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Metabolism of Neoplastic Tissue VII. Effects of Dinitrophenol and Fluoride on Glucose Oxidation in Tumor Homogenates* CHARLES E. WENNERf AND SIDNEY WEINHOUSE (Lankenau Horpital Re8earch In8titute and the 1n3t@tuiefor Cancer Re8earch, Phüadelphia 11, Pa.) As yet, the high aerobic and anaerobic glycoly his co-workers as a result of an extensive investi sis, an outstanding characteristic of neoplastic gation of phosphate metabolism in normal and cells, remains unexplained. This phenomenon has neoplastic tissues (12, 14—16).Potter and Lyle been variously attributed to disturbances of res (15) found that homogenates of such nonneoplas piration, to deficiencies of electron transport en tic tissues as liver, heart, and kidney consumed zymes or co-factors, and to a lack of the citric acid oxygen in the presence of oxalacetate and exhibit cycle (2, 4, 14, 23). However, none of these hy ed relatively low rates of splitting of organic phos potheses has decisive experimental support, and phate. On the other hand, homogenates of the against them can be cited the observations that Walker 256 carcinoma did not absorb oxygen but tumors as a class possess the same electron trans split ATP rapidly. It was reasoned that excessive port components and consume oxygen about as ATP breakdown in the Walker tumor did not al rapidly as do moderately active normal tissues, low maintenance of ATP-dependent oxidative and oxidize fatty acid and carbohydrate interme processes. A key observation in the formulation of diates via the citric acid cycle (10, 24). this theory was the divergent behavior of fluoride, In a search for other explanations for the high which inhibited oxygen consumption in kidney glycolysis of tumor cells, the possibility that this homogenates but stimulated oxygen uptake in type of tissue differs from the non-neoplastic in its homogenates of the Flexner-,Jobling tumor (11). capacities for synthesis and breakdown of phos This finding was explained on the basis of the well phate bonds deserves consideration. The participa known inhibition by fluoride of phosphatase ac tion of phosphorylated intermediates as well as tion; it was reasoned that oxidative activity is lim nucleotides, such as adenosine di- and triphos ited in tumors by excessive dephosphorylation and phate (ADP and ATP), in many steps of glucose is therefore enhanced by fluoride, whereas oxida catabolism implies that the balance between phos tive activity in normal tissues is limited by exces phorylative and dephosphorylative rates might sive phosphorylation and is therefore inhibited by strongly influence the rate of glycolysis. The rapid fluoride. As a further test of this theory, Siekevitz growth of tumors undoubtedly depends on a varie and Potter (19) studied the effects of 2,4-dinitro ty of synthetic processes which require the utiliza phenol (DNP) and fluoride on the oxidation of tion of phosphate bond energy. Thus, the high pyruvate and fumarate in homogenates and mito synthetic activity associated with neoplasia may chondria of various normal and neoplastic tissues. be a primary factor affecting the balance between It was anticipated that DNP, which accelerates phosphate uptake and release, affecting, in turn, ATP breakdown, would act antagonistically to the rate of glucose catabolism. fluoride. No consistent patterns of response to The hypothesis of an altered phosphate metabo these agents were observed, however, and it was lism in neoplasia has been advanced by Potter and concluded that possible differences in phosphoryla tive and dephosphorylative activities may have * Aided by grants from the National Cancer Institute, Public Health Service, the American Cancer Society, and the been largely overshadowed by individual varia United States Atomic Energy Commission, Contract No. tions in enzymatic constitution within each tissue AT(30-1)777. type. t Most of this work was conducted during the tenure, by In view of the importance of this problem to a C. E. W., of a Damon Runyon Memorial Fund Fellowship. proper understanding of carbohydrate metabolism Received for publication January 7, 1955. in neoplasia, a further test of this hypothesis 497 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1955 American Association for Cancer Research. 498 Cancer Research seemed desirable, particularly a test of the effects thickness.― They are reported as patoms of labeled glucose of dinitrophenol and fluoride specifically on the carbon converted to CO2. calculated from the formula: oxidative catabolism of glucose. An available tis @iatomsglucose carbon oxidized = sue preparation, suitable for this study, is the c.p.m barium carbonate X @Mbarium carbonate whole, isotonic homogenate. Inasmuch as this preparation will oxidize glucose rapidly, when suit - c.p.m. glucose as barium carbonate ably fortified, and does not require an external MaieriaLt.—Diphosphopyridine nucleotide (DPN) was source of high energy phosphate as a driving force obtained from the Pabst Laboratories; it was found to be 85 (27),anymarkeddifferencesbetweentissuesin per cent pure by the enzymatic procedure of Racker (17). their capacities for synthesis and breakdown of fl,4-Dinitrophenol was a recrystallized commercial product. Hexokinase was supplied by the Pabst Laboratories as a dry, phosphate bonds should be manifested in differ lyophilized powder from autolyzed yeast. Cytochrome c, ences in oxidative response to fluoride and dinitro adenosine triphosphate (AlP), fumaric acid, sodium fluoride, phenol. The measurement of the incorporation of and nicotinamide were commercial preparations. Uniformly radioactivity from C'4-labeled glucose into respir labeled glucose-C1' was obtained from the Nuclear Instrument atory carbon dioxide in these experiments pro and Chemical Corporation on allocation by the Isotopes Divi. sion of the United States Atomic Energy Commission. vides a more precise criterion of the oxidation of Tumors.—With two exceptions, the tumors used in this this substrate than can be supplied by manometric study have been used in previous studies (26). One exception data alone and thus allows a more critical evalua was the LettréEhrlich ascites carcinoma (1), a hyperdiploid tion of the effects of these inhibitors on glucose tumor with a chromosome mode of 45-47 carried in Swiss strain mice. This tumor was used because it is relatively free of blood @ catabolism specifically. cells. The other was the Krebs ascites carcinoma, described by Klein and Klein (7). MATERIALS AND METHODS Incubation of tiwse.—The animals, young, adult, well RESULTS nourished rats or mice from our stock colony, were decapitated, Intact cells.—Before experiments were conduct and the tissues were rapidly excised. The peripheral tissue of ed with homogenates, a preliminary survey was the tumors was carefully removed, and any grossly necrotic made to ascertain the effect of DNP on glucose portions were discarded. When homogenized tissue prepara tions were used, the experimental procedure was the same as oxidation in tumor slices and in whole ascites cells. @ described previously (%7).The tissue was homogenizedfor As shown in Table 1, there was considerable van minutes by the Potter-Elvehjem procedure (13) in 6 volumes of ation in the consumption of oxygen by the tumor an ice-cold isotonic medium containing KCJ, 0.133 as, and phos cells, ranging from 1 to about 7 @iMinthe DNP phate buffer (pH 7.4), 0.O1@3ii. An aliquot of the suspension, free system. These values correspond to Q, values representing 86 mg. of tissue unless otherwise specified, was added to Chilled Warburg vessels containing the other ions, (,d oxygen/mg dry tissue/hour) of from 1.4 to 10, factors, and substrates constituting the basic medium. This and are thus in the range observed in previous in consisted of the followingsubstances in their final concentra vestigations (4). Despite this variation, the pat tions: MgSO4, S X 10—'M; fumarate, 7 X 10' M; cytochrome terns of response to increasing DNP concentration C, 4 X 10' M; phosphate buffer (pH 7.4), 6 X 10-' at; KC1, @ 0.14 M; diphosphopyridine nucleotide, X 10' M; and water were similar. There was a variable stimulation in to make the final volume 1.6 ml. For the experiments with the range, 7 X 10@ to 7 X 10' M, whereas at slices, approximately flOO mg. of tissue was sliced with the 3 X 10@ M inhibitions in oxygen consumption, Stadie-Riggs slicer (@1), and was suspended in @.8ml. of cal ranging from 18 to 56 per cent, were observed. The cium-free Ringer phosphate solution (8) contained in Warburg one exception was the Lettrétumor, in which stim flasks. Substrate and other requirements were added to give a final volume of 3.0 ml. ulation was still evident at the highest DNP level. For carbon dioxide absorption, a filter-paper roll, soaked This pattern of augmentation and inhibition of with 0.1 ml. of 10 N CO,-free sodium hydroxide, was placed respiration in the range of DNP concentrations in the center well. After attachment to the manometers, the herein employed is typical of a wide variety of ani flasks containing tissue slices were filled with oxygen. All mal, plant, and microbial cell types (20). flasks were equilibrated for 10 minutes at 38°C.; the manom eter stopcocks were then closed, and the flasks were shaken Data on glucose oxidation, as determined from for 50 minutes. Dilute sulfuric acid was then tipped in from the incorporation of C'4 into respiratory C02, closely side bulb to stop the reaction and liberate the bound CO2. paralleled oxygen consumption. Invariably, glu After being shaken for 10 minutes to allow complete absorption cose oxidation was enhanced at the intermediate of CO2 by the alkali, the flasks were removed, the filter paper and washings were transferred to a stoppered flask, 0.3 m@ of DNP levels and inhibited at the highest level. sodium carbonate was added as carrier, and the carbonate was Thus, it appears that intact tumor cells exhibit precipitated by the addition of excess 1 M barium chloride essentially the same oxidative response to DNP solution.

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