Enzymes of Uracil Catabolism in Normal and Neoplastic Human Tissues1 / Fardos N

[CANCER RESEARCH 45, 5405-5412, November 1985]

Enzymes of Uracil Catabolism in Normal and Neoplastic Human Tissues1 / Fardos N. M. Naguib,2 Mahmoud H. el Kouni, and Sungman Cha

Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

ABSTRACT to homogeneity from rat liver cytosol (9). Dihydropyrimidinase (5,6-dihydropyrimidine amidohydrolase, Enzymes of the pyrimidine base catabolism, dihydrouracil de- EC 3.5.2.2) is the second enzyme of pyrimidine base degrada hydrogenase (EC 1.3.1.2), dihydropyrimidinase (EC 3.5.2.2), and tion. It is responsible for the reversible hydrolytic ring opening of /3-ureidopropionase (EC 3.5.1.6) were compared in the cytosolic dihydrouracil and dihydrothymine and is located in the cytosol extract of several normal and neoplastic human tissues. The fraction (12-15). activity was measured by following the catabolism of [6-14C]- /3-Ureidopropionase (A/-carbamoyl-j8-alanine amidohydrolase, uracil to dihydrouracil, carbamyl-/3-alanine, and /3-alanine. Sub EC 3.5.1.6) is the last of the pyrimidine degradative enzymes. It strate inhibition, hysteresis, allosterism, and the lack of dihydro splits the /3-ureidopropionic acid (carbamyl-ÃŸ-alanine) or /3-urei- pyrimidinase are pointed out as special problems in assaying doisobutyric acid formed by dihydropyrimidinase from dihydrour enzymes of pyrimidine degradation. The activity of dihydrouracil acil or dihydrothymine into 0-alanine or /9-aminoisobutyric acid, dehydrogenase has been demonstrated in several human extra- ammonia, and carbon dioxide (Chart 1). /3-Ureidopropionase is hepatic tissues and tumors. The enzyme is rate limiting in extra- also located in the cytosol (14, 16, 17). The reaction catalyzed hepatic solid tumors but not in their normal counterparts. Some by this enzyme is irreversible. of these solid tumors contain greater amounts of activity than Reports on dihydrouracil dehydrogenase activity in the various do their normal equivalents, which encourages the use of inhibi extrahepatic tissues are contradictory. The kidney was reported tors of this enzyme in conjunction with treatment of these tumors to be the only extrahepatic tissue to contain dihydrouracil dehy by 5-f luorouracil. Because of the lack of a pattern in dihydrouracil drogenase (4,18-20). However, this activity was later reported dehydrogenase activity between tumors and normal tissues, the to be present in rat thymus, intestinal mucosa, spleen, kidney, enzyme is not a good marker for tumorigenicity. Dihydropyrimi brain cortex, skeletal muscles, heart, lung, stomach, and bone dinase, on the other hand, is highly active in all solid tumors marrow (21,22); in mouse colon and colon tumors (23); in human studied but not in their normal counterparts; therefore, we sug kidney and kidney tumors (24); and in normal and neoplastic gest that dihydropyrimidinase can serve as a good marker of human colon, lung, and stomach (25). The early discrepancies in tumorigenicity as well as a target for cancer chemotherapy of the literature could be attributable to the assay conditions used human solid tumors. to determine enzyme activity. Higley and Buttery (26) reported that none of the standard assays developed by Grisolla and INTRODUCTION Cardoso (5), Smith and Yamada (10), or Fritzson (6) proved satisfactory. In mammalian systems the pathway for the catabolism of In the present study while determining dihydrouracil dehydro uracil, thymine, and their analogues (Chart 1) is via degradative genase activity in normal and neoplastic tissues from different reduction by which the pyrimidine ring is first reduced at positions human organs, we have initially experienced some difficulties in 5 and 6 with hydrogen from NADPH, then opened between obtaining optimal conditions for the measurements of this en positions 3 and 4, and finally split between position 1 and 2 to zyme activity. We found that substrate inhibition by uracil, en yield the corresponding <â€¢>'aminoacid, carbon dioxide, and am zyme hysteresis, and allosterism were the main factors behind monia. this difficulty. This prompted us to study the kinetic parameters Dihydrouracil dehydrogenase (5,6-dihydrouracil: NADP+ oxi- of this enzyme in the cytosol of various tissues and organisms. doreductase, EC 1.3.1.2) is the first and purportedly rate limiting We herein demonstrate that the enzyme from the various enzyme of this chain of three reactions. It is responsible for the sources studied displayed substrate inhibition by uracil, and that reversible reduction of both uracil and thymine to dihydrouracil under appropriate assay conditions, the activity of dihydrouracil and dihydrothymine, respectively (1-4). This enzyme is also dehydrogenase can be detected in various normal and neoplastic responsible for the breakdown of the widely used antineoplastic human tissues. However, no definitive pattern in the relationship agent 5-fluorouracil, and the radiosensitizing agents 5-bromo- between enzyme activity in the normal and in corresponding and 5-iodouracil, thereby limiting their therapeutic effectiveness. neoplastic tissues could be established. Dihydropyrimidinase, on Cytosine and its analogues are not substrates for this enzyme the other hand, was absent from or showed little activity in and have to be converted to uracil before entering the reductive normal extrahepatic tissues as well as all neoplastic lymphoid degradation pathway. Most reports locate dihydrouracil dehydro tissues tested. In contrast all solid tumours tested had apprecia genase activity in liver cytosol (2, 5-9). However, an additional ble dihydropyrimidinase activity. A preliminary report has been dehydrogenase activity in the paniculate fraction has also been presented (27). reported (10,11 ). Dihydrouracil dehydrogenase has been purified

'Supported by Grants CA-31706 and CA-13943 awarded by the National MATERIALS AND METHODS Cancer Institute, Department of Health and Human Services, and Grant CH-136 from the American Cancer Society. Chemicals. [6-"C]Uracil (56 mCi/mmol) was purchased from Moravek 2To whom requests for reprints should be addressed. Biochemicals, Brea, CA; [6-14C]5-fluorouracil (55 mCi/mmol) was from Received 3/18/85; revised 6/9/85; accepted 7/22/85.

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+ NH, -. CO,

K = H UKACIl OIHYDROURACIt Â«-tWEIOOntOPIOIUTE .-â€¢AlANIÂ« R=CH, THVMIHE DIHYDItOTHVMINE 0- UMEIDOIKMUTVKATf Â«-AMINOIKWUTYRATE

R= F flUOROURACIL DIHYMOFLUOROUKACIL Â«-FLUORO-Â«-UREIOOFItOPIONATE .. HUORO s ALAMINÃ• Chart 1. Pathway of pyrimidine base catabolism in mammalian systems.

Amersham Corp., Arlington Heights, IL; uracil, dihydrouracil, carbamyl- blood of a patient with immunoglobulin plasma cell leukemia (34); K-562. 0-alanine, /3-alanine, NADPH, and ninhydrin, were from the Sigma Chem an undifferentiated blast cell line established from the pleural fluid of a ical Co., St. Louis, MO; Polygram CEL 300 UN/UMand silica gel G/UVuM patient with chronic myelocytic leukemia (35); KG-1, a mixture of pre TLC3 plates were from Brinkmann Instrument Co., Westbury, NJ; Om- dominantly myeloblasts and promyelocytes derived from the bone mar nifluor was from New England Nuclear, Boston, MA; dimethylamino- row of a patient with erythroleukemia (36); and HL-60, a promyelocytic benzaldehyde was from Aldhch Chemical Co., Milwaukee, Wl, and all cell line established from the peripheral blood of a patient with acute other chemicals were from Fisher Scientific Co., Boston, MA. promyelocytic leukemia (37). Normal Tissues. Normal human organs were obtained from autop RWLy-1, is a lymphoma line established and characterized by Wie- sies, peripheral blood lymphocytes were prepared from the blood of mann." It was isolated from the pleural effusion of a patient with mixed histiocytic-lymphocyticnon-Hodgkin's lymphoma. In tissue culture, about volunteers by the method of Boyum (28), and normal murine organs were obtained from Swiss albino (CD-1) mice or Lewis rats (Charles 50% of RWLy-1 cells have the morphological appearance of small River Breeding Laboratories, Wilmington, MA). Mice were killed by malignant lymphocytes and the other 50% have the appearance of large cervical dislocation and rats by decapitation. The organs were washed histiocytes. Cell surface IgM can be detected by immunofluorescence in with ice-cold normal saline (0.9% NaCI solution) before homogenization. greater than 70% of the cells. RWLy-1 is an Epstein-Barr virus-free cell Solid Tumors. Carcinomas of the colon, established from biopsies, line. were grown as xenografts in nude mice: DLD-1, a carcinoma of the Preparation of Extracts. Cells in suspension were collected by cen- sigmoid colon, morphologically heterogeneous, varying from moderately trifugation. The cells were washed by resuspending in the homogeniza to poorly differentiated (29); clone A, a subclone of DLD-1, producing tion buffer (20 mw potassium phosphate (pH 8) containing 1 rriM EDTA poorly differentiated adenocarcinomas (29); clone D, another subclone and 1 mw mercaptoethanol). After washing twice the pellet was homog of DLD-1, producing moderately differentiated adenocarcinomas (29); enized in 2 volumes of buffer, using a polytron homogenizer (Brinkmann). DLD-2, a well differentiated adenocarcinoma of the sigmoid colon; HCT- The homogenate was then centrifugea at 105,000 x g for 1 h at 4Â°C. 15, a moderately well differentiated adenocarcinoma of the sigmoid colon The supernatant fluid (cytosol) was collected and used as the source of (29); HOT-3, a metastasis to the ovary of a patient with a sigmoid colon enzyme. Other tissues were homogenized (1:2-3, w/v) in the homoge primary cancer, producing well differentiated adenocarcinomas (30); nization buffer and the cytosol was prepared as described for neoplastic lntob-3, derived from a metastasis to the omentum of a patient with a cell lines. When used for kinetic parameter estimations, HL-60 cells were colorectal primary and producing well differentiated adenocarcinomas; treated for 7 days with the maturational agent W,A/-dimethylformamide and OM-1, derived from a metastasis to the omentum of the same (10.8%) before extracting the cytosolic fluid. This treatment was neces patient that gave rise to HOT-3, also producing well differentiated ad sary to increase the level of dihydrouracil dehydrogenase activity some enocarcinomas (30). 10-fold (38). DAN, a carcinoma of the pancreas, was grown in culture. This cell line Enzyme Assays. Dihydrouracil dehydrogenase activity was deter was derived from a metastasis to the liver of a patient with pancreatic mined by measuring the sum of the products, dihydrouracil, carbamyl-j3- primary cancer. Histologically the specimen is described as a squamous alanine, and 0-alanine, formed from [6-14C]uracil. The standard reaction cell carcinoma (31 ). mixture contained 10 mM potassium phosphate (pH 8), 0.5 mw EDTA, Carcinomas of the pancreas, derived from liver biopsies from patients 0.5 mW mercaptoethanol, 2 mw dithiothreitol, 5 mw MgCI2, 25 //M with primary cancer metastatic to the liver, were grown as xenografts in [6-'4C]uracil (56 mCi/mmol), 100 MM NADPH, and 25 n\ cytosol (5 mg nude mice; RWP-1, a liver metastasis from a primary duct cell of the protein/ml) in a final volume of 50 n\. Incubations were carried out at head of the pancreas; and RWP-2, a moderately well differentiated duct 37Â°Cfor 5 to 30 min, except where stated otherwise. The reaction was cell adenocarcinoma of pancreatic origin (32). terminated by immersing the reaction tubes (1-ml Eppendorf tubes) in a LX-1, a carcinoma of the lung, was grown in culture. This cell line was boiling waterbath for 1 min, the reaction tubes were then frozen at established from a metastasis to the arm of a patient with an inoperable -20Â°C for at least 20 min before any further manipulations were under primary oat cell carcinoma of the lung. Histologically the specimen is taken. Proteins were removed by centrifugation and 5 Â¡Aof the super described as a nodule of poorly differentiated carcinomas (33). natant fluid were spotted on cellulose TLC plates which were prespotted HLN-3, a salivary gland carcinoma, was grown as xenografts in nude with 5 /il of a standard mixture of 10 mw uracil, carbamyl-,Â¡-alanine, ÃŸ- mice. This carcinoma was established by Dr. D. L. Dexter and colleagues alanine, and 25 mw dihydrouracil. The plates were then developed from a cervical lymph node metastasis following appearance of an overnight in the top phase of a mixture of n-butanol:water:ammonium unspecified salivary gland primary site. hydroxide (90:45:15, v/v/v). Uracil was identified by UV quenching and HST-2, a carcinoma of the stomach, was grown as xenografts in nude /3-alanine by spraying with 0.2% ninhydrin in 95% ethanol. Dihydrouracil mice. This carcinoma was established by Dr. D. L. Dexter and colleagues and carbamyl-|8-alanine were visualized by dyeing with 5% dimethylami- from a portion of the stomach of a patient with a moderately well nobenzaldehyde in 50% ethanol:1 N HCI, after dihydrouracil has been differentiated adenocarcinoma. hydrolyzed to carbamyl-/J-alanine by spraying with 0.5 N KOH in 50% Neoplastia Hematopoietic Tissues. Leukemic cell lines were grown ethanol. R( values for dihydrouracil, uracil, and carbamyl-/3-alanine plus in culture: ARH-77, a B-lymphoblast line established from the peripheral /3-alanine were 0.46, 0.23, and 0.09, respectively. Spots were cut out

3 The abbreviation used is: TLC, thin layer chromatography. 4 M. C. Wiemann. details to be published elsewhere.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES and placed in vials subsequently filled with 20 ml of Omnifluor-based scintillant. Radioactivity was counted in a Packard Tri-Carb Model 460 scintillation counter. The presence of dihydropyrimidinase and /3-ureidopropionase activities was inferred from the formation of carbamyl-/3- alanine and 0-alanine, respectively, in the dihydrouracil dehydrogenase 0.03 assay described above. On cellulose TLC plates, carbamyl-j-alanine and /3-alanine do not separate from one another. Therefore they were sepa X rated from each other on silica gel TLC plates. The plates were developed 9 E in chloroform:methanol:formic acid (65:18:1, v/v/v). The R( values for â€¢v uracil, carbamyl-.i-alanine. dihydrouracil, and .Â¡-alaninein this system ^c were 0.66, 0.55, 0.24, and 0.13, respectively. This system was not used 10.02 routinely for the separation of all uracil catabolites because of the frequent o difficulty in visualizing dihydrouracil after dyeing with dimethylaminobenz- aldehyde. Protein Estimation. Protein concentrations were determined by the method of Bradford (39) as described by the Bio-Rad Laboratories (40), using bovine ^ -globulin as a standard. 0.01

RESULTS

Dihydrouracil Dehydrogenase Activity versus Time and Amount of Enzyme. Chart 2 showsthat in mouseliver,withina 0.01 0.02 0.03 certain limit of incubation time and protein concentration, dihy l/(Urocil), juM-' drouracil dehydrogenase activity was linear with respect to time Chart 3. Activity of dihydrouracil dehydrogenase in human liver cytosol. Plot of after an initial lag and to amount of enzyme. The time lag is a 1/velocity versus 1/uracil at two fixed concentrations of NADPH of 100 JIM(â€¢)and distinctive characteristic of hysteresis (41). Similar results were 500 UM(â€¢)â€¢Thereaction mixture was incubated at 37Â°Cfor 10 min. Points, mean from three determinations. The kinetic parameters estimated from the straight line obtained with the promyelocytic line HL-60 and human and rat portion of each curve were: Km = -580 and 14 ^M uracil, respectively; and Vâ€žÂ»= livers (data not shown). In contrast in peripheral blood lympho -513 and 155 pmol/min/mg protein, respectively. cytes dihydrouracil dehydrogenase activity (120 pmol/min/mg) was strictly linear with respect to time from 0-30 min. Substrate Inhibition by Uracil and NADPH. Charts 3 and 4 o.o2r show that high concentrations of uracil inhibited dihydrouracil dehydrogenase from human and rat livers, respectively. Inhibition 'S by high concentrations of uracil was also detected with extracts E v from the promyelocytic cell line HL-60, DAN, LX-1, and mouse C E liver (data not shown). Chart 5 shows that high concentrations >. of the cofactor NADPH also inhibited dihydrouracil dehydrogen ase from HL-60 enzyme. Moreover the concentration of either 20.01

100 x 15 mg 80 0.1 1/lUrocil), 60 Chart 4. Activity of dihydrouracil dehydrogenase in rat liver cytosol. Rot of 1/ velocity versus 1/uracil at a fixed concentration of NADPH of 100 Â»u.The reaction mixture was incubated at 37Â°Cfor 5 min. Points, mean from three determinations. The kinetic parameters estimated from the straight line portion of the curve were: 40 Km = 6 UM uracil and Vâ„¢,= 242 pmol/min/mg.

substrate or cofactor mutually influenced the concentration at 20 which the other became inhibitory. With a higher concentration of the cofactor (1 mMor above), inhibition was observed at lower substrate concentrations. Table 1 also lists the concentrations of uracil and NADPH at which optimal rates were observed, as 20 40 60 5 IO well as the conditions under which these estimates were ob TIME, min P ROTEI N, mg/ml tained. Chart 2. Activity of dihydrouracil dehydrogenase from mouse liver. Assay con ditions are as described in "Materials and Methods." Points, mean from three Determination of the Apparent Km and Vmâ€žinVarious determinations. A, activity with time shows hysteresis (time lag); B. linearity with Tissues. It was not possibleto measuredihydrouracildehydro amount of enzyme. genase activity in various tissues at one optimal substrate con-

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES centration. Indeed the standard assay conditions chosen were centration had to be raised to 0.5 mu (approximately 5.5 x the slightly inhibitory to the HL-60 enzyme; nonetheless these con estimated K,,,value for NADPH) in order to obtain a Kmvalue for ditions were adopted because they were appropriate for the uracil, otherwise the double reciprocal plot gave a negative Km enzymes from most other tissues tested. The linear portion on value. Negative Km values were also observed with human the double reciprocal plot was used to determine the apparent platelets and mouse kidney extracts. These results indicate that Kmvalues for uracil and NADPH in the different tissues presented dihydrouracil dehydrogenase from these sources is of the allo- in Table 1, along with other kinetic parameters. The data should steric type. The depletion of NADPH by other liver dehydrogen- be taken as approximate values, since the enzyme used was ases has been assessed and ruled out. not purified. Nonetheless the results in Table 1 show that the Km Dihydrouracil Dehydrogenase Activity in Normal and Neo- values for uracil [5.8 Â±0.7 (SD) fM] and NADPH [9.6 Â±0.6 UM] plastic Human Tissues. Table 2 shows the levels of dihydrour estimated for rat liver enzyme are compatible with the Kmvalues acil dehydrogenase activity in various normal and neoplastic reported for uracil [1.8 ^M] and NADPH [11 ^M], with purified rat human tissues. Of the normal tissues, peripheral blood lympho liver enzyme (9). The differences in estimated Kmvalues between cytes and liver contained the highest activities. Of the neoplasms, various tissues suggest that, at least in humans, there may exist both the leukemic cell line, KG-1, and the pancreatic carcinomas, more than one isozymic form of this enzyme. DAN and RWP-1, had relatively high levels of activity, equivalent Chart 3 shows that in the human liver extract, NADPH con- to that of normal liver, yet lower than that of normal lymphocytes. When compared with their normal counterparts no significant 0.05 difference (P > 0.05) was observed between normal intestinal mucosa and colon tumors or between normal lung and the lung tumor LX-1. In contrast the activity of the pancreatic tumors DAN and RWP-1 were significantly higher (P < 0.01) than that of the normal pancreas. Therefore we could not assign any distinctive pattern to enzyme activity in tumors relative to the corresponding normal tissues. Formation of Dihydrouracil, Carbamyl-0-alanine and 0-Ala- nine from "C-Uracil by Various Human Tissues and Organs. Table 2 also shows the relative amounts of dihydrouracil, carbamyl-/3-alanine, and 0-alanine formed from uracil by the cytosolic extract of different human tissues and organs. In normal liver 90% or more of the products formed were carbamyl-0-alarÃ¯me and n'-alanine, while in normal lung and pancreas only 5-8% of the total product formed was carbamyl-j8-alanine and /3-alanine. In normal peripheral blood lymphocytes and intestinal mucosa, the catabolism of uracil could not be detected beyond dihydrour acil, indicating a very low or a lack of dihydropyrimidinase activity. As for the neoplasms, all of the human leukemic lines tested as well as the lymphoma line RWLy-1 accumulated their products as dihydrouracil. In contrast all solid tumours catabolized uracil I/(NADPH), >uM-Â« to carbamyl-(8-alanine and /3-alanine. These results indicate that Charts. Activity of dihydrouracil dehydrogenase in HL-60. Ptot of 1/velocity dihydropyrimidinase is highly active in all of the solid tumors versus 1/NADPH at a fixed concentration of uracilof 100 MM.The reaction mixture was incubated at 37Â°Cfor 5 min. Points, mean from three determinations. The tested, unlike their normal counterparts. Furthermore the present kinetic parameters estimated from the straight line portion of the curve were: Km= results also show that in solid neoplasms, with the exception of 2 MMNADPHand Vâ„¢,= 45 pmol/min/mg. the pancreatic tumor RWP-1, the pattern of uracil catabolism

Table 1 Kinetic parameters of dihydrouracil dehydrogenasefrom various tissues Kmvalues for uracil were determined using 100 MMNADPH and uracil concentrations ranging from 4 to 100 MM.Kmvalues for NADPH were obtained using 25 MM uracil and NADPH concentrations ranging from 4 to 50 MM.

HL-601.1 lymphocytes18.7 liver14.3Â± liver5.8 liver9.3 Â±0.2*39.3 1.46155.1 Apparent(MM)Vâ€žÂ» Km(uracil) Â±1.8137 Â±0.7241.9 Â±0.9724.7 (pmol/min/mg)Vâ€ž^Kâ€ž[Uracil] Â±1.735.7501.7Â±0.1C45.2.3Â±4.97.3>80ND"NDNDNDHumanÂ±3.010.817587 Â±10.141.7409.6 Â±18.978.3>100900

rate)Apparent(MMat optimum

(MM)Vâ„¢,Km(NADPH) .4 Â±21.738.7 Â±0.6220.6 Â±1.90016,600 (pmol/min/mg)Vâ€ž,/Km[NADPH] Â±0.626.15Human Â±7.00.4NDRat Â±3.923.080Mouse Â±33,10018.2ND

(MMat optimum rate)Human " Mean Â±SD. 6 Determinedat 500 MMNADPH. ' Determinedat 100 MMuracil " ND, not determined.

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Table 2 Activity of dihydrouracil dehydrogenase and distribution of the products formed from uracil by the extracts of various human tissues Assay conditions and tissue sources are those described in "Materials and Methods.'

SpÃ©cifieactivity formedCarbamyl-0-alanine47850066444846555636385231958565400000.(â€¢AlanmeI2bb0030384344393837354835635404100000cof product protein)TissuesNormalLiverPancreasLungIntestinal (pmol/min/mg

ofvariationwithin

Â±SO21 samples0.01-0.040.18-0.380.04-0.090.05-0.180.01-0.020.09-0.220.10-0.470.11-0.150.10-0.850.05-0.070.14-0.500.10-0.370.36-0.470.03-0.300.01-0.160.01-0.090.07-0.170.07-0.150.36-0.470.09-0.300.03-0.260.02-0.050.12-0.500.16-1.30Dihydrouracil11929510010041891066272803385745100100100100100%

.5Â±6.04.3 Â±1.35.2 Â±2.87.1 mucosaLymphocytesNeoplasmsSolid Â±6.9106.3 Â±21.53.8

tumorsColonDLD-1Clone

Â±0.42.9 AClone Â±1.33.7 DDLD-2HCT-15HOT-3lntob-3OM-1LungLX-1PancreasDANRWP-1RWP-2SalivaryÂ±0.40.9 Â±0.22.4 Â±0.52.8 Â±2.81.9 Â±1.64.0 Â±3.215.4

Â±12.124.3 Â±5.3e16.3 Â±2.1Â°4.1 Â±0.15.1 glandHLN-3StomachHST-2Hematopoietic Â±1.84.0

Â±3.33.5 tumorsLeukemic cellsARH-77K-563KG-1HL-60Lymphoma Â±2.37.8 Â±2.625.4 Â±6.71.9 Â±0.31.2 cellsRWLy-1" Â±0.2N*322222222222233222233333Coefficient tested.*N, number of samples Counted with car bamyl-, i-alanmeMean Significantly (P < 0.01) different from normal pancreas. resembles that of the liver, in that the major product formed (70- to the results of others (4, 18-20) who could not demonstrate 100%) was carbamyl-ii-alanine and /8-alanine as opposed to their dihydrouracil dehydrogenase activity except in livers and kidneys normal equivalents, where the major product formed (90-100%) of various animals. Our results indicate that the failure of those was dihydrouracil. investigators (4, 18-20) to demonstrate this activity in extrahe- Activity toward Fluorouracil. Table 3 shows the activity of patic and extrarenai tissues could be due to complications in dihydrouracil dehydrogenase from various tissues toward uracil their assay system arising from substrate inhibition, hysteresis, or 5-fluorouracil as well as the distribution of the 5-fluorouracil allosterism, and the absence of dihydropyrimidinase from the catabolites. The results indicate that in all tissues tested, 5- catabolic pathway of pyrimidine bases. fluorouracil is a better substrate for dihydrouracil dehydrogenase Dihydrouracil dehydrogenase from various tissues and organs than uracil. Dihydrofluorouracil too may be a better substrate for is inhibited by both substrates, uracil and NADPH (Charts 3-5). dihydropyrimidinase than dihydrouracil. Table 3 also shows dif We have reported substrate inhibition by uracil (27), and Queener ferences in the ratio of dehydrogenase activity toward 5-fluo ef al. (21) observed substrato inhibition by NADPH. Pero ef al. rouracil relative to that toward uracil in human liver and peripheral (42) showed the same phenomenon occurring with thymine in blood lymphocytes, which supports our suggestion that the human platelet extracts. Inhibition of dihydrouracil dehydrogen lymphocyte enzyme may be a different isozyme form from that ase by its substrates appears to be a general characteristic of of the liver. Similar results were observed with the enzymes from this enzyme, although the substrate concentration at which the liver and kidney of the rat (Table 3). inhibition occurs may differ from one tissue or animal to the other. For example we found that optimal substrate concentra tions established for extracts from mouse liver ([uracil] = 0.25 DISCUSSION mw; [NADPH] = 3 mw) were inhibitory for human extrahepatic The present results demonstrate the occurrence of dihydrour tissues. Whether or not substrate inhibition plays a role in the acil dehydrogenase activity in all the tissues tested, in agreement regulation of pyrimidine base catabolism in vivo remains to be with the results of some investigators (21-25). This is contrary determined.

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Tables Activity of dihydrouracil dehydrogenase toward uracil or 5-fluorouracil and distribution of the products formed from 5-fluorouracil Assay conditions are those described in "Materials and Methods."

Substrate % of product formed from 5-fluorouracil (pmol/min/mg protein)

Fluoroureidopropionate Tissue Uracil 5-Fluorouracil Ratio Dihydrofluorouracil + fluoro-.i-alanine Â±5.06 Human liver Â±2.5 Human lymphocytes 88.8 Â±6.2 216.7 Â±6.2 2.4 100 0 Rat liver 95.9 Â±6.9 400.0 Â±2.9 4.2 0 100 98a Rat kidney26.4 29.1 Â±3.7110.3 45.9 Â±7.04.2 1.51 299 uracil.hRatio of enzyme activity toward 5-fluorouracil to that toward Moan -*- Cn

Chart 2A shows that the mouse liver enzyme definitely dis dropyrimidinase may be the rate limiting enzyme in human extra- played a lag period, indicative of hysteresis (41). Similar results hepatic tissues and hematopoietic neoplasms, as 90% or more were observed with enzymes from HL-60 and human and rat of the total product formed was dihydrouracil. livers (data not shown). However, the peripheral blood lympho Dihydrouracil dehydrogenase degrades the widely used anti- cyte enzyme did not exhibit any such time lag, and among all cancer agent 5-fluorouracil more efficiently than it does the the tissues tested, only human lymphocytes lacked this charac natural substrates uracil and thymine (Table 3, Refs. 9, 22, 46, teristic. Therefore it may be considered that, in humans, the and 47). Nevertheless few inhibitors for this enzyme are presently lymphocyte has an isozyme different from that of the liver. available, even though it has been reported that the antitumor In addition to hysteresis some human tissues, e.g., liver and activity of 5-fluorouracil can be potentiated by 5-cyanouracil (48) platelets but not lymphocytes, showed negative Km values on and 5-diazouracil (49). The reason that (Â»administration of di regular double reciprocal plots. This is indicative of allosterism, hydrouracil dehydrogenase inhibitors with 5-fluorouracil has not /.e., a sigmoid curve on the velocity versus concentration plot. In been popular is the fact that it was generally accepted that human liver this allosterism disappears when the concentration tumors lack or possess very little of this activity (50-52). This of NADPH is increased to 0.5 Ã•TIM(Chart 3). The absence of assumption was made on the basis of studies on mouse and rat allosterism from the lymphocyte and its presence in the liver tumors (18,19, 22, 23, 53, 54) which may not represent human enzyme support our contention that dihydrouracil dehydrogen tumors. Dihydrouracil dehydrogenase is present in all of the ase may have more than one isozymic form. This contention is human tumors studied (Table 2; Refs. 24 and 25). Furthermore further supported by the observed differences in Km values for our results (Table 2) and those of others (24, 25) show that in all the enzyme from various tissues (Table 1) as well as in the ratio the human tumors studied, with the exception of kidney (24) and of the activity toward 5-fluorouracil to that toward uracil between liver (25) tumors, dihydrouracil dehydrogenase activity was human liver and lymphocytes, and between rat liver and kidney equivalent or higher in the tumors than in their normal counter (Table 3). The existence of two isoenzymes for dihydrouracil parts. Maehera et al. (25) showed that human colon, stomach, dehydrogenase was reported in rat liver cytosol (8). and lung tumors had similar activity to that of normal tissues. In It could be argued that our results on enzyme activities in addition our results show that in certain tumors such as DAN, organs obtained from autopsies may not represent the actual RWP-1, or KG-1, the activity is high and equivalent to that of activity in fresh tissues. However, the enzymes of pyrimidine normal liver (Table 2). Therefore we suggest that active search base catabolism from human liver and lymphocytes were quite for inhibitors of dihydrouracil dehydrogenase activity may be stable when stored at -10Â°C, or after repeated freezing and useful in the treatment of, at least, these types of tumors with thawing, for over 1 week. These enzymes were also stable when 5-fluorouracil or similar compounds. stored at -70Â°C for several months. Similar remarks were made The catabolism of uracil did not proceed substantially beyond for the enzyme from rat liver (43). Furthermore we found that dihydrouracil in normal human pancreas, lung, intestinal mucosa, the activity toward 5-fluorouracil in human liver autopsy speci peripheral blood lymphocytes, all of the leukemic lines tested, mens (14.1 nmol/min/g tissue) is comparable to that (16.9 Â±2.5 and the lymphoma line RWLy-1 (Table 2). These results dem nmol/min/g tissue) reported in liver biopsies obtained from pa onstrate the presence of little if any dihydropyrimidinase activity tients (25). In addition hysteresis and allosterism have been also in these tissues. Similar results were reported with human pe observed in nonautopsy organs, e.g., mouse liver and kidney, ripheral blood platelets (42); rat brain extract (55); sliced or respectively. We therefore believe that, although human livers minced rat spleen, bone marrow, lymph node, adrenal, testis, were obtained from autopsies and as such could have had thymus, lung, brain, heart muscle, skeletal muscle, skin, and a altered enzymatic activity, this possibility is quite unlikely. few tumors (13, 16, 56); and with perfused intestine, perfused It was reported that dihydrouracil dehydrogenase is the rate kidney and eviscerated preparations of the rat (57). ,8-Ureidopro- limiting enzyme for pyrimidine base catabolism in rat liver (2-4, pionase was present in all tissues which had dihydropyrimidinase 43). On the other hand dihydropyrimidinase was suggested to activity (Table 2). be the rate limiting enzyme in rat hepatocytes (44, 45) and ÃŸ- The absence of dihydropyrimidinase may be the major factor ureidopropionase in mouse liver (14). Our present results (Table for the failure of some investigators (18-20) to detect dihydrour 2) suggest that dihydrouracil dehydrogenase is the rate limiting acil dehydrogenase activity in extrahepatic tissues. These inves enzyme in human liver and all solid tumors tested with the tigators were using the amount of 14CO2released from [2-14C]- exception of the pancreatic carcinoma RWP-1. In contrast dihy uracil as an estimate of dihydrouracil dehydrogenase activity in

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. ENZYMES OF URACIL CATABOLISM IN HUMAN TISSUES these tissues. In contrast we measured enzyme activity as the imidinedehydrogenase.Biochim. Biophys. Acta, 25: 430-431,1957. amount of 14C-labeled dihydrouracil, carbamyl-/J-alanine, and ÃŸ- 6. Fritzson, P. Properties and assay of dihydrouracil dehydrogenase of rat liver. J. Biol. Chem., 235: 719-725,1960. alanine formed from [6-14C]uracil. This allowed us to detect a 7. Goedde, H. W., Agarwal, D. P., and Eickhoff, K. Purification and properties of dihydrouracildehydrogenasefrom pig liver. Hoppe-Seyter'sZ. Physiol.Chem., lack of dihydropyrimidinase but not dihydrouracil dehydrogenase 357:945-951,1970. from certain tissues, when the accumulation of radioactive di 8. Hallock, R. O., and Yamada, E. W. Visualizationof dihydrouracil dehydrogen hydrouracil but not carbamyl-(8-alanine and /3-alanine was ob ase activity after disc gel electrophoresis.Anal. Btochem.,56: 84-90,1973. 9. Shiotani, T., and Weber, G. Purification and properties of dihydrothymine served. Had we measured dihydrouracil dehydrogenase activity dehydrogenasefrom rat liver. J. Btol. Chem., 256: 219-224,1981. as a function of the amount of CO2 released from [2-14C]uracil, 10. Smith, A. E., and Yamada, E. W. Dihydrouracildehydrogenase of rat liver. J. we would have reached the erroneous conclusion that this Btol. Chem., 246: 3610-3617,1971. 11. Hallock, R. O., and Yamada, E. W. Pyrimidine reducing enzymes of rat liver. enzyme is absent from extrahepatic tissues, since the absence Can. J. Biochem., 54:178-184,1976. of dihydropyrimidinase from a tissue will not allow the formation 12. Fink, R. M., McGaughey, C., Cline, R. E., and Fink, K. Metabolism of inter mediate pyrimidinereduction products in vitro. J. Biol. Chem., 278:1-7,1956. of CO2, regardless of the presence of dihydrouracil dehydrogen 13. Grisolia, S., and Wallach, D. P. Enzymic interconversion of hydrouracil and ÃŸ- ase. ureidoproptonicacid. Biochim. Biophys. Acta, 78: 449,1955. One of the most striking finding in this study is the reappear 14. Sanno, Y., Holzer, M., and Schimke, R. T. Studies of a mutation affecting pyrimidinedegradation in inbred mice. J. Btol. Chem., 245: 5668-5676,1970. ance or increase in dihydropyrimidinase activity in all the solid 15. Maguire, J., and Dudley, K. H. Partial purification and characterization of tumors tested when compared with normal tissues (Table 2). In dihydropyrimidinasefrom calf and rat liver. Drug Metab. Dispos., 6: 601-605, fact a greater qualitative difference between the tumors and the 1978. 16. Caravaca,J., and Grisolia, S. Enzymic decarbamylationof carbamyl .f-alanine normal tissues is found in dihydropyrimidinase rather than in and carbamyl /3-aminoisobutyricacid.J. Btol. Chem., 237: 357-365,1958. dihydrouracil dehydrogenase activity (Table 2). This coincides 17. Campbell, L. L. Enzymatic conversion of W-carbamyl-/3-alanineto0-alanine, carbon dioxide, and ammonia.J. Biol. Chem., 235: 2375-2378,1960. with our suggestion that in all normal human extrahepatic tissues 18. Canellakis, E. S. Pyrimidine metabolism. III. The interaction of the catabolic tested, dihydropyrimidinase rather than dihydrouracil dehydro and anabolic pathways of uracil metabolism. J. Bid. Chem., 227: 701-709, genase is the rate limiting enzyme in the pyrimidine base cata- 1957. 19. Potter, V. R., Pilot, H. C., Ono, T., and Morris, H. P. The comparative bolic pathway. We therefore suggest that future studies be enzymology and cell origin of rat hepatomas. I. Deoxycytidylate deaminase directed at dihydropyrimidinase and at developing inhibitors of and thymine degradation. Cancer Res., 20:1255-1261,1960. this enzyme. This suggestion is made more interesting by the 20. Barret, H. W., Munavalli, S. N.. and Newmark, P. Synthetic pyrimidines as inhibitor of uracil and thymine degradation by rat-liver supernatant. Biochim. finding that dihydrofluorouracil contributes to the toxicity of 5- Biophys. Acta, 97:199-204,1964. fluorouracil, as the former persisted in blood circulation long after 21. Queener, S. F., Morris, H. P., and Weber, G. Dihydrouracil dehydrogenase the latter had been cleared (58). Fluroro-/3-alanine, on the other activity in normal, differentiating, and regenerating liver and in hepatomas. Cancer Res., 37:1004-1009,1971. hand, had no toxic effect at all (58). This would suggest that 22. Ikenaka,K., Shirazaka,T., Kitano,S., and Fujii,S. Effect of uracilon metabolism dihydrofluorouracil may function as a more stable depot form of of 5-fluorouracilin vitro. Gann, 70: 353-359,1979. 5-fluorouracil. Furthermore the present study shows that in solid 23. Weber, G. Colon tumor: enzymology of the neoplastic program. Ufe Sci., 23: 729-736,1978. tumors, in contrast to their normal counterparts, dihydrouracil 24. Weber, G. Recent advances in the design of anticancer chemotherapy. On dehydrogenase, the enzyme responsible for the conversion of cology (Basel),37 (Suppl. 1V 19-24.1980. 25. Maehera, Y., Nagayama, S., Okazaki, H., Nakamura, H., Shirazaka, T., and dihydrofluorouracil to fluorouracil, is rate limiting. This indicates Fuji, S. Metabolism of 5-fluorouracil in various human normal and tumor that dihydropyrimidinase has a higher activity than does dihy tissues. Gann, 72: 824-827,1981. drouracil dehydrogenase in these tumors; hence it is important 26. Higley, B., and Buttery, P. J. Effects of dietary RNA on some enzymes of pyrimidinemetabolismin the rat. Nutr. Rep. Int., 27: 303-313,1983. to inhibit dihydropyrimidinase in order to increase the toxicity of 27. Naguib, F. N. M., el Kouni, M. H., and Cha, S. Dihydrouracil dehydrogenase dihydrofluorouracil in such tumors. activity in human tissues. IUPHR9th InternationalCongress of Pharmacology, In conclusion dihydropyrimidinase is highly active in all solid London, 1817, 1984. 28. Boyum, A. Isolation of lymphocytes, granutocytes and monocytes. Scand. J. tumors studied but not in their normal counterparts; therefore Immunol.,5 (Suppl.5):9-15,1976. we suggest that dihydropyrimidinase can serve as a good marker 29. Dexter, D. L., Barbosa, J. A., and Calabresi, P. N,N-Dimethylformamide- of tumorigenicity as well as a target for cancer chemotherapy of induced alteration of cell culture characteristics and loss of tumorigenicity in cultured human colon carcinomacells. Cancer Res., 39: 1020-1025,1979. the human solid tumors. 30. Spremulli, E. N., Scott, C., Campbell, D. E., Libbey. N. P., Shochat, D., Gold, 0. V., and Dexter, D. L. Characterization of two metastatic subpopulations originating from a single human colon carcinomas. Cancer Res., 43: 3828- ACKNOWLEDGMENTS 3835,1983. 31. Chu, M. Y., Naguib, F. N. M., Iltzsch, M. H., el Kouni, M. H., Chu, S-H., Cha, We wish to thank Drs. F. W. Burgess, K. C. Agarwal, M. Y. Chu, D. L. Dexter, S., and Calabresi, P. Potentiatton of 5-fluoro-2'-deoxyuridine antineoplastic and M. C. Wiemann of Brown University and Roger Williams General Hospital, activity by the uridine phosphorylase inhibitors benzylacydouridine and ben- Providence,Rl, for providing the peripheralblood lymphocytes, platelets,tumor cell zyloxybenzylacyclouridine.Cancer Res., 44:1852-1856,1984. lines, tumor xenografts, and autopsy materials used in this study. We also wish to 32. Dexter, L. D., Matook, G. M., Meitner, P. A., Bogaars, H. A., Jolly, G. A., extend our thanks to Ann Hollimanand Norma Messier for their excellent technical Turner, M. D., and Calabresi, P. Establishment and characterization of two assistance. human pancreatic cancer cell lines tumorigenic in athymic mice. Cancer Res., 42: 2705-2714,1982. 33. Leith, J. T., Dexter, D. L., DeWyngaert, J. K., Zeman, E. M., Chu, M. 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Fardos N. M. Naguib, Mahmoud H. el Kouni and Sungman Cha

Cancer Res 1985;45:5405-5412.

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