Isozymes of Pyruvate Kinase in Liver and Hepatomas of the Rat1

[CANCER RESEARCH 34, 1439-1446, June 1974] Isozymes of Pyruvate Kinase in Liver and Hepatomas of the Rat1

Francis A. Farina,2 Jennie B. Shatton, Harold P. Morris, and Sidney Weinhouse

The Fels Research Institute and the Department of Biochemistry, Temple University School oj Medicine, Philadelphia. Pennsylvania IV140 (F. A. F., J. B. S.. S. W.\, and the Department of Biochemistry. Howard University School of Medicine. Washington. D. C. 20001 [H. P. M .\

SUMMARY 23, 32, 41, 57). These alterations involve the replacement of those isozymes that are under dietary and hormonal control Pyruvate kinase (PK) (EC 2.7.1.40) isozymes were assayed by the host, and that have important metabolic functions in in normal rat liver and a series of transplantable rat the adult differentiated liver by other isozymes which are hepatomas ranging widely in growth rate and degree of normally either low in, or absent from, the adult tissue. As differentiation, with the use of gradient elution by chloride part of an ongoing study of this phenomenon, we have ion from columns of DEAE-cellulose. In agreement with examined the alteration of PK3 (EC 2.7.1.40) isozymes in other studies, three noninterconvertible forms were found in the Morris hepatomas (30. 31), a series of chemically rat tissues: isozyme I, the major form in adult rat liver: induced, transplantable rat hepatomas ranging widely in isozyme II, the sole form in heart and skeletal muscle: and growth rate and degree of differentiation. This enzyme isozyme III, the sole form in poorly differentiated hepato occupies a key position in the metabolism of cells and, as we mas, the major form in normal kidney and lung, and the pointed out previously (27, 28. 57), high levels of this minor form in adult liver. In normal adult rat liver, PK I enzyme may profoundly affect the carbohydrate metabo makes up 90% of the total activity at 57 units/g tissue, and lism and may be a determining factor in the high aerobic is decreased to 30 units/g after a 48-hr fast. Carbohydrate glycolysis of tumors by way of competition with the refeeding restores the normal PK I level in 24 hr and respiratory system for the available ADP. In this study, we increases its activity to twice the normal level after 72 hr. report data on the activity levels and the molecular forms of this enzyme in the "spectrum" of Morris hepatomas and Little orno change occurs in PK III, which remains at 3 to 6 units/g. In regenerating liver after partial hepatectomy, PK compare these with similar data on fetal and adult normal I was slightly below the normal fasted level, but PK 111at 72 liver of the rat [preliminary reports of this work have hr had about twice the normal activity. Fetal liver of 19 to appeared (12, 13, 28, 57)]. 21 days had equal PK I and PK III levels, whereas early Tanaka et al. (49) first demonstrated the existence of fetal liver or whole fetus contained almost entirely PK III. multiple forms of PK in rat tissues and established the Although one highly differentiated hepatoma, 9618A. has presence of 2 forms in rat liver. One of these (PKL) was the a PK isozyme pattern similar to that of liver, other well- and form shown previously to be dependent on diet and insulin highly differentiated hepatomas had much lower total (24, 49. 55, 56), whereas the other form (PKM) had an activities than liver, with a preponderance of PK III. In electrophoretic mobility and kinetic as well as immunologi- contrast, the rapidly growing, poorly differentiated hepato cai properties similar to those of skeletal muscle PK. Since mas had extremely high total PK activity, virtually all then, a host of studies has appeared that point to the consisting of PK III. These results provide further evidence presence, depending on the method of separation used, of as of a profound alteration of gene expression in hepatomas many as 6 isozymes (9, 10, 33, 48), but it is difficult to resulting in the loss of a specific liver-marker isozyme with establish the exact number, owing to the ease with which loss of differentiation and its replacement in poorly differen they can undergo various modifications (6, 17, 22, 26, 47, tiated hepatomas by high activities of an isozyme normally 52). It appears from more recent work that there are only 3 very low in the normal liver. distinct molecular forms (19, 20, 21), each of which probably undergoes conformational changes or intertype hybridization (8, 17, 19). INTRODUCTION That liver neoplasia involves alterations of PK isozyme It is becoming increasingly evident that liver neoplasia is pattern was suggested by several previous studies. Imamura associated with profound alterations in isozyme pattern (10, and Tanaka (20), using acrylamide gel electrophoresis, found that the major form in the AH 130 ascites hepatoma 'This work was aided by Grants CA-10729, CA-10916. and CA-12226 was identical with the minor (M) form in liver. Walker and from the National Cancer Institute, and by Grant BC-74 from the Potter (53), also using starch gel electrophoresis, found an American Cancer Society. increased level of the minor liver form in the preneoplastic 2Part of this work was taken from a thesis submitted to the Graduate Council of Temple University, June 1970, in partial fulfillment of the requirements for the Ph.D. degree. Present address: Department of Clinical 3The abbreviations used are: PK, pyruvate kinase; Kos, the concentra Chemistry, Temple University Hospital. tion at half-maximal velocity without implying Michaelis-Menten kinetics; Received October 23. 1973: accepted March 7, 1974. FDP, fructose 1,6-diphosphate.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. F. A. Farina et al. liver, in the hyperplastic liver nodules, and in thÃ©hepatomas Cellulose. Tissue was homogenized in an equal volume, w/v, that develop after feeding of the carcinogen 3'-methyl- of a solution of 0.5 M sucrose 25 mM Tris HC1, 2.5 mM 4-dimethylaminoazobenzene to rats. Taylor et al. (51) re EDTA, 4 mMacetylcysteine, and 2.5 mM MgCl2 adjusted to ported that the poorly differentiated Morris hepatoma pH 7.5 while cooling in the ice-salt bath, and the resultant 3924A had a PK that differed kinetically from the rat liver suspension was centrifuged at 105,000 x g for 1 hr at 0Â°. isozyme. In our laboratory we found that the liver type was The clear supernatant solution was applied directly to a strongly held by DEAE-cellulose, whereas the isozymes of column. 600 x 11 mm, containing a depth of 400 mm of the Novikoff and Morris 3924A hepatomas. which were DEAE-cellulose, prepared according to the method of present in extremely high activity, were not bound by the Peterson and Sober (34) and equilibrated with a pH 7.5 resin (13). The use of DEAE-cellulose chromatography has solution containing 0.25 Msucrose, 10 mMTris HC1, 1 mM now made it possible to verify the existence of 3 forms of the EDTA, 1 mM MgCl2, and 4 mMacetylcysteine. The column enzyme in rat tissues and to assay quantitatively the was then washed with at least 50 ml of the same solution, activities of each in a series of normal and neoplastic tissues. and elution was conducted with a linear KC1gradient, with 500 ml of this solution in Chamber 1 and 500 ml of the MATERIALS AND METHODS same pH 7.5 buffer solution containing 0.5 M K.C1 in Chamber 2 of a Buchler Varigrad gradient maker. With Tumors used in this study were induced originally in the use of a Buchler polystatic pump, the rate of flow was Buffalo and ACI rats by feeding of carcinogenic chemicals. adjusted to 60 ml/hr, and 5-ml fractions were collected. Complete descriptions of their induction, properties, Chloride was determined gravimetrically by precipitating growth rates, and histolÃ³gica!characteristics have been re AgCl and weighing. Fractions were assayed as described ported previously (30, 31). They were maintained by serial above except for the use of 5 instead of 13 mM phospho transplantation in Buffalo or ACI rats, either i.m. or s.c., enolpyruvate. At the peaks, where enzyme activity was at either the National Cancer Institute or at Howard Uni high, the eluates were diluted with 1% bovine serum albu versity, Washington, D. C., and were routinely shipped to min containing 0.5 M sucrose, 10 mM Tris-HCl, 10 mM Philadelphia for enzyme studies. The Novikoff hepatoma malate, 1 mM EDTA, 4 mM acetylycysteine, and 1 mM was maintained in Philadelphia by serial transplantation in MgCl2 at pH 6.O. When necessary, fractions were concen female Sprague-Dawley rats purchased from the Holtz- trated by vacuum dialysis against the homogenization mann Company, Madison, Wis. Transplantation was buffer solution, using Sartorius collodion bags supplied by either by i.m. or s.c. routes or, where the tumor grows in the Science Essentials, Anaheim, Calif. These preparations ascites form, i.p. Animals were routinely fed a commercial are stable for at least 1 month when kept frozen. rat pellet diet, except in the dietary experiments. All Use of DEAE-Cellulose Chromatography for Assay of PK chemicals were the purest available commercial products. Isozymes. To expedite assays of nonadsorbing and adsorb PK Assays. The routine assay procedure is based on those ing forms of PK, a micromodification of the DEAE-cel of BÃ¼cherand Pfleiderer (7) and LlÃ³rente et al. (26), lulose column procedure was used. The resin was thor with modifications as indicated throughout this paper. Ani oughly washed with 0.01 MEDTA solution, then treated by mals were decapitated and bled; tissues were rapidly ex repeated suspension and dÃ©cantationofa solution of 0.25 M cised and homogenized in a motor-driven coaxial homoge- mannitol in 50 mM triethanolamine at pH 7.5. To separate nizer with a Tenon pestle, with 1 volume w/v of a pH 7.5 the nonadsorbing from the adsorbing forms, the fractiona- solution containing 0.5 M sucrose, 25 mM Tris, 2.5 mM tion was carried out in glass and plastic tubes approximately EDTA, 4 m.Macetylcysteine, and 2.5 mM MgCU. After 0.5 x 8 cm, usually the barrel of a 2-ml disposable syringe. centrifugation for 1 hr at 105,000 x g in a Spinco Model L The bottom was plugged with glass wool and the tube was ultracentrifuge at 2Â°,the supernatant layer was carefully then packed with 2 ml of the washed DEAE-cellulose. removed and stored at 0Â°.This preparation was diluted Several successive 5-ml portions of the ice-cold mannitol/ appropriately to give a AA of 0.03 to 0.1/min with a pH triethanolamine buffer solutions were washed through the 6.0 buffer solution containing 1% bovine serum albumin, column. The receiver was then replaced with a graduated 0.5 M sucrose, 10 mM Tris, 10 mM malÃ©ate,1mM EDTA, centrifuge tube packed in ice, and a 0.2-ml portion of a 4 mM acetylcysteine, and 1 mM MgCl2. For liver and well- 105,000 x g supernatant of a 33 or 50% w/v tissue and poorly differentiated hepatomas, respectively, the homogenate was applied to the top of the column and usual dilutions were 100-, 25 to 50-, and 200-fold. The assay washed through with small portions of the mannitol/tri- mixture contained, in a 0.4-ml volume, 50 mMglycylglycine, ethanolamine buffer solution until 5 ml was collected. The pH 7.5; 100 mM KC1; 10 mM MgCl2;0.15 mM NADH; 5 or receiver was then replaced and the elution was repeated 13 mMphosphoenolpyruvate; and 0.75 unit lÃ¡clatedehydro- with 5 ml of the same buffer solution containing 0.25 MKC1. genase. The reaction was started by the addition of ADP to The adsorbed and nonadsorbed fractions were then assayed, a final concentration of 2 mM and, with a Gilford spectro- as already described, with 13 mM phosphoenolpyruvate. photometer. readings were made for about 10 min at a This procedure effectively separated the nonadsorbed from temperature of 25Â°.Under the conditions used, the reaction the adsorbed enzymes. Molecular weights of the isozymes rates were linear with time and were proportional to the isolated by DEAE-celhilose fractionation were determined amount of enzyme, and blanks without substrate or enzyme by elution from Sephadex G-200 according to the proce were negligible. dure of Andrews (5). A column of 2.5 x 100 cm was filled to Separation of Isozymes by Chromatography on DEAE- a depth of 88 cm with Sephadex G-200 equilibrated with a

1440 CANCER RESEARCH VOL. 34

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. PK in Liver and H epatamos buffer, pH 7.5. composed of O.I \i KC1, 10 mvi Tris HC1, I mM EDTA, and 1 m\i MgCl2. Purified standard proteins, i.e., hemoglobin, yeast hexokinase. liver alcohol dehydro- genase, and rabbit muscle aldolase. covering the range 32,000 to 160,000 were dissolved in 1 ml of the buffer, placed on the column, and eluted with the same buffer. The various PK specimens were applied in the same way. The molecular weight was estimated from the straight line 025 obtained by plotting log molecular weight of the standard proteins against the ratio, elution volume/void volume. 015 005 Â£-o RESULTS LU O. co 50

Separation and Identification of PK Isozymes by Gradient Â§ 40 Elution from DEAE-Cellulose. Although PK isozymes have t 'O been separated by electrophoresis (47, 49, 54) and by 025 O 20 < isoelectric focusing (9, 10. 18), our objective required a more 015 convenient, reliable, and rapid procedure that would sepa IO rate the forms of this enzyme from crude tissue prepara 009 tions, and would give reasonably quantitative recoveries. 60 This has been achieved by means of gradient elution with SO Cl from DEAE-cellulose. Using the methods described in "Materials and Methods." recoveries of applied activity 40 30 ranged between 75 and 100%. and only 3 distinct forms can 025 be identified. As shown in Chart \A, 2 forms are present in 20 015 rat liver. One form is not held by the resin and is eluted in IO 005 the void volume. It is a minor form, representing about 10% J of the total activity in livers of adult, carbohydrate-fed rats, 100 200 300 Â«00 but in 19- to 20-day fetal rat liver this is a major form, VOLUME ELUTED, ML representing about 60% of the total activity (Chart IÃŸ).As Chart 1. Chromatography of rat normal tissue PK iso/ymes on shown in Chart 1/4, the major form in adult liver is tightly DEAE-cellulose. Whole-tissue supernatant fractions obtained by centrifu- bound to the resin and is eluted at Cl concentrations gation of broken cell preparations at 105,000 x g were placed on between 0.10 and 0.15 M.This form is also present in fetal DEAE-cellulose columns and eluted with the KC1gradient, as described in the text. Successive 5-ml fractions were collected and assayed as described liver and, as shown in Chart \B, always appears as a double in the text with the use of 5 mvi phosphoenolpyruvate by the routine peak, one of which Ã©lÃ»tesslightlybefore the adult liver procedure. Activities are given in units (AmÃ³lesNADH disappearing per form. min) per 5-ml fraction. A, normal rat liver: B. 19-to 20-day fetal liver: C, Chart 1C shows that in rat skeletal muscle there is a skeletal muscle. Straight lines intersecting the right ordinales refer to CI single form which is also held by the resin, but which Ã©lÃ»tes before the liver form, at a Cl~ concentration of 0.01 to 0.05 concentration. Mer. necessarily associated with a single tissue type, we feel it is These 3 forms are the only ones seen in either normal or more appropriate to avoid this connotation, and have neoplastic tissues of the rat. All 3 forms, when rechromato- therefore adopted the terminology used by Walker and graphed separately or together, show absolutely no evidence Potter (54) of numbering these forms as I, 11,and 111,in the of interconvertibility or alteration in their elution properties inverse order of their degree of binding to DEAE-cellulose. from DEAE-cellulose. These data are in agreement with Thus, the major liver form is PK 1, the skeletal muscle form those of Imamura et al. (21), who separated the PK is PK II, and the minor liver form is PK III. isozymes by acrylamide gel electrophoresis, as well as with Elution patterns of the hepatomas under study are shown those of Middleton and Walker (29), who used cellulose in Chart 2. The highly differentiated, very slow-growing acetate electrophoresis and DEAE-cellulose chromatogra- Morris hepatoma 9618A gave a pattern very similar to that phy; and with those of Ibsen and Trippet (18), who used of adult liver, with most of the activity in type I and minor isoelectric focusing. Different investigators have used vari activity in type III (Chart 2/4). All of the well-differentiated ous terminologies for the designation of these PK isozymes. hepatomas had low activities of both types, as seen for It is clear that our nonbound form corresponds to the M2 Hepatoma 96I8B in Chart IB. In contrast, all of the form of Imamura et al. (20). the PK IN of Walker and fast-growing, poorly differentiated hepatomas exhibited Potter (54), and the K form of Ibsen and Trippet (18); our only type III, as a single, sharp peak in the void volume tightly bound form corresponds to the major liver form of exemplified by the Novikoff ascites hepatoma pattern in these authors, and the loosely bound form corresponds to Chart 2C. their skeletal muscle form. Because these isozymes are not Range of PK Isozyme Composition in Rat Liver. To

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. F. A. Farina et al. provide a baseline for quantitative comparative studies on modification of liver PK is due solely to changes in the L PK isozymes in hepatomas, a study was made of the range form, and this is corroborated in Table 1. These data show of normal variation of activity in rat liver. To assay the 2 that after a 48-hr fast, there was no significant change in PK isozymes, a micromodification of the DEAE-cellulose type III activity, whereas type I activity dropped by about elution procedure was used, as described in "Materials and 50% on a per g liver basis, and by 75% on a total liver basis. Methods." Tanaka et al. (49) previously showed by both On repletion with a high carbohydrate diet, there was a electrophoretic and immunochemical criteria that dietary remarkable restoration of type I activity, which by 48 and 72 hr was over twice that of the normally fed rats, while little if any change occurred in type 111activity. As shown in SO the last column, the mean type I/type IM activity ratio was

40 I1 in the normally fed rats, dropped to 6 in the 48 hr-fasted rats, and reached extremely high values of over 30 in the ,30 carbohydrate-refed rats. 025 Regenerating livers, through the 24- to 72-hr period of 20 0.15 rapid regeneration, displayed PK activities that were at the IO low, fasting normal levels, and activities in the whole liver O 05 were also quite low, reflecting the low liver weights, which ID ranged from about 3 to 6 g over the 72-hr regeneration ce 40 ÃœJ period as compared with about 10 g for the normal rats. Ã»_ 30 However, in contrast to the dietary effects, type III PK 025 â€¢¿ increased to more than double the normal level during the 20 i period of rapid proliferation at 48 to 72 hr posthepatectomy. >â€¢ OI5 During this period, the type I/type III ratio was below 3. H IO > 0.05 Although the total activity in 19- to 20-day fetal liver was O fairly constant at about 30 units/g, there was a much higher < 200 proportion of type III and a lower proportion of type I than in adult liver. The mean ratio of the 2 forms was about 160 unity: however, there was a wide spread of individual ratios,

120 ranging from 0.7 to 1.8. This wide variance is not surprising 025 since this is a period during which isozyme III is rapidly eo decreasing and isozyme I is increasing. Using an indirect 0.15 40 method of calculation, based on differences in activities at 0.25 mvi phosphoenolypyruvate and differences in inhibition OO5 by Cu2*, Middleton and Walker (29) obtained a type I/type O 100 200 300 400 III ratio of about 1.5 at this time of prenatal development, VOLUME ELUTED.ML. with total activities about the same as ours. Chart 2. Chromatography of rat hepatoma PK isozymes on DEAE-cel PK Isozyme Composition in Hepatomas. With this back lulose. Designations are the same as for Chart 1. A, Hepatoma 9618A; B, ground of information on normal liver, the PK isozyme Hepatoma 9618B; C, Novikoff ascites hepatoma. activities for hepatomas are presented in Table 2. Three

Table I PK isoiymes oj normal adult, fetal and regenerating liver Normal rats were fed a commercial pellet diet ad libitum. Carbohydrate-fed rats were given a diet consisting of 80% sucrose, 10% casein, and 10%cellulose (Alphacel). Male rats weighing between 220 and 230 g were chosen for the dietary study and for partial hepatectomy. Activities are expressed in units (AmÃ³lesNADH oxidized per min at 25Â°)perg liver or per whole liver. Ranges are given in parentheses. The isozymes were separated by the micro-DEAE-cellulose procedure as described in "Materials and Methods," and were assayed with 13 mMphosphoenolpyruvate.

activityPer isozymesType of of DietaryconditionNormal48-hr rats633333337Totalg70(53-90)35(33-37)79(73-88)151wholeliver754(652-825)203(188-212)550(480I, perg57(41 III, perg5.1 I/IllII.

70)30(28-31)76(70 (3.56.8)4.7(4.2-5.0)3.3(2.116.42335264.82.92.31.1 fastRefed24-hr48-hr72-hrRegenerating24-hr48-hr72-hrFetal

670)1660(1560 82)140(136-144)1614.2)4.1(3.1 (147-153)175(169 1710)1500(1450 5.1)6.2(6.1-6.3)5.2(4.8-5.5)9.5(7.9-10.9)10.5(6.9 180)34(32-37)38(37-38)37(36-38)30(181550)112(109-118)184(150 (158165)25(23

26)28(26-30)24(22-26)15(9.0-25)Type 220)220(179 262)Activity 16.0)13(5.7-23)Ratio. (19 to 21 days)No. 39)Per

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Table 2 PK isozymes in rat hepaiomas Values are given in units per g tissue, with ranges in parentheses. Isozymes were separated by the micr-DEAE-cellulose procedure described in "Materials and Methods," and were assayed with the use of 13 mM phosphoenolpyruvate.

isozymesType % of nftumors21134Nn rÂ»fassays575827Total tumor"HepatomasHighlyof activity33(16-44)96(76

differentiated96 7Well18A, Generation 140)13(3 differentiatedPoorly 25)259(150 differentiatedMrv 460)I269432(8?)Ill7466892 "The tumor numbers and generations used for these experiments are listed as follows: highly differentiated: 961 IB (Generations 2 to 6), 7787 (Generations 7 and 8). and 96I8A (Generation 7); well differentiated: 5I23A (Generations 60 to 71), 5123C (Generations 58 to 62), 5123TC (Generation 66), 5123D (Generations 53 to 59). 7793 (Generations 14to 16),7794A (Generations 18 to 21), 7795 (Generation 23), 7800 (Generations 27 to 34), 8999 (Generation 4), 9108 (Generation 11), 9121 (Generation 10), 9633 (Generations 4 and 5), and Reuber H-35 (Generations 43 to 48); poorly differentiated: 3683F (Generations 342 to 379), 3924A (Generations 219 to 248). 7288C (Generations 102 to 122); and Novikoff ascites (generation unknown, > 1000). hepatomas classified histologically as highly differentiated purified by DEAE-cellulose chromatography, gave data were assayed. One of these, Hepatoma 9618A, is in a special identical with those of Imamura el al. (20) with respect to category in growing extremely slowly, requiring up to 1 year Ko.s for phosphoenolpyruvate and ADP, allosteric activa between transfers, having a normal chromosome number tion by FDP, and inhibition by ATP. However, we observed and karyotype, and high levels of glucokinase (44) and a difference in molecular weight. According to Imamura et liver-type phosphorylase activity (39). Its PK isozyme al. this isozyme, obtained in highly purified form from the pattern was virtually identical with that of adult liver, with AH-130 Yoshida sarcoma, had a molecular weight of 94% of type I and only 6% of type III activity. The other 2 216,000. Although we also observed similar values ranging highly differentiated hepatomas, 961 IB and 7787, had total from M.W. 150,000 to 200,000 for different preparations of activities characteristic of normal fasting liver but had only the Novikoff PK III, determined according to the method 26% of their total activity as type I. The 13 well-differen of Andrews et al. (5), when 4 mM FDP was present in the tiated hepatomas had very low total activities, with a type elution buffer, values of 105,000 to 125,000 were obtained I/type III ratio of about 0.5. It is evident that the major when FDP was omitted. These preliminary data suggest difference between normal liver and the well-differentiated that the conformation changes induced by FDP may involve hepatomas is a loss of most of the type I isozyme. aggregation of subunits. It is likely that these low-molecu In contrast, there was a drastic reversal in the isozyme lar-weight forms of PK III are the same as the 2 forms of composition of the poorly differentiated hepatomas. In kidney PK found by Ibsen and Trippet (18) to have these tumors, which grow sufficiently rapidly to kill their molecular weights of 116,000 and 178,000. hosts within 1 month or less, the total activities were very high, from 3 to 7 times that of normal rat liver, and they had DISCUSSION nearly or completely lost the type I isozyme. The value of 8% for PK I given in Table 2 is certainly too high and may Identity of PK HI with PK Isozymes in Other Tissue. be an artifact. As shown in Chart 2C, by DEAE-cellulose Despite the massive and confusing literature on the various chromatography, only type III was evident in the Novikoff forms of PK in animal tissues, owing to its pleotropic nature ascites hepatoma, which exemplified other poorly differenti (18, 22, 47, 54), it appears that in the rat only 3 nonintercon- ated hepatomas. In the micromethod used in these studies, vertible forms exist (17, 20, 54). PK IIÃ•, separated by elution of type III was not always complete when applied in chromatography on DEAE-cellulose, appears to be identi high activity, and it was found that the enzyme that cal with the tumor form observed by Imamura and Tanaka appeared to be held back appeared mainly in the void (20) and Farron et al. (14) by means of gel electrophoresis; volume on rechromatography (12). Thus, we can conclude this form has been further identified with a form observed in that with decreased differentiation and increased growth tissues such as kidney, adipose tissues, fetal tissue, and rate, there was a virtually complete disappearance of the PK cultured liver cells by means of electrophoresis (18. 20, 33, isozyme which is the major form in adult liver, this form 54) or by isoelectric focusing and DEAE-cellulose chroma having been replaced by high activity of a form normally tography (29, 33)." The present study thus amplifies and very low in adult liver. 4This microprocedure did not separate the muscle (PK II) from liver Properties of the Novikoff Ascites Hepatoma Isozyme, PK isozyme (PK I), but this was not necessary since the only adsorbed form in III. A determination of the kinetic constants of isozyme III, liver and the hepatomas was the liver form. A more refined micro-DEAE- obtained from the Novikoff ascites hepatoma partially cellulose fractionation has been developed (J. B. Shatton and S. Wein-

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1974 American Association for Cancer Research. F. A. Farina et al. extends these previous studies in confirming, by means of tion which would presumably maintain the isozyme in the DEAE-cellulose chromatography, the identity of the hepa- active 1Mb configuration. We may add that the embryo, toma PK III with those of other fetal and adult rat tissues, another tissue having characteristically high aerobic gly in confirming the existence of 3 isozymic forms, and in colysis (3), had very high PK III activity (J. B. Shatton providing quantitative data on the activities of these forms and S. Weinhouse, unpublished data), and since tumors in normal liver and in a series of hepatomas. generally have high PK activities (45) that are mainly or The pattern of PK isozyme alteration in hepatomas is entirely PK III, there is a likelihood that this regulatory similar to those patterns observed for the glucose ATP mechanism plays a role in their high aerobic glycolysis. phosphotransferases (44), the aldolases (2), and the phos- Significance of Fetal PK Isozyme Expression in Hepato phorylases (39). Highly differentiated hepatomas retain an mas. The presence of a fetal PK isozyme pattern in poorly isozyme pattern similar to that of liver, with a preponder differentiated hepatomas has already been noted by Tanaka ance of PK I. In less differentiated hepatomas, there is a et al. (50), Schapira et al. (42), and Farron et al. (14) and is variable loss of PK 1 without remarkable change in PK III; in accord with previous observations on other enzymes (23, however, with further loss of differentiation and increased 32, 39, 41, 57). The presence of embryonic antigens in many growth rate, PK I is nearly or completely lost, while PK 111 different tumors types (1,4, II, 15, 25, 37, 46) is now well attains extremely high activity. established, and there seems to be little doubt that, whether Role of PK III in Tumor Glycolysis. We have already measured as isozyme or antigens, there is a misprogram- pointed out (13, 28) that the high PK activity of poorly ming of protein synthesis in cancer such that genes, active in differentiated hepatomas may be an important determinant fetal state but inactivated during normal embryonic devel of the high aerobic glycolysis of these tumors. Recent opment, become reactivated. Granting this, however, many information on PK III, together with the results of this questions remain, among which are whether the fetal expression represents a "programmed," systematic ret study, lends further plausibility to this concept. It has been known for several years that the major liver isozyme, PK I, rogression of adult, differentiated cells, or whether only exists in 2 interconvertible forms. One of these is highly certain primordial cells become neoplastic, or are selected allosteric with a high K0.5 for phosphoenolpyruvate, a low out during subsequent neoplastic development, or whether KÂ¡for ATP and alanine, and a powerful activating effect of the transformed primitive stem cells undergo incomplete FDP, which abolishes the alanine and ATP inhibition and differentiation as suggested by Pierce (35) or as envisioned drastically lowers the K0.5 for phosphoenolpyruvate. The by Potter (37) in the phrase, "oncogeny is blocked ontog other form is insensitive to FDP, but already has a very low eny." When the isozyme data are considered in the light of KO.Sand is not appreciably inhibited by ATP or alanine (6, the fact that the fetal liver PK III is also present in such 26, 43). In a recent paper, Walker and Potter (54) demon adult tissues as kidney, adipose tissue, lung, and brain (17), strated that the PK III in cultured liver cells also exists in and that fetal liver phosphorylase is also present in adult 2 interconvertible forms. One of these forms (Ilia) has kidney and brain (40) and fetal liver aldolase A (41, 46, 57) a relatively high K0.5 of 0.33 mM and was inhibited by is widely distributed among adult tissues, it would appear ATP, alanine, and phenylalanine at low concentrations. that the fetal manifestation may be fortuitous. It may more The other form, Illb, had a low K0.5 and was not inhibited accurately represent a disturbance in the mechanism that by ATP, and was inhibited much less by alanine and phen controls the site specificity of protein synthesis. Further ylalanine. A crucial difference between the allosteric forms examples of a similar phenomenon are widespread in the of PK III and PK I is that, whereas the allosteric PK I is clinical literature, namely, the ectopie polypeptide hormone strongly activated by FDP, the allosteric PK Ilia is actu synthesis by tumors of nonendocrine tissue (11, 16, 38). ally converted to the nonallosteric PK 1Mb, either by FDP Despite the close association of these phenomena with or Mg2+ ions. Walker and Potter point out the similarity of cancer, a necessary relationship is not yet clearly estab this system to the interconversion of forms of the PK III of lished. The switchover of isozyme pattern does not occur adipose tissue (36) and kidney (18), and suggest that this full blown with the induction of hepatomas but rather interconversion may be a factor in the regulation of glucose occurs in a phased sequence. Highly differentiated hepato metabolism. According to the work of Walker and Potter mas retain liver marker isozymes, and only in the poorly (54),5 cells grown in glucose have all of the PK III in the b differentiated tumors is the hepatic isozyme fully switched or active form, presumably owing to the resultant high in- off and the nonhepatic isozyme fully switched on. These tracellular level of FDP. It is conceivable, therefore, that considerations raise the further question as to whether these the high aerobic glycolysis in the poorly differentiated alterations of gene expression are the cause or result of hepatomas is enhanced not only by the high activity of PK neoplasia or are merely an accompaniment of rapid cell III, but also by the glucose-induced rise in FDP concentra- proliferation. Answers to these and other questions remain for further investigation. house, unpublished data) and is being used for PK Â¡sozymeassays in nonhepatic tissues and tumors. 5Recently, Walker and Potter (54) reported that cultured liver cells ACKNOWLEDGMENTS have only PK III. In preliminary unpublished studies (J. B. Shatton. G. Williams, E. Farber, and S. Weinhouse), we found that in several liver cell lines PK III was the main form but was also accompanied by variable, We acknowledge with thanks the assistance of Albert Williams. small amounts of PK I. Charity M. Jackson, and Louise Lawson.

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Francis A. Farina, Jennie B. Shatton, Harold P. Morris, et al.

Cancer Res 1974;34:1439-1446.

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