cells. IV. The Phosphorylation of Thymidine. Biochem. Cytol., 10:403â€”10,1961. Biochim. et Biophys. Acta, 45:101â€”10,1960.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1965 American Association for Cancer Research. Some Factors Affecting the Phosphorylation of Thymidine by Transplantable Rat Hepatomas

Josef Bukovsky and Jay S. Roth

Cancer Res 1965;25:358-364.

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