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Phosphofructokinase Regulation in Some Transplantable Thyroid Tumors1

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Phosphofructokinase Regulation in Some Transplantable Thyroid Tumors1 [CANCER RESEARCH 39, 2796-2801 , July 1979] 0008-5472/79/0039—0000$02.00 Phosphofructokinase Regulation in Some Transplantable Thyroid Tumors1 Maria-Fiore Meldolesi2 and Paolo Laccetti Centro di Endocrinologia ad Oncologia Sper,mentale del C. N. A., Instituto di Patologia Generale dellUniversitá di Napoli, II Facoltádi Medicina, Via S. Pansini 5, I- 80 131 Naples, Italy ABSTRACT ferent properties, i.e. , electrophonetic mobility, affinity to DEAE cellulose, and immunological properties (1, 7, 10—13,16, 20, Some kinetic, molecular, and nate-limiting properties of par 22, 23). We recently reported that nearly pure phosphofruc tially purified phosphofructokinase from normal rat thyroid and tokinase from a transplantable rat thyroid tumor (line 1—8in three transplantable rat thyroid tumors have been studied, and Wollman's classification) showed kinetic and molecular prop interesting differences have been found. Citrate inhibition of erties different from those of the nearly pure rat thyroid enzyme tumor phosphofnuctokinase and its reversal by cyclic adeno (19). We suggested that these differences might partially ac sine 3':S'-monophosphate were lower than those of the normal count for the lack of regulatory control exerted by the tumor enzyme. A direct relationship between tumor growth rate and enzyme on the glycolytic pathway with respect to normal thy the decrease of citrate inhibition was observed. The diethyl roid phosphofructokinase (18). Kinetic studies comparing the aminoethyl cellulose affinity properties of the rat thyroid phos properties of normal and tumor phosphofructokinases showed phofructokinase were slightly but consistently different from that the tumor enzyme was less sensitive to inhibition by ATP those of the three tumors; differences observed among the and by citrate and that the reversal of citrate inhibition of tumor tumor enzymes with regard to these properties were likewise phosphofructokinase by 3':S'-cyclic AMP was less effective, related to their respective growth rates. When the amounts of whereas the protective effect of fnuctose-6-P was stronger. lactate, pyruvate, and glycerol 3-phosphate produced from Despite these kinetic and regulatory differences, the electro each of the substrates of the glycolytic pathway were meas phonetic mobilities of both normal and tumor enzymes were ured, it was found that, in contrast to results obtained with quite similar, and an examination of the respective DEAE normal thyroid extracts, no rate-limiting function was associ cellulose affinity properties showed only small, although con ated with phosphofructokinase activity in tumor extracts. It was sistent, differences. concluded that the modulation of phosphofructokinase by its In order to investigate further if the differences in the kinetic allostenic effectons was similar in all three tumors, although and regulatory properties existing between normal and tumor different from that of the enzyme from normal rat thyroid; it was phosphofructokinase were restricted to only one line of tnans proportional to tumor growth rate and probably related to plantable thyroid tumor, we examined 3 other lines of Woll molecular and regulatory modifications. man's transplantable thyroid tumors which have different growth rates and different histological patterns (28). INTRODUCTION In the present report, we show that the previously described modifications of phosphofructokinase derived from tumor line Phosphofnuctokinase3 is a well-known complex regulatory 1—8are present in all of the other 3 lines of thyroid tumors. enzyme the activity of which is under the control of a large The extent of the defect is directly related to the growth rate of number of allostenic effectons, among which are 5'-AMP, 3':S'- the particular tumor studied. cyclic AMP,4 ADP, fructose-i ,6-P2, phosphoglycerates, phos phoenolpyruvate, citrate, ammonium ions, and the 2 sub stnates, ATP and fructose-6-P (16). The effectiveness of these MATERIALS AND METHODS substances on phosphofructokinase activity differs widely, since there are many phosphofructokinase isozymes with dif Auxiliary enzymes, coenzymes, substrates, and calf thymus DNA were from Boehninger, Mannheim, Germany; bovine se 1ThisworkwassupportedbytheConsiglioNazionaledelleRicerche,00100 rum albumin, EDTA, and dithiothreitol were from Sigma Chem Rome, Italy. ical Co., St. Louis, Mo.; microgranular DEAE-cellulose (DE-52) 2 To whom requests for reprints should be addressed. was from Whatman, Maidstone, England; Sepharose 4B was 3 Enzymes: aldolase (D-fructose 1 ,6-bisphosphate:o-glyceraldehyde-3-phos phate lyase, EC 4. 1.2.13): cytochrome oxidase (ferrocytochrome c:oxygen oxi from Pharmacia, Uppsala, Sweden. All other chemicals were doreductase, EC 1.9.3. 1): enolase (2-phospho-D-glycerate hydro-lyase, EC of reagent grade. 4.2. 1.11): fructose-i ,6-diphosphatase (D-fructose-1,6-bisphosphate 1-phospho The thyroid tumor lines used throughout these studies were hydrolase, EC 3.1.3.11): glyceraldehyde-3-P dehydrogenase [D-glyceraldehyde 3-phosphate:NAD@oxidoreductase (phosphorylating), EC 1.2.1.12]: glycerol-3- developed in Fischer rats by Wollman (28) and designated as phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD@ 2-oxidoreductase, lines 1-3, 1-SG, and 16-S. The tumors were passaged in male EC 1.1.1.8): hexokinase(ATP:D-hexose-6-phosphotransferase, EC 2.7.1 .1): lac tate dehydrogenase (L-lactate:NAD@oxidoreductase, EC 1.1.1.27); phospho Fischer rats by s.c. implantation and reached an average size fructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1 .11); of about 2 g in 11, 45, and 70 days, respectively. The tumors phosphoglycerate kinase (ATP:3-phospho-o-glycerate 1-phosphotransferase, were kindly supplied by Dr. S. H. Wollman (National Cancer EC 2.7.2.3); phosphoglycerate mutase (2,3-bisphospho-D-glycerate:2-phospho D-glycerate phosphotransferase, EC 2.7.5.3): pyruvate kinase (ATP:pyruvate 2- Institute, NIH, Bethesda, Md.). Normal thyroid glands were O-phosphotransferase, EC 2.7.1 .40): triosephosphate isomerase (D-glyceralde obtained from male Fischer rats (Charles River Breeding Lab hyde-3-phosphate ketol-isomerase, EC 5.3. 1.1). oratories Inc., Wilmington, Mass.). 4 The abbreviations used are: 3':5'-cyclic AMP: cyclic adenosine 3':5'-mono phosphate: fructose-i ,6-P2; fructose-i ,6-bisphosphate; P, phosphate. Assay of Phosphofructokinase Activity. Phosphofructoki Received May 8, 1978: accepted April 3, 1979. nase activity was determined by measuring the rate of fructose 2796 CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1979 American Association for Cancer Research. Phosphofructokinase Regulation in Thyroid Tumors 1,6-P2 formed at pH 8.2 and 22°in 1 ml of medium, using the nase (8), aldolase (6), glyceraldehyde-3-P dehydrogenase (3), conditions described by Kemp (10), including both 6 m@iMgCl2 phosphoglycerate kinase (5), phosphoglycerate mutase (4), and S mM ammonium sulfate. One unit of enzyme was defined enolase (2), pynuvate kinase (25), glycerol-3-P dehydrogenase as that amount of phosphofructokinase which converted 1 @moI(1 4), fructose-i ,6-diphosphatase (24), and cytochrome oxi of fructose-6-P to fructose-i ,6-P2 in 1 mm under the above dase activities (27) were determined at 22°and expressed as conditions and in the presence of 1 m@fnuctose-6-P and 1 mr@i nmol of substrate utilized by the 22,000 x g supernatant x ATP. NADH oxidase activity was assayed in the same reaction mg (wet weightY1 X min'. mixture but in the absence of fructose-6-P and was subtracted Protein concentration was determined by the method of from the phosphofructokinase activity. Lowry et a!., (15) with crystalline serum albumin as standard; In all experiments comparing the kinetic properties of normal DNA was measured by the method of Webb and Levy (26); and tumor phosphofructokinases, an amount of enzyme was lactate formation by thyroid hemilobes on by tumor slices was added in each assay that would give a velocity of 0.01 @imolof for 1 hr at 35°in Knebs-Ringer-bicanbonate buffer, pH 7.5, and fructose-i ,6-P2 formed per ml of the standard assay mixture was measured enzymatically (9). and per mm; this was defined as V@(10). Experiments at pH 7.2 were in the presence of 35 mM tniethanolamine buffer (pH RESULTS 7.2), 0.1 mM dithiothreitol, 0.2 mM NADH, 1.0 m@ATP, aldol ase (0.2 unit/mI), tniosephosphate isomerase (0.1 unit/mI), The comparison of several parameters, such as DNA content, glycerol-3- P dehydrogenase (0. 1 unit/mI), fructose- 6- P, lactate production, and tissue dry weight of thyroid hemilobes MgCI2, and other additions (19) stated in the legends to the and tumor slices, showed that lactate production was higher tables and charts. The sequence of addition of inhibitors and and tissue dry weight was lower in tumors versus normal the use of dialyzed auxiliary enzymes were as previously de thyroid (Table 1). The DNA content of the 2 more rapidly scnibed (19). growing tumors was higher than that of normal thyroid. Normal Preparation and Partial Purification of Normal and Tumor thyroid extracts contained less endogenous lactate [8 nmol x Phosphofructokinases. The enzymes from either normal rat mg (wet weightY ‘1thandid that of tumor lines I 6-5, 1-SG, and thyroid on from tumor lines 16-5, 1-5G, and 1-3 were partially 1-3 [14, 19, and 24 nmol x mg (wet y1 , respectively]. purified (123-, 192-, 107-, and 125-fold, respectively) only Kinetic Properties of Normal and Tumor Phosphofructo through the first 3 steps of the previously reported procedure kinases. Below pH 7.4, mammalian phosphofructokinases (19). The final enzyme preparations had specific activities of showed cooperative kinetics and were sensitive to a wide range 11.0, 10.4, 13.2, and 12.4 units/mg, respectively. of activators and inhibitors, among which were citrate, 3':5'- Separation of the Isozymes of Phosphofructokinase by cyclic AMP, and also the 2 substrates ATP and fructose-6-P, Chromatography on DEAE-cellulose Column.
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