Control of the Adenylate Charge in the Morris Â€Œminimal Deviationâ€• 1

[CANCER RESEARCH 33, 51â€”56,January1973J

Control of the Adenylate Charge in the Morris â€œMinimal Deviationâ€• 1

WayneE. Criss Department ofObstetrics and Gynecology, University ofFlorida CollegeofMedicine, Gainesville,Florida 32601

SUMMARY adenylate kinase reaction) and the energy charge of the adenylate system were performed. The Q's for liver tissue and The activity of several enzymes that contribute to the groups of highly and poorly differentiated hepatomas were cellular adenylate energy charge and the change in the total 1.42, 1.88, and 2.06, respectively. The calculated adenylate pool of adenine nucleotides were measured in groups of highly charges were 0.87 in normal liver tissue, 0.89 in a group of and poorly differentiated Morris â€œminimal-deviationâ€•highly differentiated hepatomas, and 0.90 in a group of poorly hepatomas. differentiated hepatomas. The activities of several substrate-level phosphorylating It would appear that the poorly differentiated hepatomas enzymes were determined: cytoplasmic adenylate kinase, have acquired the potential to maintain a high energy charge pyruvate kinase, and phosphoglycerate kinase; mitochondrial without the need for large mitochondrial involvement. adenylate kinase and nucleoside diphosphokinase. Tabulation of these enzymatic measurements were made by setting up a ratio of cytoplasmic to mitochondrial nonoxidative INTRODUCTION phosphorylation potential. The ratio was 0.44 in normal liver tissue, 0.50 in the group of highly differentiated hepatomas, The adenylate system, consisting of AK2 (EC 2.7.4.3), and 22.2 in the group of poorly differentiated hepatomas. AMP, ADP, and ATP, has been compared to an The activities of several enzymes that are involved in the electrochemical storage cell in its ability to accept, store, and transportation of reducing equivalents across the supply energy (7). The energy charge of the adenylate system mitochondrial membrane were made: cytoplasmic malate may be a central signal in the control of glycolysis, dehydrogenase, glyceraldehyde phosphate dehydrogenase, gluconeogenesis, lipogenesis, and terminal oxidative lactate dehydrogenase, glucose 6-phosphate dehydrogenase, phosphorylations (10â€”12, 28, 29, 47). Atkinson has defined the energy charge as one-half of the average number of flavin adenine dinucleotide- and nicotinamide adenine anhydride-bound phosphate groups per adenosine moiety. dinucleotide (NAD@)-glycerophosphate dehydrogenase; When only AMP is present, there is a net charge of zero or mitochondrial malate dehydrogenase, flavin adenine complete discharge. When only ATP is present, there is a net dinucleotide- and NAD@-glycerophosphate dehydrogenases. Cytoplasmic NAD@-malate dehydrogenase was over 500 units charge of 1. Atkinson and his colleagues have shown that certain key enzymes that participate in ATP-regenerating in normal liver and less than 100 units in the group of pathways show plots of enzyme activity against energy charge poorly differentiated hepatomas. Cytoplasmic NAD @- with negative slopes that increase with charge ; certain enzymes glycerophosphate dehydrogenase was 120 units in liver that participate in biosynthetic or other ATP-utilizing tissue and about 20 units in the group of poorly differentiated pathways show plots of enzyme activity against energy charge hepatomas. NAD@-malate dehydrogenase is a key enzyme in with positive slopes that increase with charge (for reviews, see the malate-aspartate shuttle system; NAD@-glycerophosphate Refs. 8, 9, 11, and 26). Thus, the energy charge of the dehydrogenase is a key enzyme in the a-glycerophosphate adenylate system could provide the cell with a very sensitive shuttle system. intracellular regulatory control mechanism. Comparison of the ratio of the activities of NAD@-g1ycero Obviously, many enzymes and certain metabolites make phosphate dehydrogenase versus NAD@-lactate dehydrogenase varying contributions to the energy charge within a cell. allows for the examination of the competing hydrogen Compartmentation would probably be an important factor in accepting systems of phospholipid synthesis versus anaerobic determining location and levels of the components of the metabolism. The ratio was 0.5 in liver tissue and 0.1 in the adenylate pool (ATP, ADP, AMP) and raises the possibility of group of poorly differentiated hepatomas. a distinct energy charge in distinct subcellular units. In the Measurement of adenosine mono-, di-, and triphosphates cytoplasm, substrate-level phosphorylating enzymes such as and calculations of Q (a reaction parameter based on the

2 The abbreviations used are: AK, adenylate kinase; PGK, 1 This work was supported by NIH Grants CA-10439, CA-10906-08, phosphoglycerate kinase; PK, pyruvate kinase; MDH, malate and CA-i 1818; Grant F71UF from the Florida Division of the dehydrogenase;GADPH, glyceraldehyde 3-phosphate dehydrogenase; American Cancer Society; and Grant P-202 from the National Division GPDH, glycerophosphatedehydrogenase;LDH, lactate dehydrogenase; of the American CancerSociety. G6PDH, glucose 6-phosphate dehydrogenase; NDK, nucleoside ReceivedApril 28, 1972; acceptedOctober 3, 1972. diphosphokinase.

JANUARY 1973 51

hexokinase-glucokinase , phosphofructokinase , PGK , PK, etc., 370 in a Beckman Model DU spectrophotometer equipped would directly contribute to the cytoplasmic adenylate charge. with a Gilford recorder. All measurements were continuously The energy charge within the mitochondria would depend monitored at 340 nm by following the oxidation and/or upon the efficiency of the oxidation-reduction shuttle reduction of the pyridine nucleotides. systems of malate-aspartate (59, 60), oxaloacetate (22), MDH (L-malate:NAD@ oxidoreductase, EC 1.1.1.37) was a-glycerophosphate (58), the coupling to the adenine measured as in the work of Skilleter et a!. (49). @ nucleotides, and transport of ADP inward and AlP outward GADPH (D -glyceraldehyde 3-phosphate :NAD oxido (27, 34). It has also become apparent that there is a close link reductase, EC 1.2.1 .12) was measured by the method of between the nicotinamide and adenine nucleotide systems in Furfine and Velick (20). the cell (52, 60). Now, with the postulate that the adenine FAD-GPDH [L-glycerol 3-phosphate:(acceptor) oxidore nucleotides contribute directly to the metabolic control of the ductase, EC 1.1.99.5] activity was assayed with an oxygen Pasteur effect (1 1, 19, 39) and with one of the most notable electrode (23). and controversial features of the neoplastic cell being the NAD @-GPDH (L -glycerol 3-phosphate :NAD@ oxidore inability of the Pasteur effect completely to inhibit its ductase, EC 1.1 .1.8) was measured as by White and Kaplan glycolysis (4, 42, 54, 56), it becomes an important and (58). challenging problem to investigate the contribution of the NAD@-LDH (L-lactate:NAD@ oxidoreductase, EC 1.1.1.27) adenylate charge to metabolic regulation (especially of activity was determined by the method of Pesce et a!. (41). glycolysis) in the neoplastic cell. NADP@-G6PDH (D-glucose 6-phosphate :NADP@ oxido This paper and the following one (16) report the changes in reductase, EC 1.1 .1.49) was measured as described by Criss the levels of several enzymes that contribute to the cellular and McKerns (18). adenylate energy charge and the change in the total cellular NDK (ATP:nucleoside diphosphate phosphotransferase, EC pool of adenine nucleotides in Morris â€œminimal-deviationâ€• 2.7.4.6) was determined by the method of Ratliff et a!. (44) hepatomas and in Novikoff hepatomas (solid and ascites). with GDP as the phosphate acceptor and coupling the production ofADP as described by Adam (1). AK (ATP:AMP phosphotransferase, EC 2.7.4.3) activity was MATERIALS AND METHODS measured as described by Criss et al. (17). PGK (ATP:3-phospho-D -glycerate l-phosphotransferase, EC Animals and Tumors. Normal male adult rats (CFN) were 2.7.2.3) was determined by the method of Rao and Oesper purchased from Carworth Farms, New City, N. Y.; maintained (43) with the production of ADP as described by Adam (1). on laboratory chow; and sacrificed when approximately 250 g. PK (ATP:pyruvate phosphotransferase, EC 2.7.1.40) All tumors, except the Novikoff hepatoma, were transplanted activity was measured as by Reynard et a!. (45). in Bethesda, and the rats were shipped to our laboratories. The Preparation of Cellular Homogenates for Nucleotide Assay. rats were maintained on laboratory chow until the tumors The rats were kified by cervical dislocation; the liver or tumor were 1 to 3 cm in diameter. The origin and properties of many was exposed, clamped, and removed with pre-nitrogen-cooled of the Morris â€œminimal-deviationâ€•hepatomas have been Wollenburger tongs and immersed into liquid nitrogen through described (35â€”37). The tumors were examined histologically which CO had been bubbled. The procedure was completed in by Dr. David Meranze of the Fels Research Institute. 13 sec. The frozen tissue was powdered with a pre-nitrogen Reagents and Materials. Biochemical reagents and glassware CO-cooled mortar and pestle, weighed, and added to 5 were purchased from Fisher Scientific Co., Fairlawn, N. J.; A. volumes of cold 5% HC1O4. The tissue was then homogenized H. Thomas Co., Philadelphia, Pa.; Scientific Products, in a Potter-Elvehjem homogenizer with motor-driven pestle at Evanston, Ill.; Mallinckrodt Chemical Works, McGraw, Ill.; and 00 with acetone and Dry Ice. The extract stood for 15 mm and Corning Glass Works, Corning, N. Y. The coupling enzymes was then centrifuged at 10,000 X g for 15 mm at 4Â°.The and various substrates and nucleotides were purchased from supernatant was removed and analyzed for adenine nucleotides Sigma Chemical Co., St. Louis, Mo.; Boehringer-Mannheim as described below. Corp., Mannheim, Germany; P-L Biochemicals, Milwaukee, Extraction and Measurement of Adenine Nucleotides. The Wis.; Schwarz/Mann, Orangeburg, N. Y.; Worthington Bio adenine nucleotides were determined by 2 methods. Aliquots chemical Corp., Freehold, N. J.; and Calbiochem, Los Angeles, (I 00 p1) of the supernatant from the centrifugation were Calif. spotted on Whatman No. 3MM filter paper which previously Preparation of Cellular Homogenates for Enzymatic Assay. had been soaked in 0.5 M citrate buffer at pH 3.3. All rats were decapitated by guillotine and exsanguinated. The High-voltage electrophoresis was carried out in the citrate liver or tumor was quickly removed and placed in cold 0.25 M buffer at 1500 V for 3 hr at 2Â°.The paper was dried for 1 hr at sucrose. It was subsequently homogenized, and cellular 80Â°;theadeninenucleotidespotsweredetectedwithUVlight, fractions were prepared as described by Criss (14). cut into small pieces, and incubated in 3 ml of 0.75 M The variously prepared extracts were both assayed NH@HCO3 solution at 37 for 16 hr. Eluent from the spots immediately and frozen. The latter was accomplished by was assayed spectrophotometrically at 259 nm for adenine spraying with Freon small vials containing 1-ml samples. The nucleotides, and the concentration of each was calculated with frozen samples were stored at â€”87Â°andlater assayed. a molar extinction coefficient of 15 .4 X 106 . The eluent from Measurements of Enzymes. All enzymes were measured by the spots was also assayed by methods previously described by spectrophotometric methods. The assays were performed at Adam (1) and Criss (17). Recovery was from 85 to 97%.

52 CANCER RESEARCH VOL. 33

RESULTS NAD@-MDH's, key enzymes in the malate-aspartate shuttle (30, 49), and the GPDH's, key enzymes in the glycero Substrate-Level Phosphorylation. We examined the ability phosphate thuttle (25 , 48), in both normal rat liver and Morris of normal liver and highly and poorly differentiated Morris hepatomas. Cytoplasmic NAD@-MDH was about 100 units in a hepatomas to phosphorylate ADP at the substrate level by group of poorly differentiated Morris hepatomas compared to measuring the activity of AK, PK, PGK, and NDK. The results 500 units in normal liver (Table 2). Cytoplasmic NAD @-GPDH in Table 1 show greatly increased levels of PK and PGK in the was about 20 units in the poorly differentiated hepatomas cytoplasm and large decreases in the activities of AK and NDK compared to 120 units in normal liver (Table 2). The in the mitochondria of the poorly differentiated hepatomas decreased levels of these key cytoplasmic enzymes for both compared to levels in normal liver. The calculated ratio of the malate-aspartate and glycerophosphate shuttle systems cytoplasmic to mitochondrial substrate-level nonoxidative could reduce the ability of the Morris hepatoma mitochondria phosphorylation potential via these enzymes was 0.44 in the to utilize reducing equivalents produced outside the liver tissue, 0.50 in the highly differentiated tumors, and 22.2 mitochondria, if the lowered levels of these enzymes are in the poorly differentiated hepatomas. determined to be rate limiting. Intracellular Hydrogen Transport Systems. Cytoplasmic Measurement of the activities of cytoplasmic NAD@-GPDH reducing equivalents apparently cannot pass directly into the and NAD@-LDH in normal and malignant tissue allow mitochondria but cross the mitochondrial membranes via comparison of competing hydrogen-accepting systems for shuttle systems (13). We have measured the activity of the phospholipid synthesis and anaerobic metabolism (13).

Table 1 Substrate-levelphosphorylation in liver and hepatomas Cytoplasmic:mitochondrial phosphorylation potential ratio: liver, 0.44; highly differentiated hepatomas, 050; poorly differentiated hepatomas, 22.20.

enzymesAKPKPGKpâ€¢pâ€¢@zAKNDKPP.Liver47@8b45Â±1134Â±10126252Â±2034Â±5286HighlyCytoplasmic enzymesMitochondrial

differentiated hepatomas 96l8A 8999 40 Â±10 42Â±9 43 Â±14 210Â±16 41 Â±11 39Â±10268Poorly778745Â±10 44Â±1159Â±1453Â±1139Â±1133Â±13133251Â±27 226Â±2736Â±8

differentiated hepatomas 3924A 3683F 37Â±9 395Â±39 189Â±29 13Â±4 5Â±3 Novikoffhepatoma (solid)42Â±10 44 Â±12372Â±48420 Â±46203Â±27231 Â±3864421Â±8 37 Â±103Â±1 7 Â±329

a Average phosphorylation potential. b @rmoles product/min/g tissue at 37Â° (mean Â±S.E.).

Table 2 Intracellular hydrogen transport systems

enzymesNAD@- Cytoplasmic enzymesMitochondrial

GPDHLiver510Â±43a134Â± MDHNAD@- GAPDHFAD GPDHNAD@- GPDHNAD@- LDHNADP G6PDHNAD@- MDHFAD GPDHNAD4-

3QHighly 22a12@4b123Â± lsa231 Â±32@10Â± 3@277Â± l9a499k 37b6Â±

differentiated hepatoma 96i8A Â±46 Â±18 9 Â±10 21 Â±4 28 Â±49 Â±3 8999 503Â±53 146Â±21 19Â±6 116Â±13 218Â±14 15Â±6 310Â±19 399Â±39 5Â±2 7Â±3Poorly7787497 478Â±36127110Â±916Â±10Â±3109 88Â±11204Â±236Â±1911 13Â±5294Â±259Â±33487541Â±659

differentiated hepatoma 3924A 3683F 116Â±29 109Â±16 12Â±5 10Â±4 222Â±21 27 Â±10 301Â±39 508 Â±42 7 Â±3 Novikoff87Â±18 106Â±8124Â±24118Â±1817Â±1114Â±432Â±915Â±4198Â±17209Â±2418Â±529 Â±9288Â±27354Â±32490Â±36576 Â±445Â±27 Â±3

a Mmoles product/min/g tissue at 37Â° b [email protected] /min/g tissue at 37Â°

JANUARY 1973 53

Table 3 QandadenylatechargeofliverandMorrishepatomas

nucleotide levels(smoles/g tissue)QAdenylate

chargeATPADPAMPLiverTissueAdenine

0.021.420.869Highly(14)a2.47 Â±0.200.51 Â±0.030.15 Â±

differentiated hepatomas 9618A (11) Â±0.23 Â±0.04 Â±0.03)@ 0.031.880.887Poorly8999 (9)2.63 2.78 Â±0.260.470.43 Â±0.060.140.14 Â±

differentiated hepatomas 3924A (27) Â±0.23 Â±0.05 Â±0.02 3683F (18)2.90 2.72 Â±0.240.420.44 Â±0.060.130.14 Â±0.032.060.896

a Numbers in parentheses, number of tumors used to determine the adenine nucleotide levels.

NAD@-GPDH activity was decreased and NAD@-LDH activity used for macromolecular synthesis and growth. Many of the was unchanged in the Novikoff hepatoma (Table 2). The ratio original observed biochemical characteristics of neoplasia of NAD@-GPDH to NAD@-LDH was 0.5 in liver and 0.1 in the revolved around altered forms of energy metabolism: (a) high group of poorly differentiated hepatomas. The decrease in the rates of anaerobic glycolysis; (b) presence of aerobic ratio of these 2 enzymes, which are probably competing in the glycolysis; (c) low rates of respiration (4, 13, 42, 54, 56, 57). same NADH pool, would support the observations of Warburg (54) proposed the first substantial theory of decreased lipid synthesis and increased ability to convert carcinogenesis such that the cancer cell had faulty respiration glucose to lactate in these tumors (13, 38, 55, 57). and had to develop its fermentative metabolism to survive. Only minor changes were observed in the activities of However, development and study of the Morris â€œminimal NAD@-GADPH, cytoplasmic and mitochondrial FAD-GPDH, deviationâ€• hepatomas over the last 12 years have provided NADP@-G6PDH, and the mitochondrial NAD@-GPDH. ample data to illustrate that these 3 original biochemical Comparison of Adenylate Charge and Adenylate Equilib findings are not necessary in certain tumors (e.g. , limited rium. The total quantities of ATP, ADP, and AMP were number of highly differentiated hepatomas) and can be measured in normal rat liver and a group of highly and poorly observed in certain normal tissues (e.g. , leukocytes, intestinal differentiated Morris hepatomas (Table 3). These values were mucosa, retina). The common observation of altered energy used to calculate the reaction parameter of the AK reaction metabolism in highly developed tumors, however, is still (Q) andtheenergychargeoftheadenylatesystemasbelow: paramount. The poorly differentiated Morris hepatomas have undergone - [AMPJ [ATPJ numerous enzymatic and isozymic shifts and/or deletions (15, Q- [ADPJ2 55). These changes may have resulted in altering the potential of metabolic pathways for the competition of common [ATP] + 1/2EADPI metabolites. Shortly after Warburg's hypothesis on carcino Adenylate charge @ [ATP] + [ADPI [AMP] genesis, Johnson (24) and Lynen (32) suggested that the Pasteur effect was mediated by pathway competition. They independently postulated a competition for substrates or Keq for the AK-catalyzed reaction would be equal to Q at metabolites between the transphosphorylating enzymes of equilibrium. The calculated Q for liver tissue, 2 highly glycolysis and the respiratory enzymes of mitochondrial differentiated Morris hepatomas, and 2 poorly differentiated oxidative phosphorylation. Recently, Lo et a!. (31) Morris hepatomas were 1.42, 1.88, and 2.06, respectively. demonstrated a competition for metabolites between the Using highly purified rat liver AK in vitro, we observed the enzymatic systems involved in nonoxidative (glycolyzing) and Ke of the AK reaction to be 1.18. This is in good agreement oxidative (respiratory) phosphorylation. These latter studies wit?i the 1.2 that was determined when bovine liver AK was were interpreted to suggest that a powerful glycolytic system used (33). Obviously, AK is closer to equilibrium in rat liver could carry out glycolytic phosphorylation at the expense of than in the tumor cells. The calculated adenylate charge was respiratory phosphorylation. In support of this concept, we determined to be 0.869 in normal liver, 0.887 in a group of have examined the potential of several enzymes in these highly differentiated hepatomas, and 0.896 in a couple of pathways and have observed that a theoretical ratio of poorly differentiated Morris tumors. All of these values are in cytoplasmic to mitochondrial nonoxidative phosphorylation the most sensitive part of the regulatory range in the energy potential shifts from 0.4 in normal liver to 22 in the poorly charge of the adenylate system (8, 10, 12, 28, 47). differentiated liver tumors. Thus, mitochondrial nonoxidative phosphorylation could be favored in liver, whereas DISCUSSION cytoplasmic nonoxidative phosphorylation could be favored in the hepatomas. The tumor cell has apparently developed an almost Warburg's hypothesis was originally interpreted to suggest unlimited capacity for the production of energy that may be the possibility that tumor mitochondria were malfunctional.

54 CANCER RESEARCH VOL. 33

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56 CANCER RESEARCH VOL. 33

Wayne E. Criss

Cancer Res 1973;33:51-56.

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