Enzymic Capacities of Furine De Novo and Salvage Pathways for Nucleotide Synthesis in Normal and Neoplastic Tissues1

[CANCER RESEARCH 44,2475-2479, June 1984]

Enzymic Capacities of Furine de Novo and Salvage Pathways for Nucleotide Synthesis in Normal and Neoplastic Tissues1

YutakaNatsumeda,2NoemiPrajda,3JohnP. Donohue,JohnL. Glover,andGeorgeWeber4

Laboratory for Experimental Oncology [Y. N., N. P., G. W.], Department of Urology [J. P. D.Â¡,and Department of Surgery [J. L G.], Indiana University School of Medicine, Ifioiondpolis, 'noidnd 46223

ABSTRACT of the amidophosphoribosyltransferase, which is lower than that of the salvage enzymes, is linked with transformation as it is The enzymic capacities of the de novo and the salvage path increased in all examined tumors. The high activity and affinity ways for purine nucleotide synthesis were compared in rat in to PRPP of the purine phosphoribosyltransferases indicate the normal, differentiating, and regenerating liver, and in three important role which salvage enzymes might play in circumvent hepatomas of widely different growth rates. The activities of the ing the action of inhibitors of de novo purine biosynthesis in key de novo and salvage enzymes were also determined in cancer chemotherapy. mouse lung and Lewis lung carcinoma, in human kidney and liver, and in renal cell carcinoma and hepatocellular carcinomas. A precise and reproducible assay was worked out for meas INTRODUCTION uring the activities of adenine phosphoribosyltransferase (EC Previous studies in this laboratory demonstrated that the 2.4.2.7) and hypoxanthine-guanine phosphoribosyltransferase activities of the key enzymes of IMP de novo synthetic pathway, (HGPRT; EC 2.4.2.8) in crude liver and hepatoma systems. amidotransferase,5 and of IMP utilizing pathways for AMP and Kinetic studies on the salvage enzymes were carried out in the GMP, adenylosuccinate synthetase (EC 6.3.4.4), adenylosucci- crude 100,000 x g supernatant fluid from normal liver and rapidly nate lyase (EC 4.3.2.2), IMP dehydrogenase (EC 1.2.1.14), and growing hepatoma 3924A. In both tissue extracts, Michaelis- GMP synthetase (EC 6.3.5.2), were increased in all tumors Menten kinetics was observed for adenine phosphoribosyltrans examined (1, 3-5, 9-11,15-17). The increased capacity for de ferase and HGPRT. The reciprocal plots for 5-phosphoribosyl-1- novo purine nucleotide synthesis should confer selective advan pyrophosphate (PRPP) of liver and hepatoma enzymes gave tages to the neoplastic cells and provide a biochemical basis for apparent Kâ€ž,sof2 /tw for adenine phosphoribosyltransferase and drug selectivity in chemotherapy. Antimetabolites which inhibit 4 UM for HGPRT, showing two orders of magnitude higher de novo synthesis and utilization of IMP have been used in the affinities for PRPP than that of the rate-limiting enzyme of ote experimental and clinical treatment of cancer. However, recent novo purine synthesis, amidophosphoribosyltransferase (EC studies in rat pointed out that the nucleotide synthesis by salvage 2.4.2.14) (Kâ„¢= 400 to 900 (IM). The apparent Kâ„¢values for pathways might contribute, in part at least, to preserve the adenine of liver and hepatoma adenine phosphoribosyltransfer nucleotide pools in the transplanted hepatoma against the anti- ase were 0.6 to 0.9 /Â¿M,respectively. For both liver and hepatoma glut amine agent, acivicin (2, 21). Investigations in cultured hep HGPRT, the reciprocal plots for hypoxanthine and guanine atoma cells also revealed that dipyridamole, a transport inhibitor yielded the same Kmof 3 Â¿Â¿M. of nucleosides and bases, depressed the nucleotide pools and The specific activities of purine phosphoribosyltransferases killed the cancer cells as a single drug and synergistically with were markedly higher than that of amidophosphoribosyltransfer acivicin (15,18, 23). These results emphasize the importance of ase in rat thymus, spleen, testis, bone marrow, colon, liver, the salvage pathways in chemotherapy. The present study was kidney cortex, lung, heart, brain, and skeletal muscle, but were undertaken to compare the enzymic capacities of de novo and lower in the small intestine. salvage pathways for purine nucleotide synthesis in normal, In hepatomas and regenerating and differentiating liver, the proliferating, and neoplastic tissues. activities of the salvage enzymes were 2.1- to 32-fold higher than that of amidophosphoribosyltransferase. The purine phos phoribosyltransferase activities were also higher than that of MATERIALS AND METHODS amidophosphoribosyltransferase in Lewis lung carcinoma (8.2- Materials. [8-14C]Adenine (54 mCi/mmol) and [8-14C]hypoxanthine (53 to 32-fold), human renal cell carcinoma (3.5- to 22-fold), and to 55 mCi/mmol) were purchased from Amersham/Searle Corp., Arling hepatocellular carcinoma (3.4- to 30-fold). The high activities and ton Heights, IL. [8-14C]Guanine (56.7 mCi/mmol) was obtained from New the high affinity to PRPP of the purine phosphoribosyltransfer England Nuclear, Boston, MA. Sodium salt of PRPP was from Sigma ases might explain the lack of linkage of the behavior of these Chemical Co., St. Louis, MO, and all other reagents were of the highest enzymic activities with proliferation in normal, regenerating, dif quality available. ferentiating, or neoplastic tissues. In contrast, the specific activity Animals and Tumors. The maintenance of tumor-bearing and normal rats, the killing of animals, the excision of tissues, and the studies on 1Supported by Grants CA 13526 and CA 05034 from the National Cancer regenerating and differentiating liver were conducted as reported earlier Institute, NIH. 2 Visiting Assistant Professor at Indiana University School of Medicine. Perma (22). Liver tumors representing a wide spectrum of proliferation rates, hepatomas 16, 9618Am, and 3924A, were investigated. The very slowly nent address: Department of Biochemistry, Yokohama City University School of Medicine, Yokohama, Japan. 3 Visiting Associate Professor at Indiana University School of Medicine. Perma 5 The abbreviations used are: amidotransferase, amidophosphoribosyltransfer nent address; National Institute of Oncology, Budapest, Hungary. ase; HPRT, hypoxanthine phosphoribosyltransferase; PRPP, 5-phosphoribosyl 1- 4 To whom requests for reprints should be addressed. pyrophosphate; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine Received December 22,1983; accepted March 12,1984. guanine phosphoribosyltransferase; GPRT. guanine phosphoribosyltransferase.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. V. Natsumeda et al. growing hepatoma 16, the intermediate 9618Am, and the rapidly growing it was not inhibited by excess levels of PRPP up to 2 ITIM.For 3924A took 52, 8, and 3 weeks after s.c. inoculation, respectively, to liver and hepatoma, the double-reciprocal plots yielded apparent reach a diameter of 1.5 cm. For investigation of organ distribution of the Kmof 2 MM- enzymic activities, male Wistar rats, weighing 180 to 220 g, were used. The effects of PRPP on liver and hepatoma HGPRT activities Samples from the intestines were obtained by carefully scraping off the are compared in Chart 2. The enzyme activity in both normal mucosa; the muscle layer was not involved. and neoplastic tissues was saturated at a PRPP concentration C57BL/6J x DBA/2J F, mice carried s.c. implants of Lewis lung carcinoma. The human renal cell carcinoma and hepatocellular carcino of about 50 MM,and it was not inhibited by PRPP levels up to 2 mas were obtained from patients who underwent resection at the hos rriM. In liver and hepatoma, an apparent Kmof 4 MMwas observed. pitals of Indiana University. The histologically normal parts of the host Effect of Concentrations of Adenine, Hypoxanthine, and tissues were used for controls. Guanine. APRT activity in liver and hepatoma was saturated at Assay of Purine Phosphoribosyltransferase Activity. Twenty % an adenine concentration of approximately 10 MM(Chart 3). The homogenates (w/v) of freshly excised tissues were prepared in 0.15 M enzyme activities were not inhibited by adenine concentrations KCI solution adjusted to pH 7.4 with 2% KHCO3 solution. The homoge- up to 0.4 mM. Apparent Kâ€ž,swere0.6 MMfor liver APRT and 0.9 nate was centrifuged at 100,000 x g for 30 min at 4Â°.The clear, lipid- MMfor hepatoma APRT. free supernatant was used for the enzyme assays. No loss in the activity The substrate velocity curves with hypoxanthine (Chart 4) and of APRT, HPRT, or GPRT was observed during storage of the super natant for at least 4 weeks at -20Â°. with guanine (Chart 5) for HGPRT both in liver and hepatoma Enzyme assays were based on separation of the product 14C-labeled were hyperbolic, and enzyme activities were saturated at sub nucleotide from the substrate 14C-labeled base and from other reaction strate concentrations of approximately 20 MM.The HGPRT activ products by high-voltage electrophoresis. The reaction mixture contained ities were not inhibited by excess levels of hypoxanthine or 70 mm Tris-HCI, 1 rriM PRPP, 4 mm MgCI2, and [14C]purine base in a final volume of 25 Â¡A.The final concentration of 14C-labeled base was either 0.39 ITIM [8-14C]adenine, 0.30 mw [8-14C]hypoxanthine, or 0.29 ITIM[8-14C]guanine. The final pH of the reaction mixture was 7.4. Reaction HEPATOMA 392Â«A was performed at 37Â°and terminated by the addition of 5 Â¡Aof 2.1 N HCIOÃat 0, 5, and 10 min. After 30 min in an ice bath, the mixture was neutralized with 5 Â»ilof2.21 N KOH. After centrifugaron, a 20-//I aliquot was spotted on Whatman No. 3MM chromatography paper (WH-330) with 30 nmol of each punne base, nucleotide, and nucleoside as camers. To determine the possible interference with the assay by other enzymic activities such as 5'-nucleotidase, xanthine oxidase, and guanine de- ammase, parallel reactions without PRPP were carried out as described by Kizaki and Sakurada (7). High-voltage electrophoresis was performed at 3000 V for 30 min in 50 mw sodium borate buffer (pH 9.0). The separation of purine compounds by high-voltage electrophoresis was as follows (the migration towards the anode is given in cm): (a) for APRT -600 -WO -200 0 200 WO 600 800 1000 assay, adenine (0.9), adenosine plus inosine (4.2), AMP (6.7); (b) for I/S (mM)-1 HPRT assay, hypoxanthine (2.6), inosine plus xanthine (4.4), IMP (6.7); and (c) for GPRT assay, guanine (1.1), guanosine plus xanthine (4.3), 0.02 0.0Â« 0.06 O.OB 0.10 GMP (7.0). UV-absorbing areas were cut out and counted in a liquid scintillation counter. Chart 1. Effect of PRPPconcentration on APRT activity in liver and hepatoma Assay of Amidotransferase Activity. This enzyme activity was mea 3924A. The activity was measured under the standard assay conditions described in "Materials and Methods," except that PRPPconcentration was varied. sured as described by Prajda ef al. (10). Protein was determined by a standard method (8) with bovine serum albumin as standard. Expression and Evaluation of Results. The enzyme activities were calculated in nmol of product formed per hr per mg protein (specific activity). The results were subjected to statistical evaluation by the f test for small samples. Differences between means giving a probability of less than 5% were considered as significant.

RESULTS AND DISCUSSION

Comparison of Kinetic Conditions of Purine Phosphoribo- syltransferases in Liver and in Hepatoma. To develop a stand ard assay for purine phosphoribosyltransferase activity in the crude system and to establish that the enzyme activity measured reflected the enzyme concentration, the kinetic properties of purine phosphoribosyltransferases were determined in extracts from normal liver (ACI/N rats) and rapidly growing hepatoma 3924A (carried in ACI/N rats). 0.02 0.0Â« 0.06 O.Oe 0.10 Effect of PRPP Concentration. Chart 1 shows the effect of PRPP concentration on APRT activity in normal liver and in Chart 2. Effect of PRPPconcentration on HGPRTactivity in liver and hepatoma 3924A. Hypoxanthine was used as the substrate, and the activity was measured hepatoma 3924A. The enzyme activity in both tissues was under the standard assay conditions described in "Materials and Methods,' except saturated at a PRPP concentration of approximately 20 MM,and that PRPPconcentration was varied.

2476 CANCER RESEARCH VOL. 44

HEPATOMA 392IJA

HEPATOMA 3924A

1400 -1000 -600 -200 0 200 GOO 1000

-Â«00 -200 0 200 Â«00 600 800 1000 I/S (mM)-1 0 0.02 0.00 O.OC 0.0Â« O.IO 0.12 0.14 0.16

AOENINE i-vi 0.02 0.04 0.06 0.08 0.1

Chart 3. Effect of adertineconcentration on APRT activity in liver and hepatoma GUANINE (mM) 3924A. The activity was measured under the standard assay conditions described in "Materials and Methods," except that adenineconcentration was varied. Chart 5. Effect of guanine concentration on HGPRT activity in liver and hepa toma 3924A. The activity was measured under the standard assay conditions described in "Materials and Methods," except that guanine concentration was varied.

APRT and HGPRT, compete for PRPP with the purine de novo rate-limiting enzyme, amidotransferase, for which the apparent KmSrange from 400 to 900 /tw in liver and hepatoma (10). The PRPP content was recently determined in liver (5.6 Â±0.4 nmol/ g) and in hepatoma 3924A (10.3 Â±0.7 nmol/g) (19). The steady state concentrations of PRPP would confer an advantage on the salvage enzymes over the amidotransferase. HGPRT competes with xanthine oxidase for hypoxanthine and also with guanine deaminase for guanine. The apparent K,,,s for the catabolic enzymes have already been determined in this

-100 -200 0 200 400 Â«00 BOO 1000 laboratory, yielding K,,,s for hypoxanthine of xanthine oxidase in I/S ImM)-' liver, 4 IJM;and in hepatoma 3924A, 3 //w6 and Kms for guanine of guanine deaminase in liver, 7 /IM; and in hepatoma 3924A, 8 0 0.02 0.04 0.0Â« 0.06 0.10 0.12 0.14 0.16 /IM/ Thus, the affinities for hypoxanthine and guanine of the HYPOXANTHINE (mM) purine catabolic enzymes were similar to those of HGPRT in liver Chart 4. Effect of hypoxanthine concentration on HGPRT activity in liver and and in hepatoma. Therefore, the ratios of the enzymic activities hepatoma 3924A. The activity was measured under the standard assay conditions described in "Materials and Methods," except that hypoxanthineconcentrationwas should govern the direction of the routing of the metabolites. varied. Purine Phosphoribosyltransferase and Amidotransferase Activities in Various Rat Organs. Table 1 shows the activities of the purine synthetic enzymes in 12 different rat organs. guanine up to 0.4 mM. For both liver and hepatoma HGPRT for Amidotransferase activities were generally high in organs of high hypoxanthine and guanine an apparent Kmof 3 /Â¿Mwasobserved. cell renewal (thymus, testis, bone marrow, and mucosa of small Effect of pH. The pH profiles for liver and hepatoma APRT intestine) and low in heart and skeletal muscle, whereas the had a similar pattern with a broad optimum at pH 7.5 to 10. highest HGPRT activities were in heart and testis, and the lowest APRT activity at physiological pH 7.4 was 90% of that observed were in small intestine and skeletal muscle. The APRT activities at the optimum pH 8.5. The pH profiles for liver and hepatoma were highest in colon and spleen, and the lowest were in skeletal HGPRT were similar, although the enzyme exhibited different pH muscle, brain, and testis. Thus, the activities of the purine optima with the different substrates: (a) hypoxanthine, pH 10 to phosphoribosyltransferases were not linked with high cell-re 10.5; and (b) guanine, pH 8.5 to 9.5. At pH 7.4, HPRT activity newal rates, but the activities of the salvage enzymes were was 50%, and GPRT activity was 75% of the activities observed markedly higher than that of amidotransferase. An exception at the optimum pH. was the small intestine, where the activity of amidotransferase Through systematic kinetic studies, a standard assay was was higher than that of HGPRT. established for determination of purine phosphoribosyltransfer- Purine Phosphoribosyltransferase Activities in Normal, Dif ase activities in liver and hepatomas. In the standard assay of ferentiating, and Regenerating Liver and in Hepatoma. The APRT and HGPRT with hypoxanthine and guanine as substrates behavior of purine Phosphoribosyltransferase activities in prolif in liver and hepatoma 3924A, good proportionality was achieved erating and neoplastia tissues was studied in 3 hepatomas of with incubation time up to 12 min and with amount of enzyme vastly different proliferation rates, and in developing and regen- added up to 10 ng of protein. Affinities of Purine Salvage, de Novo, and Catabolic En 6N. Prajda and G. Weber, unpublishedobservations. zymes for Shared Substrates. The purine salvage enzymes, 7H. Kizaki and G. Weber, unpublishedobservations.

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Tabtel Furine phosphoribosyltransferaseand amidotransferaseactivities in rat organs The activity was measured in 100,000 x g supernatant from tissue homogenates as described in "Materials and Methods." Enzyme activity (nmol/hr/mg protein)

pathwaysHPRT235 novopathwayAmidotransferase182 TissuesThymusSpleenTestisBone Â± 8a(107f842 Â±17(58)388 Â±40(63)1290 (272)64 Â± 9(170)273 Â± 7(96)463 Â±70(101)1540 (96)174(260)118(176)112(167)72(107)67(100)86(128)47 Â± 1(55)499 Â±6(114)280 Â±50(120)782 marrowSmall Â±25(101)464 Â± 6(69)42 Â±31(61)80 intestineColonLiverRenal Â±58(94)11 Â± 6(10)118Â± Â± 5(6)295 90 Â±85(240)495 5(29)405 Â±17(23)1280 Â±12(100)615 Â±8(100)227 Â±23(100)763 cortexLungHeartBrainSkeletalÂ± 5(124)559 Â±11(56)140 Â±62(60)460 Â±81(113)694 Â±13(35)533 Â±45(36)1760 (70)16 Â±76(140)257 Â±10(132)338 Â±69(138)1350 (24)43 Â± 1(52)150Â± Â± 4(83)57 Â±41(105)211 (64)10 muscleAPRT529 5 (30)Salvage Â± 5 (14)GPRT802 Â± 9 (16)De (15) * Mean Â±S.E.of 3 or more samples. 6 Numbers Â¡nparentheses,percentages of those observed in liver.

Table2 Purinephosphoribosyltransferaseand amidotransteraseactivities in hepatomasof different growth rates and in differentiating and regeneratingliver The activities were measured as described in "Materials and Methods."

Enzymeactivity (nmol/hr/mg protein)

Salvage pathways De noyÃ³pathway Tissues APRT HPRT GPRT Amidotransferase 539Â±19a(100)Â£> Liver (Buffalo),control for hepato 367 Â± 9(100) 1170 Â± 43(100) 69 Â±5(100) mas 16 and 9618Am

Liver (ACI/N) control for 3924A 498 Â± 2 (100) 460 Â±5(100) 1470 Â± 39(100) 75 Â±3(100) Â±30(271)c Â± 77(277)Â° Hepatoma 16 Â± 3 (99) Â±6(146)Â° 828 Â±44 (155)c 668 Â±22(182)Â° 2030 Â±150(174)Â° NO" Hepatoma9618Am 851 Â±23 (170)Â° 325 Â±11 (70)c 155 Â±5(207)Â° Hepatoma 3924A 1010Â± 32 (70f Adult liver 497 Â±13 (100) 403 Â± 9(100) 1280Â± 25(100) 67 (100) 348+12(86)c512 day)Sham-operatedNewborn liver (9 462 Â±21(93)454 1310 Â±46(102)1440 83(124)58.4 liver 12 hr Â±16 (100) Â±42(100) Â±100(100) Â±2(100) 24 hr 432 Â±16 (100) 515 Â±19(100) 1550Â± 30(100) 57.9 Â±1(100) 48hrRegenerating 532 Â±57(100)424 597 Â±29(100)525 1580(100)1410 Â±100 57.0 Â±2(100)66.5

liver 12hr Â±37 (93) Â±43(103) Â±150 (98) Â±5(114) 634 Â±44 (147)c 24 hr 528 Â±35(103) 1460 Â± 60 (94) 87.2 Â±1(151)Â° 48 hr532 593 Â±45 (111)996 566 Â±26 (95)3240 1300 Â±100 (82)101 96.3 Â±3(169)Â° * Mean Â±S.E.of 3 or more samples. " Numbers in parentheses, percentages of the relevant control activity. c Significantly different from the respective controls (p < 0.05). " ND, not determined. erating liver (Table 2). Purine Phosphoribosyltransferase and Amidotransferase In slowly growing hepatoma 16, the activities of APRT, HPRT, Activities in Mouse Lewis Lung Carcinoma, Human Renal Cell and GPRT were 99, 271, and 277% of normal liver values, Carcinoma, and Hepatocellular Carcinomas. In mouse Lewis respectively. In hepatoma 9618Am of medium growth rate, the lung carcinoma, in human renal cell carcinoma, and 2 cases of APRT and HGPRT activities were 1.6- to 1.8-fold higher than in human hepatocellular carcinoma, amidotransferase activity was normal liver. In rapidly growing hepatoma 3924A, APRT activity significantly increased over the values of the relevant normal was higher (170%), whereas HGPRT activity was lower (70%) host tissues (Table 3). The activities of the salvage enzymes than that of the normal liver. were markedly elevated in the rapidly growing Lewis lung carci The specific activities of APRT and HGPRT in newborn and noma as compared to the values of normal mouse lung. How regenerating liver were not significantly different from those in ever, there was no consistent pattern of alteration in the human adult liver and in sham-operated liver. Thus, no correlation of neoplasms. It is relevant to the main points of this paper that all purine phosphoribosyltransferase activities with tissue prolifera the activities of the salvage enzymes in normal or neoplastic tion rates was observed; this agrees with the investigations of tissues were markedly higher than those of the amidotransfer Rhoads and Morris (14). However, the novel observation we ase. Thus, the enzymic capacity of the salvage pathways was emphasize is that the activities of APRT, HPRT, and GPRT were higher than that of amidotransferase, 8.2- to 32-fold in Lewis 2.1- to 32-fold higher than that of the amidotransferase in the lung carcinoma, 3.5- to 22-fold in human kidney tumor, and 3.4- control livers and the hepatomas. to 30-fold in human hepatoma.

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Table 3 Purina phosphoribosyltransferase and amidotransferase activities in mouse Lewis lung carcinomas, human renal cell carcinoma, and hepatocellular carcinomas Enzyme activities were measured as described in "Materials and Methods.'

Enzyme activity (nmol/hr/mg protein)

pathwaysHPRT40 novo pathway TissuesMouse Amidotransferase15 Â± 0.9" (100J6 lung Â±1.5 (100) Â± 6.5(100) + 0.5(100) 981 Â±22 (2130)Â° 254 Â±1.8 (635)c 795 Â±22 (704)Â° 31 Â±0.9 (207)c Lewis lung carcinoma Human kidney 540 Â± 6.0 (100) 128 Â±3.4 (100) 385 Â±22 (100) 20 Â±0.2 (100) 672 Â± 2.6 (124)c 109 Â±3.5 (85)c 309 Â± 5.8 (80)c 31 Â±0.5(158)c Kidney tumor Human liver (Case 1) 579 Â±13 (100) 113Â±1.0(100) 296 Â± 5.0(100) 19 Â±1.1 (100) 494 Â± 4.0 (85)c 1160 Â±15 (392)c 39 Â±2.3 (205)c Hepatoma(Casel) 360 Â±1.0 (319f Human liver (Case 2) 542 Â± 9.1 (100) 115 Â±6.1 (100) 317 Â±25 (100) 21 Â±1.4(100) 29 Â±2.7 (138)Â° Hepatoma (Case 2)APRT46 541 Â±10 (100)Salvage 100 Â±0.7 (87)GPRT113 324 Â±27 (102)De * Mean Â±S.E. of 3 samples from individual lungs and lung tumors of groups of mice and 3 to 4 samples from different parts of each human tissue. " Numbers in parentheses, percentages of the control activity of the relevant normal tissue. c Significantly different from the respective controls (p < 0.05).

Biological Significance of Furine Phosphoribosyltransfer- 1982. 3. Jackson, R. C., Gouldmg, F. J., and Weber, G. Enzymes of purine metabolism ases in Furine Metabolism in Neoplastic Tissues. Earlier stud in human and rat renal cortex and renal cell carcinoma. J. Nati. Cancer Inst., ies in this laboratory showed that, in hepatomas of different 62: 749-754,1979. growth rates, the activities of key enzymes of de novo pyrimidine 4. Jackson, R. C., Monis, H. P., and Weber, G. Enzymes of the purine ribonucle- otide cycle in rat hepatomas and kidney tumors. Cancer Res., 37:3057-3065, biosynthesis and salvage increased markedly, whereas the activ 1977. ities of the degradative enzymes decreased (15). In purine syn 5. Jackson, R. C., Weber, G., and Morris, H. P. IMP dehydrogenase, an enzyme linked with proliferation and malignancy. Nature (Lond.), 256: 331-333,1975. thesis, the activities of the key enzymes of the de novo biosyn 6. Kizaki, H., Morris, H. P., and Weber, G. Behavior of inosine phosphorytase thesis of IMP and of the adenine and guanine nucleotides in (EC 2.4.2.1) activity in normal, differentiating, and regenerating liver and in creased in hepatomas (1, 3-5, 9-11, 15-17), and those of the hepatomas. Cancer Res., 40: 3339-3344,1980. purine degradative enzymes decreased (6, 9,12,13,15-17). 7. Kizaki, H., and Sakurada, T. A micro-assay method for hypoxanthine-guanine and adenine phosphoribosyltransferase. Anal. Biochem., 72:49-56,1976. The new results in this study indicate that the behavior of the 8. Lowry, 0. H., Rosebrough, N. J., Fair, A. L, and Randall, R. J. Protein activities of the purine phosphoribosyltransferases displays no measurement with the Folin phenol reagent. J. Biol. Chem., ?93: 265-275, 1951. consistent pattern in the various neoplastia tissues examined. 9. Prajda, N., Donohue, J. P., and Weber, G. Increased amidophospnoribosyt- The absence of linking of the behavior of activity of the purine transferase and decreased xanthine oxidase activity in human and rat renal salvage enzymes with normal or neoplastia proliferation might cell carcinoma. Life Sci., 29: 853-860,1981. 10. Prajda, N., Katunuma, N., Morris, H. P., and Weber, G. Imbalance of purine be accounted for, in part at least, by the 2 novel observations metabolism in hepatomas of different growth rates as expressed in behavior reported in this investigation. The activities that were orders of of glutamine-phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14). Cancer Res., 35: 3061-3068,1975. magnitude higher and the affinities for PRPP that were markedly 11. Prajda, N., Morris, H. P., and Weber, G. Glutamine-phosphoribosylpyrophos- better than those noted for the rate-limiting enzyme of purine de phate amidotransferase (amidophosphoribosyltransferase, EC 2.4.2.14) activ novo biosynthesis, amidotransferase, make it understandable ity in normal, differentiating, and neoplastia kidney. Cancer Res., 39: 3909- 3914,1979. that the reprogramming of gene expression in hepatomas does 12. Prajda, N., Morris, H. P., and Weber, G. Imbalance of purine metabolism in not require an overall step-up of activity for the purine salvage hepatomas of different growth rates as expressed in behavior of xanthine oxidase (EC 1.2.3.2). Cancer Res., 36:4639-4646,1976. enzymes. 13. Prajda, N., and Weber, G. Malignant transformation-linked imbalance: de The marked decrease in xanthine oxidase activity (12) favors creased xanthine oxidase activity in hepatomas. FEBS Lett., 59: 245-249, sparing of hypoxanthine and permits its ready recycling to IMP 1975. 14. Rhoads, A. R., and Morris, H. P. Enzymatic potential for preformed purine by action of HPRT. Moreover, the decreased guanine deaminase metabolism in hepatomas with different growth rates. J. Nati. Cancer Inst., 59: activity (18, 20) should make guanine available for salvage syn 905-910,1977. thesis of GMP through the action of GPRT. Therefore, the overall 15. Weber, G. Biochemical strategy of cancer cells and design of chemotherapy: G. H. A. Clowes Memorial Lecture. Cancer Res., 43:3466-3492,1983. purine enzymic pattern confers selective advantages to cancer 16. Weber, G., Hager, J. C., Lui, M. S., Prajda, N., Tzeng, D. Y., Jackson, R. C., cells by making them more efficient for retention and production Takeda, E., and Eble. J. N. Biochemical programs of slowly and rapidly growing human colon carcinoma xenografts. Cancer Res., 41: 854-859,1981. of precursors for synthesis of purine nucleotides. 17. Weber, G., Kizaki, H., Tzeng, D., Shiotani. T., and Olah, E. Colon tumor: These observations, showing the high capacity of salvage enzymotogy of the neoplasie program. Life Sci., 23: 729-736,1978. enzymes for recycling precursors, may explain, in part at least, 18. Weber, G., Lui, M. S., Natsumeda, Y., and Faderan, M. A. Salvage capacity of hepatoma 3924A and action of dipyridamole. Adv. Enzyme Regul., 27: 53- the apparent failure of inhibitors of the cÃenovosynthetic pathway 69,1983. of IMP production to provide lasting chemotherapeutic results. 19. Weber, G., Natsumeda, Y., Lui, M. S., Faderan, M. A., Uepnieks, J. J., and The evidence in this study provides further support for a rational Elliott, W. L. Control of enzymic programs and nucleotide pattern in cancer cells by acivicin and tiazofurin. Adv. Enzyme Regul., 22: 69-93,1984. basis for combination chemotherapy of inhibitors of the de novo 20. Weber, G., Olah, E., Lui, M. S., Kizaki, H., Tzeng, D. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1984 American Association for Cancer Research. Enzymic Capacities of Purine de Novo and Salvage Pathways for Nucleotide Synthesis in Normal and Neoplastic Tissues

Yutaka Natsumeda, Noemi Prajda, John P. Donohue, et al.

Cancer Res 1984;44:2475-2479.

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