Metabolism of Neoplastic Tissue II. a Survey of Enzymes of the Citric Acid Cycle in Transplanted Tumors*

Metabolism of Neoplastic Tissue II. A Survey of Enzymes of the Citric Acid Cycle in Transplanted Tumors*

CHARLES E. WENNER,t MORRIS A. SPIRTES, AND SIDNEY WEINHOUSE

(The Lankenau Hospital Research institute and the Instituiefor Cancer Research, and the Department of Chemistry, Temple University, Philadelphia, Pa.)

Recent isotopic tracer studies of tumor metabo on homogenates of the whole tissue or on extracts lism indicated that the neoplastic cell can carry out of acetone powders. All assays were carried out in the complete oxidation of glucose and fatty acids the zero order range of substrate concentration via the citric acid cycle (84, 85). In view of a strong and with proportionality of enzyme activity and body of evidence against the occurrence of the tissue concentrationâ€”these conditions having Krebs cycle in tumors (18, 19), further information been established by preliminary experiments. De bearing on this problem seemed desirable. Accord tails of the procedures are given in the individual ingly, a survey was undertaken of various trans sections and in the table headings. The prepara planted neoplasms for their content of citric acid tions of homogenates will be described in the indi cycle enzymes. In order to obtain a â€œprofileâ€•of vidual assays. The acetone powder extracts were enzymatic activity, approximate assays were car prepared as follows : the freshly dissected tissue ned out of those enzymes for which straightfor (pooled from four to six animals) was homogenized ward methods were available; these included for 1 minute at 2Â°C. in a Waring Blendor with 2 malic, isocitric, and a-ketoglutaric dehydroge volumes of acetone. The homogenate was filtered nases, the â€œcondensingâ€•enzyme, and the hydrases, with suction, and the residue was resuspended in fumarase and aconitase. The peripheral enzymes, 10 volumes of acetone and refiltered with suction. lactic dehydrogenase and oxalacetic carboxylase, The tightly packed cake was powdered in a mor were also assayed, but succinic dehydrogenase, tar, air-dried on filter paper, then dried thoroughly having been thoroughly investigated and found to in a vacuum desiccator over sulfuric acid and par be present in tumors by others (24, 25), was affin, and stored at less than 5Â°.The extract was omitted from the present investigation. All the prepared bystirring 1 gm. of the acetone powder tumors were found to possess the enzymatic equip with 10 ml. of water for 10 minutes at 40Â°C. and ment for the citric acid cycle. by centrifuging at 3,000 r.p.m. for 20 minutes in the angle head of a No. 2 refrigerated Internation METHODS AND RESULTS al Centrifuge. The opalescent solution was used as Tumors.â€”The neoplasms employed in this such or was further diluted if necessary. study were described in previous papers (33, 35). Substrates and other materials.â€”Pyruvic, malic, They were implanted subcutaneously and used in citric, and a-ketoglutaric acids, and adenosine the experiments when they attained a diameter of triphosphate were commâ‚¬@cialproducts. Lithium approximately 1 cm. Assays were carried out either acetyl phosphate, 60 per cent pure, was obtained a This work was aided by grants from the American Cancer through the kindness of Drs. Stern and Ochoa. Society, on recommendation by the Committee on Growth of Oxalacetic acid, 90 per cent pure by aniline citrate the National Research Council; the National Cancer Institute assay (5), was prepared from the ester by the pro of the National Institutes of Health, Public Health Service; cedure of Krampitz and Werkman (9). Isocitric and the U.S. Atomic Energy Commission, Contract No. acid was prepared by catalytic reduction of tn AT(80-1)777. A preliminary report of a portion of this work has appeared ethyl oxalosuccinate, synthesized according to the (36). method of Wislicenus and Waldmtiller (38). The t This study will constitute part of a thesis to be presented reduction was carried out with platinum at room by C. E. Wenner to the Graduate School of Temple University temperature at 3â€”4atmospheres of hydrogen, and in partial fulfillment of the requirements for the Ph.D. degree. the reduced product was saponified with the cal Receivedfor publication September 24. culated quantity of sodium hydroxide. The final 44

product consisted of a mixture of four isomers, D gm.) were intimately mixed with 0.5 gm. of alumi and t-isocitric and D- and fralloisocitric acids. As na (Alcoa No. A301, 325 mesh) by grinding to @ expected, only of the product was acted upon by gether in a mortar for a few minutes at room tem aconitase, the active isomer being presumably perature. The mixture was transferred to a Lus D-isocitric. teroid centrifuge tube of 10-ml. capacity and Reduced diphosphopyridine nucleotide was pre stirred with 0.5 ml. of M/50 phosphate buffer, pared according to the method of Ohlmeyer (17) pH 7. The suspension was centrifuged for 20 mm from commercial cozymase assaying 33 per cent utes in a Sorvall centrifuge at 12,000 r.p.m. The pure by the enzymatic method of Racker (22) em supernatant fluid, which contained the transacety . ployi@ alcohol dehydrogenase. Triphosphopyri lane, was kept refrigerated at all times and was S dine nucleotide, assaying 40 per cent pure by the freshly prepared each day of use. Sufficient co Zwischenferment assay (32), was prepared by the enzyme A is present in this extract so that no method of LePage and Mueller (11). further additions are required. Condensing enzyme.â€”Ofall the reactions of the Since only small quantities of some of the mate cycle, the one least understood until quite recently rials were available, only a few assays were per is the condensation leading to formation of the 6- formed. However, the data, given in Table 1, con carbon acid. Recent studies have clarified this process, however (16, 26, 29), and the reaction can TABLE 1 now be formulated, according to Stern and Ochoa CONDENSING ENZYME ASSAY (28), as follows: Thecomponentsoftbe auaysystemwere: Mphosphatebuffer, pH 7, 0.025 nil.; 0.08 K MgCI*, 0.05 ml.; 0.2 N cysteine (neutral. @ Acetyl-Coenzyme A + Ã³xalacetate â€”+ ised), 0.05 nil.; 0.14 oxalacetic acid, 0.05 ml.; 0.1 synthetic I\ acetyl phosphate. 0.i nil.; E. Coli extract, 0.20 ml.; acetone pow. citrate + Coenzyme A. â€˜%I) der extract of the 1@1hue and water to make a total volume of 1.00 ml. After incubation in small test tubes for 10 minutes at 25Â°the An impure concentrate of acetyl-CoA has been reactionlwas stopped by adding trichloroacetic acid to a final oon. ceutration of 5 per cent. An aliquot of the supernatant solution obtained by Lynen and Reichart (12); and Ochoa was removed for citric acid determination according to Natelaon and co-workers1 have found that this substance St of. (15). can condense with oxalacetate to yield citrate in pM citrate produced the presence of the crystalline condensing enzyme. Tissue 10 znin.X 100 mg. acetone powder For assay purposes the acetyl-CoA may be gener Normal mouse liver 1.63 ated in situ, either by an acetate-activating factor Mouse mammary carcinoma 8.30 Mouse hepatoma 2.90 which, according to the originalprocedure of Stern Mouse rhabdomyosarcoma 1.45 and Ochoa (27), catalyzes the formation of acetyl CoA from acetate, CoA and ATP; or, more con vincingly demonstrate the presence of the con veniently, by means of the following reaction densing enzyme in three mouse tumors in amounts catalyzed by a transacetylating enzyme present in at least as high as in mouse liver. The activities of E. Coli (26): these neoplastic tissues were of the same order as E. Ccli pigeon liver, reported by Stern and Ochoa (26), Acetyl phosphate + CoA â€”- Acetyl-CoA + and were of a magnitude similar to that obtained phosphate. (2) by these investigators for other normal tissues The latter procedure was used in the present (29). study. According to equation (3), representing a Aconilase and fumarase.â€”For assay of the summation of equations (1) and (2), citric acid, hydrases, advantage was taken of the recent spec trophotometric method described by Racker (21). Acetyl phosphate + (@oA+ E. Coli extract + With citrate or malate as the substrate, aconitase (conden4ng enzwn@) (3) or fumarase activity is measured by the appear oxalacetate -)@citrate + CoA + phosphate, ance of absorption at 240 m@&ofthe corresponding is formed in the presence of the condensing en unsaturated acids, cis-aconitic or fumaric. The zyme and E. Coli extract from a mixture of Acetyl test system contained, in a total volume of 3 ml., phosphate, coenzyme A, and oxalacetate. The 0.05 Mphosphate buffer, pH 7.4, an aliquot of the E. Coliextract was prepared,accordingto direc tissue, and the substrate. For aconitase assay the tions of Drs. Stern and Ochoa,2 from lyophilized enzyme was prepared by homogenizing the fresh cells supplied by these investigators. Cells (0.5 tissue with 20 volumes of 0.1 M phosphate buffer, pH 7.4, and centrifuging off the residue at 3,000 1 Private communication. r.p.m. Fumarase was assayed in an extract of an 2 We aregreatly indebtedto theseinvestigators for generous gifts of acetylphosphate, lyophilised E. Ccli, and details of the acetone powder prepared as described in the con condensing enzyme assay prior to publication. densing enzyme assay. The reactions were carried

out in quartz absorption cells, with the use of the The reactions were carried out in quartz micro Beckman DU spectrophotometer. A unit of en absorption cells of 1 cm. depth in the Beckman zyme activity is defined, according to Racker (21), model DU spectrophotometer; the enzymatic ac as the amount causing an increase in optical den tivity was measured by the change in light absorp sity (log [Ia/I]) of 0.001 per minute at room tem tion at 340 mj@resulting from the oxidation or re perature, 22Â°â€”26Â°.Thevalues obtained were cor duction of the pyridine nucleotide. The conditions rected for changes in a control cell with substrate outlined by Mehler et al. (13) were closely adhered omitted. to, except for the use of microcells requiring only As shown in Table 2, both aconitase and fu one-tenth of the volumeâ€”viz., 0.3 ml. This re suited in a considerable saving of nucleotide with TABLE 2 out appreciable sacrifice of accuracy. After several ACONITASEAND FUMARASEIN NORMAL preliminary readings, the reaction was started by AND NEOPLASTICTISSUE the addition of substrate and readings taken at 15- @ The teat systems consisted of the following: aconitase, 0.05 phosphate second intervals. In accordance with Mehler et al. buffer, pH 7.4; sodium citrate, 0.03 @;tissueextract and HsO to a total vol @ unie of S cc.; fuinarase, 0.05 phosphate buffer, pH 7.4; sodium inalate, (13), the enzymatic activity is given in units per 0.005 @;tissueextract and &O to a total volume of S cc. milligram of acetone powder, the unit of activity ACONITASE FUMARASE being the amount of enzyme giving a change in log Units/mg dry wt Units/mg dry wt (lo/l) of 0.01 per minute at room temperature TIssuE of tissue of acetone powder Normal: (22Â°â€”26Â°).Ablank determination, with substrate Liver (mouse) 33 132 omitted, was run simultaneously, and the values, Heart (rat) 72 @ Liver (rat) 26 ranging between 0 and 20 units/mg acetone pow Kidney (rat) 50 62 der, were subtracted from the gross rates to obtain Muscle (rat) 12 corrected values, which are displayed in Table 3. Neoplastic: Hepatoma (rat) 133 A considerable range in activities was observed for Hepatoma (mouse) 8.8 62 normal tissues, but in every instance the values for @ Rhabdomyosarcoma(mouse) 2 .2â€”S.O@ neoplastic tissues were within this range. The gen Mammaryadenocarcinoma [email protected]* (mouse) eral impression one obtains from an over-all survey

@ S of three determinations. of Table 3 is that the content of these dehydrog

TABLE 3 marase are present in neoplastic tissues. Though the fumarase content in tumors was comparable to DERYDROGENASE ASSAYS IN ACETONE that in normal tissues, aconitase activity was quite POWDER EXTRACTS The test media were as follows: for lactic and malic debydrogenases. @ low. Since aconitase is a very unstable enzyme 0.25 glycylglycine buffer, pH 7.4, 0.05 ml.; 1.7 X 10' DPN.Ht, 0.02 nil.; (3), it is not certain whether the low values ob 0.04 u pyruvate, 0.02 ml.. or 0.01 N oxalacetate, 0.10 ml.; I :1000 acetone powder extract, 0.04 nil.; and water to a total volume of 0.50 ml. For isocitric @ served represent a real deficiency or whether its dehydrogenase, 0.25 N glycylglycine buffer, pH 7.4, 0.03 ml.; 6.6 X 10' inactivation was more rapid in the tumor than in TPN, 0.03 ml.; 0.05 u MnCh, 0.01 ml.; 1:500 acetone powder extract, 0.10 niL; and water to 0.30 ml. normal tissue. DmrnaooanAsa Dehydrogenases.â€”In view of the long-held be (UNIrs/Mo ACETONE PowDER) lief (31) that tumors are deficient in their capacity TISSUE Lactic Malic Isocitric Normal:Heart for the oxidation of carbohydrate, it was of special (rat) interest to test the neoplasms for their content of Liver (mouse) 200 256 10.8 the dehydrogenases of the citric acid cycle, malic Kidney(rat) 104 173 66.0 15.6Neoplastic:Muscle (mouse)320 520388 33056.0 and isocitric dehydrogenases. Lactic dehydrog enase was included in this study because of its Rhabdomyosarcoma possible role in the high aerobic glycolysis of tu ( mouse) Mammary tumor (mouse) 168 380 16.0 mors. The method employed in this study was Hepatoma (rat) 288 540 [email protected]* that of Mehler et at. (13), which provides a direct Hepatoma (mouse) 108 288 14.8 @ assay of the dehydrogenase by measuring the rate Ascites (mouse)190,220 165148,2082006.7 4 8 of oxidation or reduction of the specific coenzyme. C 5aTaflgf@ofthree determinations. The reactions measured are given in the equations enases in neoplastic tissues is on the low side, but below: definitely within the range of activities displayed Pyruvate + DPN-H2 â€”+Lactate + DPN (4). by normal tissues. The data on lactic dehydrog Oxalacetate + DPN-H2 â€”â€˜Malate + DPN (5). enase are in substantial agreement with those of Isocitrate + TPN â€”+Oxalosuccinate + Meister (14), who used a spectrophotometric TPN-H2 (6). method similar to the one employed here, and

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1952 American Association for Cancer Research. WENNER et a2.â€”Metaboli,@m of Neoplastie Tissue. II 47 those for isocitric dehydrogenase were in accord rying out the oxidation of ketoglutarate was tested with previous assays of this enzyme using the by measuring the increase in oxygen consumption Thunberg technic (2). produced on addition of the keto acid to washed, Oxalacetic carboxylase.â€”Although this enzyme sedimentabie particles of tumors, prepared ac is not directly involved in the reactions of the citric cording to the procedure of Kennedy and Lehnin acid cycle, its assay was included in this study be ger (8), and fortified by additions of cytochrome cause it undoubtedly plays an important role in c, ATP, DPN, and magnesium ions.5 The results, the maintenance of the level of oxalacetate and as given in Table 5, are reported as Qo2 (@l of oxy gen consumed/equivalent of 1 mg dry weight of TABLE 4 original tissue/hour). Although a-ketoglutarate OXALACETIC CARBOXYLASE IN NORMAL oxidation was considerably more rapid in normal AND NEOPLASTIC TISSuE tissues, all the tumors displayed the ability to The test was carried out for 50 minutes at 55Â°(Nigas phase) in conven tional Warburg flasks containing the followingsubstances in the designated oxidize this acid. In view of the complexity of the ffnal concentrations: acetate buffer, pH 4.5, 0.1 w; TPN, 55 pg.; MnClt system, the significance of these quantitative dif 0.001 @;oxalacetic acid, pH 4.5, 0.0@ M; and tissue in a total volume of S ml. The rate of spontaneous decarboxylation of oxalacetic acid during the test ferences cannot be evaluated, but it can be con period (determined from a flask run simultaneously, containing all con eluded that tumors can oxidize ketoglut.arate. stituents except tissue) was subtracted from the CO@ evolved in the complete system. A blank without substrate permitted evaluation of the endogenous Cot. Thereactionrate expressedrepresentsthenet @ilofCO@evolved/mU. DISCUSSION ligram of dry tissue/hour. net pl CO2 evolved The present study provides definite evidence that neoplastic tissues possess the enzymatic Tissue mg dry tiuueX hr equipment for carrying out reactions of the Krebs Normal: cycle. Although the procedures employed were not Liver (mouse) 8.21 Liverofhepatoma-bearing rat 2.40 designed to give rigorous quantitative assays,4 the Neoplastic: results adequately demonstrate that specific en Ascites (mouse) 8.48 Hepatoma (rat) 2.45 zyinatic processes known to be involved in the Rhabdomyosarcoma (mouse) 2.17 oxidation of carbohydrates and fatty acids by Mammary gland adenocarci- 1.24 normal tissues can occur in tumors at rates which noma (mouse) Hepatoma (mouse) 0.78 are comparable to those of normal tissues. Several TABLE 5 thereby exercises control over the rate of the cycle. Its activity in whole, nondialyzed homogenates a-KETOGLUTARATE OXIDATION IN WASHED PARTICLES was assayed according to the procedure of Ven The medium contained, in a total volume of 1.6 nil., the followingsub. stances in the designated final concentrations: a-ketoglutarate, 7 X 10' nesland et a! (30). The rate of breakdown of N; MgSO4, S X 10' @;fumarate. 7 X 10' M; ATP, S X 10$ @; c,4 X 10'w; DPN, 1.9 X l0'u; phosphatebuffer,pH7.4,0 X 10'M; and oxalacetate was estimated manometrically at pH homogenateequivalent to 100mg.offreshtissue. Experiments wererun 1hour 4.5 and was corrected for spontaneous breakdown at 38Â°with air as the gas phase. by subtracting the CO2 evolved in a control flask Net 0, uptake (pl) Net containing no tissue. A further correction, for en Tissue (0.3 cc of 1:5 homog)Xhr Qo, dogenous CO2 production, was applied by sub Normal: tracting the CO2 production in the absence of sub Liver (rat) 152 7.7 strate. Kidney (mouse) 78.7 4.0 Kidney (rat) 11.8 The values given in Table 4 are expressed in Neoplastic: il/mg dry tissue/hour at 25@and are calculated Rhabdomyosarcoma (mouse) 65.1 3.3 Mammary gland adenocarci 62.7 8.2 from CO, outputs measured for the first 20 mm noma (mouse) utes. The data leave no doubt concerning the Hepatoma (rat) 46.5 2.4 presence of this enzyme in neoplastic tissues in Hepatoma (mouse) 42.8 2.1 amounts comparable to its content in normal liver cells. enzymes were found to have considerably lower a-Ketoglutark dehydrogenase.â€”Although the activities in tumors than in normal cells; this was mechanism of ketoglutarate oxidation is not yet particularly true of aconitase and a-ketoglutaric well understood, the reactions involved are prob dehydrogenase. It is possible that these enzymes ably analogous to those of pyruvate oxidation (7), (orothers)constituteâ€œbottle-necksâ€•forthecitric viz., a thiamine pyrophosphate and DPN-pro aThe method usedwas similar to onedescribedrecently by moted series of reactions leading to formation of Ackermann (1). CO2 and a succinic acid-coenzyme A complex. 4 For example, the content of an enzyme in terms of acetone Lacking specific assay procedures for the individ powder may not reflect its concentration in the fresh tissue, ual steps, the capacity of neoplastic tissues for car because of variations in water and lipid content.

acid cycle, and their low activity may account for sibly some of the low values for lactic and malic some of the phenomena which have led previous dehydrogenases reported in tumors by Lenta and investigators to conclude that carbohydrate or Riehl, using the Thunberg technic (10), may also pyruvate oxidation was disturbed in neoplastic have been due to lack of an essential electron cells (20). On the other hand, since wide differ transport factor. ences in oxygen consumption are not observed be tween intact normal and neoplastic tissues (31, SUMMARY 3,5), it seems probable that differences in en A series of transplanted mouse and rat tumors zymatic activity are due to various degrees of loss was assayed for their content of citric acid cycle or inactivation during preparation rather than to and related enzymes, and the results were com different concentrations in the intact cell. pared with a representative series of normal tis Perhaps the most significant observations sues. Lactic, malic, and isocitric dehydrogenases, made in this study were (a) that the condensing fumarase, oxalacetic carboxylase, and the â€œcon enzyme is present in neoplastic tissue and (b) that densingâ€• enzyme were found present in tumors in malic and isocitric dehydrogenases are present in amounts comparable to those in normal tissues. tumors in amounts comparable to those of normal Aconitase and a-ketoglutarate dehydrogenase were tissue. The presence of the condensing enzyme present in tumors but to lower extents than in nor definitely establishes the capacity of neoplastic mal tissues. The results warrant the conclusion tissues to form citric acid, a matter upon which that neoplastic tissues possess the enzymatic considerable doubt was cast, particularly by the equipment for the citric acid cycle. recent studies of Potter and Busch (19). On the basis of the observation of Buffa and Peters (4) REFERENCES that citrate accumulated in tissues poisoned with 1. [email protected], W. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1952 American Association for Cancer Research. Metabolism of Neoplastic Tissue: II. A Survey of Enzymes of the Citric Acid Cycle in Transplanted Tumors

Charles E. Wenner, Morris A. Spirtes and Sidney Weinhouse

Cancer Res 1952;12:44-49.

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