Creatine Kinase Activity and Isozyme Compositionin Normal Tissues and Neoplasms of Rats and Mic&

[CANCER RESEARCH 39, 492-501 , February 1979] 0008-5472/0039-0000$02.00 Creatine Kinase Activity and Isozyme Compositionin Normal Tissues and Neoplasms of Rats and Mic&

Jennie B. Shatton, Harold P. Morris, and Sidney Weinhouse2

The Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140(J. B. S. , S. WI, and The Department of Biochemistry, Howard University School of Medicine, Washington, D. C. 20001 (H. P. M.J

ABSTRACT monly considered to be present only in muscle and brain where its activity is extraordinarily high, it has been me Creatine kinase (EC 2.7.3.2) was assayed for total activity ported to be distributed widely in vertebrate tissues (15, 34, by a spectrophotometmic procedure after removal of adenyl 51). However, very little quantitative data are available to ate kinase by gel filtration and for isozyme composition by assess its possible wider physiological significance. We diethylaminoethyl cellulose chromatography. In addition to were prompted to study this enzyme for the following high activity in skeletal and heart muscle and brain of reasons. Creatine kinase undergoes striking changes in normal matsand mice, lower but significant activities were activity and isozyme composition during embryonic devel found in liver, kidney, spleen, intestine, mammary gland, opment and differentiation of muscle cells (48). It is increas lung, aorta, and adipose tissue. Except for muscle (MM), ingly evident that neoplasia is associated with abnormalities heart (MM and MB), mammary gland (MM and BB), lung of gene expression manifested by the appearance of pro (MM and BB), and aorta (MM and BB), all other tissues, teins expressed during early stages of fetal development including whole early (13- to 15-day) embryo had only the (38, 39, 50, 56). Whether gene products are measured as BB form. Although adult liven had exclusively BB, late antigens or isozymes, it appears that genes normally mac prenatal, neonatal, and early regenerating liver had MM and tivated during embryonic development are reactivated in BB forms. cancer, whereas genes coding for proteins of adult, differ All solid and ascites tumors had creatine kinase with entiated tissue are suppressed. It was of interest, therefore, activities at about the same levels as those of the respective to determine whether and to what extent creatine kinase, a tissues of origin. Two rat hepatomas, 5-19 and 5123C, and marker for muscle differentiation (48), would also display a two mouse ascites tumors, the Ehrlich and 5-37, had the patternofdisorderedgene regulationintumors. Incatalyz BB form exclusively, whereas other tumors had some MM ing the interconversion of ATP and ADP, creatine kinase as well as BB isozyme. Theme was no clear correlation must play a role in the dynamics of high-energy phosphate between either the total activity or isozyme composition formation and utilization. By mediating the intracellular and the growth rate or degree of differentiation of hepato concentrations of ATP and ADP, this enzyme should influ mas. The isozyme transitions of connective tissue tumors ence the flux of many reactions of energy metabolism and deviated in part toward the fetal pattern with inconsistent electron transport (22). In view of the distinctive glycolytic appearance of the BB as well as the MM forms. However, properties of some tumors, which may be due at least in isozyme composition of hepatomas and other tumors was part to competition between respiration and glycolysis for altered in the direction of ectopic isozyme expression, with the available ADP (9), the activity of creatine kinase and its the appearance of MM as well as the BB form characteristic isozyme composition in tumors assumes further impor of the tissue of origin. The same isozyme transition toward tance. Its high activity in muscle suggests some special ectopic expression of the MM form also occurred in pen function for this enzyme in the contractile process; if so, it natal and regenerating liver. These results demonstrate the may play a similar role in other tissues in view of the wide occurrence of creatine kinase in normal tissues and ubiquity of contractile proteins such as actin and myosin its survival in the neoplastic transformation. Its functional and contractile structures such as microfilaments and mi significance in these tissues has to await data on the crotubules in cells (31, 45, 58). The association of contrac concentrations therein of creatine. tile structures with the cell membrane, in view of many special features of tumor cell membranes (33), adds further INTRODUCTION to our interest in this enzyme in neoplasia. As part of a continuing study on the loss and retention of Creatine Kinase in Tumors. Data on creatine kinase in enzymes and alterations of isozyme composition in neopla tumors are very sparse. Its occurrence has been reported sia (41, 56), we are exploring the activities, properties, and by Ostretsova et a!. (29, 30) in chick embryo cells, where it isozyme composition of creatine kinase (EC 2.7.3.2) in a is increased 2- to 4-fold by infection with the Rous sarcoma series of normal and neoplastic tissues. This enzyme occurs virus. Salyamon et a!. (36) reported that a rhabdomyosar as dimenic isozymes of 2 different subunits, denoted as MM coma had one one-hundredth of the activity of skeletal (muscle), MB (hybrid), and BB (brain). Although it is com muscle, whereas hepatomas had severalfold higher activity than did liver. Aw and Simons (4) and Simons et al. (43)

, This study was supported by Grants CA-10916, CA-10729, and CA-i 2227 reported that cultured cells derived from solid murine from the National Cancer Institute, Department of Health, Education, and sarcoma virus-induced rhabdomyosarcomas could differ Welfare, and Grant BC-74 from the American Cancer Society. 2 To whom requests for reprints should be addressed. entiate to myotubule formation accompanied by appear Received August 28, 1978; accepted November 8. 1978. ance of creatine kinase; however, the same cells infected

492 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Creatine Kinase in Normal and Tumor Tissues with SV4O did not form myotubes and had a low creatine off and used immediately or stoned at 0Â°.@Partialhepatec kinase activity. Rona et al. (35) claimed to have found the tomy was conducted by the method of Higgins and Anden MM-type isozyme in several human brain tumors; Omenn son (12). and Henmodson (28) reported the BB form in human brain Assay.The methodof choicewasthe spectrophotomet tumors, and Schapima et al. (39) found the BB form in 2 nc method of Oliver (27), measuring the reverse reaction human rhabdomyosarcomas. Adamson (2) and Levine et al. according to the following equation. (17) found that when a tematocarcinoma underwent differ Creatine-P+ ADPâ€”â€˜Creatine+ ATP entiation new cell types appeared that had creatine kinase The formation of ATP was followed by coupling with of the BB type. To our knowledge, themeare no other data glucose, hexokinase, and glucose-6-P dehydrogenase and on its activity or isozyme composition in tumors. monitoring NADPH formation at 340 nm. The assay mixture in a 0.4-mI volume contained: 50 mM Tnis-histidine buffer (pH 6.7), 10 mM MgCl2, 0.5 mM glucose, 8 mM N-acetylcy MATERIALS AND METHODS steine, 0.75 mM NADP@,0.4 unit hexokinase, and 0.8 unit Tumors. Buffalo or ACI rats bearing the Morris hepato glucose-6-P dehydmogenase. The reaction was started by mas, the Morris 7315 pituitary tumor, and Morris Kidney addition of cneatine-P and ADP at final concentrations of 20 Tumors MK1 , MK2, and MK3 (24) implanted s.c., i.m. , or and 2 mM, respectively, and sufficient supemnatant solution i.p. were shipped from Washington to Philadelphia at pe was suitably diluted to give a @Aofabout 0.1/min. After 2 niodic intervals as needed. The 5-19 rat hepatoma was mm, the @Awaslinear with time and was proportional to originally induced by ethionine feeding (42) and has been the volume of supemnatant added. Readings were made for transplanted serially at intervals of 2 weeks. Primary mam 10 mm, and to ensure proportionality with enzyme concen many tumors were induced in rats by a single p.o. injection tration, all assays were routinely conducted with 1 and 2 of 15 mg dimethylbenz(a)anthnacene as described by volumes of supennatant. Exactly similar preparations were Gruenstein et al. (11). Novikoff hepatomas are carried assayed with cneatine-P omitted. This blank measures the routinely by serial transplantation in Sprague-Dawley rats adenylate kinase activity (EC 2.7.4.3) in accordance with obtained from The Holtzmann Co. , Madison, Wis. , and the reaction: Ehnlich and Sarcoma 37 tumors are carried in Swiss mice 2ADPâ€”@AMP+ATP by i.p. implantation as ascites cells or by s.c. or i.m. The creatine kinase activity is taken as the difference implantation as solid tumors. The mouse MCA sarcomas between the complete system and the blank; and these are were early generation transplants of primary tumors in the values that are depicted in the tables, calculated in units duced by topical application of 3-methylcholanthrene (32) (@mol ATP formed pen mm) pen g tissue, on pen 100 mg and were generous gifts of Richmond Prehn. The C-1300 cytosol protein [determined according to the method of mouse neuroblastomas were obtained by s.c. implantation Waddell (52)]. of cultured cells obtained from Klaus Hummeler of the Separation of Creatine Kinase from Adenylate Kinase. Children's Hospital Cancer Center, Philadelphia, Pa. , into Although this procedure is highly satisfactory for creatine A/J mice obtained from The Jackson Laboratory, Bar Ham kinase assay when its activity is high compared with that of bor, Maine. DBA/2J mice beaning the SaD2 fibmosarcoma adenylate kinase, as in muscle or brain, the values are and the BW 10139 nhabdomyosamcomas carried in CBA/J seriously compromised when, as in many normal and neo mice were purchased from The Jackson Laboratory. Re plastic tissues, the adenylate kinase activity is compana agents and enzymes were obtained from Sigma Chemical tively high, resulting in very high blank values. To connect Co., St. Louis, Mo. for adenylate kinase, Oliver (27) used 10 mM AMP in the Preparation of Tissues. Rats and mice were decapitated, assay system as an inhibitor. Although this is no doubt and organs and tumors were excised rapidly and washed satisfactory for tissues relatively high in creatine kinase with 0.15 M NaCI. Excess moisture and necrotic material, if activity, we found that the inhibition was incomplete and any, were removed by blotting and blunt dissection, speci variable and that in our hands it did not yield satisfactorily mens were placed in neutral formalin for histological ex reproducible estimations when adenybate kinase activity amination, and the remainder was weighed and homoge was relatively high.4 nized immediately in an ice-cooled, motor-driven coaxial homogenizer in an equal volume (w/v) of a solution of 0.5 M 3 Although creatine kinase is present in mitachondria of some tissues, particularly in heart, brain, and skeletal muscle (14, 25), the activity is law sucrose, 25 mM Tmis, 2.5 mM MgCI2, 2.5 mM EDTA, and 4 compared with that in the cytasol. To determine residual creatine kinase mM N-acetylcysteine adjusted to pH 7.5. Ascites cells were after centrifugation at 105.000 x g, the residual pellets were rehomagenized with 1 volume per g original tissue of 0.9 PANaCI solution containing 0.5% drawn by syringe from the penitoneab cavity. The suspen Triton x-ioo, 5 mM EDTA, and 4 mp,iN-acetylcysteine (pH 7.0), and recentri sions were centrifuged at 3400 rpm for 10 mm, the super fuged at 105,000 x g . Recoveries of additional enzyme ranged from 1 to natant was discarded, and the cells were resuspended in an 21%, in a wide range of normal and neoplastictissues,with mast under 10%. The electraphoretic patterns were identical with those of the soluble enzyme equal volume of the above-mentioned solution. While cool fractions, and we assume that the residual activity is due mainly to entrained ing in ice, the cells were broken by homogenization as soluble activity, far which no correction was made. above for 1 mm. Cell breakage was virtually complete, and 4 Creatine kinase can also be assayed in the forward direction by coupling the formation of ADP with pyruvate kinase and lactate dehydragenase and the yield of enzyme was not increased by repeated freezing measuring the oxidation of NADH spectrophotametrically at 340 nm as and thawing prior to homogenization. The crude homoge described by Tanzer and Gilvarg (46). This avoids interference by adenylate kinase but has a high blank, due to ATPase activity, and as reported nates of solid tissues or cultured cells were centrifuged at originally by Nodaetal. (26)the reaction occurs at one-third to one-tenth the 105,000 x g for 1 hr at 2Â°,and the supemnatant was drawn rate of the reverse reaction.

FEBRUARY1979 493

To make an unequivocal estimation of creatine kinase isozyme was eluted in the first 2 fractions, together with activity in the presence of high adenylate kinase activity and small, if any, amounts of residual adenylate kinase. After to quantitate creatine kinase isozyme composition, we the fourth fraction was collected, the remaining 0.13 M KCI separated creatine kinase from adenybate kinase by filtra at the top of the column was drawn off and was replaced by tion through Ultrogel AcA-44 and chromatographed the 0.20 M KCI. Three successive fractions collected at this Ultrogeb filtrate, freed of adenylate kinase, on DEAE-cellu concentration sufficed to remove the MB hybrid without lose with a KCI gradient. Ultrogel AcA-44 is described by its eluting the BB form. The latter was then completely eluted distributor, LKB Industries, to consist of beads of 60 to 140 with 1.0 M KCI by collection of five 4-mI fractions. If the @mdiameter in the swollen state, containing 4% each of columns were not overloaded with protein, the separations acrylamide and agamose, which separates proteins of a were highly satisfactory with total recovery between 80 and molecular weight of from 12,000 to 130,000. From a curve 100%, as shown in Chart 2. All of the quantitative data on of Ve/VOversus molecular weight constructed from data isozyme composition in Tables 1 and 2 were obtained by obtained by Ultrogel AcA-44 filtration of hemoglobin, chy assay of individual fractions collected in this manner. motrypsinogen, and RNase, we found the creatine kinases Gel Electrophoresis. Creatine kinase isozyme patterns had molecular weights of approximately 80,000 (53) and thus obtained were confirmed qualitatively by starch gel thus were readily separable from adenylate kinase, whose electrophoresis, essentially as described by us for separa molecular weights mangefrom 23,000 to 68,000 (37). tion of hexokinase isozymes (41). By adding 100 mM P1 p5 A column 2 x 125 cm was filled to a height of 100 cm with di(adenosine-5')pentaphosphate , an effective inhibitor of the gel. After equilibration with a pH 7.5 buffer solution adenylate kinase (19), during color development, any resid containing 100 mM KCI, 10 mM Tnis, 1 mM MgCl2, 1 mM ual bow activity of adenylate kinase was adequately sup EDTA, 4 mM N-acetylcysteine, and 0.02% sodium azide, a pressed, and no discernible bands appeared in the creatine volume of the supemnatant solution containing approxi P blank. mateby50 to 100 units of creatine kinase was applied to the top of the column. The enzymes were eluted with the same buffer at a flow rate of approximately 50 ml/hm, and 3-mb C fractions were collected and assayed for both creatine 0

kinase and adenylate kinase activity as described above. a The separation of creatine kinase from adenylate kinase E was rather sharp, yielding a single fraction with creatine In kinase activity, eluting between 100 and 150 ml, from which 4'

substantially all of the adenylate kinase was removed. With C the exception of liver, the adenylate kinase eluted as a :D single, sharp peak between 160 and 190 ml. With liver U supemnatants, as shown in Chart 1, a small blank peak 4 appeared between 80 and 100 ml, before elution of the creatine kinase. This is probably adenylate kinase isozyme Ill, which has been reported to be a polymer (37). Recover Eluate, ml ies of creatine kinase were in the range of at least 80 to Chart 1. Separation of rat liver creatine kinase from adenylate kinase by 100%, and in most instances somewhat higher activities filtration on Ultrogel AcA-44. A crude homogenate of rat liver was prepared, were found in the gel filtrate than in the crude supemnatant, and approximately 20 ml were applied to the column followed by elution as described in the text and collection and assay of successive 3-mI fractions. presumably due to an inhibitor in the whole supemnatant. ., creatine-Ppresent;0, creatine-Pabsent. Separation of Creatine Kinase Isozymes. Fractions from the Ultrogel filtration containing the creatine kinase activity were concentrated to a volume of about 1 ml by vacuum dialysis at 0Â°against the homogenization buffer. A portion of the enzyme concentrate was then submitted to a micro DEAE-cellubose chromatography procedure similar to that used previously for separation of pyruvate kinase isozymes (7). By trials with samples of each of the 3 purified creatine U, kinase isozymes, we found that sharp and complete sepa C rations of the MM, MB, and BB forms could be obtained by U the following method. Columns (6 x 250 mm) were filled to a height of 30 mm with washed DEAE-ceblubose,and these were equilibrated with 0.13 M KCI in a pH 7.4 buffer solution containing 0.25 M sucrose, 10 mM Tnis, 2 m@iEDTA, 2 mrvi MgCI2, and 4 mM N-acetylcysteine. Samples containing approximately 3 units of activity in volumes from 5 @.dto1 ml were applied to the top of the column, followed by 4ml fractions successive manual applications of 0.13 M KCI. Four 4-mI Chart 2. Separation of creatine kinase isozymes by chromatography on DEAE-cellulose with increasing concentrations of KCI as described in the fractions were collected and assayed with and without text. â€¢,skeletalmuscle, MM; 0, heart, MB; C, brain, BB isozyme. Peaks are creatine phosphate as described above. All of the MM estimated to give a best fit to a symmetrical curve.

494 CANCER RESEARCH VOL. 39

Table 1 Creatine kinase activities and isozyme composition and adenylate kinase activities in normal rat and mouse tissues

ValuesinTissueCreatine are given in (@moIof ATP formed per mm) per g tissue or p 100 mg cytosol protein. Isozyme composition is given percentage of total aunitsctivity recovered from the DEAE-cellulose columns.en kinaseNo. kinaseAdenylate compositionUnits/100 mg of Units/100mg super supemnatant BBRatLiver13 assays Units/g natant proteinIsozyme proteinMMMB Units/g

3Kidney5 18 Â± 3a 17 Â± 310026 Â± 4 25Â± 11Muscle5 39 Â± 4 54 Â± 710049 Â± 9 66 Â± 105Mammary 3143 Â±364 6150 Â±1300100160 Â±29 363 Â± glandPregnancy3 19Lactation3 137 Â± 76 291 Â± 16981-93 7-1921 Â±10 34 Â± 2Heart7 109 Â± 38 166 Â± 5761 3914 Â± 0.4 23 Â± 56Brain5 414 Â± 89 841 Â± 15489 11139 Â±28 283 Â± 32Aorta2 240 Â± 62 740 Â± 12510066 Â±14 169 Â± 38Intestinal 33,21 14618 829,6 4Spleen3 mucosa4 78 Â± 10 150 Â± 210011 Â± 2 35 Â± 3Lung5 60 Â± 5 74 Â± 710013 Â± 2 16 Â± 8Adipose3 55 Â± 12 88 Â± 2516-53 47-8425 Â± 5 36 Â± 5Whale 5 Â± 1.4 48 Â± 121002.7 Â± 0.7 27 Â±

embryo13to15-day2 9.1,12.419-day2 21,26 41,1181002.0,6.3 28.8MouseLiver6 23,27 73,8842 8 508.7, 7.2 22.8,

2Kidney3 7.1 Â± 1.7 7.8 Â± 1.638-68 32-6217 Â± 3 19 Â± 7Muscle3 2.6 Â± 1.4 4.2 Â± 2.220 8037 Â± 5 60 Â± 88Brain3 3194 Â±494 7962 Â± 821100322 Â±47 806 Â± 14Lung3 207 Â± 30 803 Â± 10110038 Â±38 147 Â± 19a 61 Â± 16 107 Â± 3775 3 2233 Â± 8 57 Â± Mean Â±SE.

Hybridization of MM and BB Forms. The separated MM Values for mouse tissues, except for a comparatively low and BB forms were hybridized by treatment with 5 M urea. value for kidney, closely paralleled those of mattissues. Approximately 4 units each of MM and BB isozymes in a Specific activities calculated pen 100 mg protein in the volume of 0.2 ml were mixed with an equal volume of 10 M supemnatant fraction, shown in Table 1, Column 4, panal urea and dialyzed overnight at 4Â°against 100 ml of the leled the specific activities pen g tissue, except of course buffer solution used for tissue homogenization, a modifi for adipose tissue where the value per 100 mg protein was cation of the procedure described by Watts et a!. (54). within the range of activities of other normal tissues. Controls were run simultaneously without urea. The enzyme Adenylate Kinase. Since the cmeatine-Pblanks provided mixture was then submitted to electrophomesis on stanch an approximation of the adenylate kinase activity, these gel, as already described. data are included in Table 1, Columns 8 and 9, calculated per g tissue and pen 100 mg soluble protein, respectively. RESULTS These values varied much more narrowly than those for creatine kinase, extending from 14 to 322 units/g; but, as Quantitative data on creatine kinase activity in a series of for creatine kinase, highest values were obtained for muscle normal tissues of the rat and mouse and in a variety of solid and brain, and lower values were found for all of the other and ascites tumors corrected for adenylate kinase activity tissues. Isozyme composition for adenylate kinase was not as described in â€œMaterialsandMethods' â€˜arecollected in determined. Tables 1 and 2. Table 1, Column 3, reveals that creatine Isozyme Compositionof Creatine Kinase in Normal kinase is present in all of the normal tissues assayed. As Tissues. Columns 5 to 7 depict the isozyme composition, expected, activity was highest in rat skeletal muscle, at over as determined in each instance by DEAE-cellubose chroma 3000 units/g, followed in decreasing order by heart and togmaphy after removal of adenylate kinase by filtration on brain, with respective mean values of 414 and 240 units/g. Ultrogel AcA-44. These patterns were verified qualitatively Activities were somewhat l6wer for the rat mammary gland in each instance by starch gel electrophoresis, as described of pregnancy and lactation; still lower for intestinal mucosa, in the experimental section. As observed previously by spleen, lung, kidney, embryo and liver; and lowest for others (5, 44, 47, 51), rat skeletal muscle had only the MM adipose tissue. Although low by contrast with muscle, the form, and this form was also predominant, but accompa activities in these tissues were fan from negligible; even in nied by the MB form, in rat heart. The MM form was also liven, the activity of 18 units/g is higher than that of such predominant, together with lesser activities of the BB form, enzymes as glucokinase, abdolase, @tndphosphofructoki in the mammary gland of pregnancy and lactation; and both nase that are involved in important hepatic functions (40). MM and BB forms were found in lung and aorta. All other

FEBRUARY 1979 495

J. B. Shatton et a!.

L()CO C) C\J C@JL() Lt) â€˜ItCsJC@J Lt)(Q @â€œJ N- L0C@J@t E 0 .E +1 +1 +1 +1 +1 +1 +1 0 4) +1 +1 +1 +1 +1 +1 +1 +1 +1

4) @ c@Jâ€œJ'CO [email protected] @Lt)L() N- 0) N-@CON-'4 CO Cl) c%JC') CsJ @sJN@C'4 C') ,,t@J C'J â€œJ' CS C Câ€•J C C') .@ C,) a) CS (0(0 â€œs' 0 >â€˜ C Cl) @-CV)C@)C')0 C'J C')Lfl T@ â€˜,J' QQ@@Q@%J C@J E a) V +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 0 â€˜4 C,) Cl) â€˜- C') C 0 r@-@-(0C')C')C')C) oLnLn C') C') â€˜@c@orâ€”c') E C'@,@-r@- â€˜- C') @- â€˜4 LO

C,, 0 C 00000 0 0 0 0 @ .u a Â¶-@r_w_@_0 0 0 0 0 C@J0I'â€”â€˜-@ CO 0 CO C,, @- ,-@-LnLnN â€˜4 LU IIIII 9) C,) C') @-N- C@J C') @ a CO C') â€˜@â€˜Câ€•- â€˜4 N- C') 0. 0 .@ E 0 LU C) 0 C,) a) E 0 >â€˜ N-0)C')COO) Cl) LOLO'4 0) @ N C')LOCOLOC%J LO@ N- C,) â€˜- @0) CO @ 0 â€˜- II I @ @0 Cl) i@d,F- I 0)COC') â€˜@â€˜ @ C%J C',J â€˜4 0 L() 0 C@) 0 a) Cl) 0) Lt)C')N- C')CSJ0 Câ€•J Fâ€” 0) 0) c')râ€”c@@ .@ â€˜4Lt) CsJCsJ@- CsJ C@) 0 ..-0C2 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1+1+1+1+1 CO C') 0) 0) N- 0) N- 0 CO CO â€˜4 â€˜@â€˜C'JCOL0'@' CO @ .@ a)'- â€” â€˜- â€˜- N- â€˜4 â€˜4 CO CO C',J CO@-C')CsJL() @ .@ 2 ,- C') C') () @.t 0. Ca c%J 4) C,'J C@) 0â€•@' CS

0 LO Câ€•JC') CSJ Câ€•)CO CON- CO CO @-N-C')C@J0) CS C@J'4 0) +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 0 Cl) .@4) C'@ C CS @COLOLO@-@'4 @e,@:8 C@J CO C@JCOC')00 C,) â€˜4 â€˜- 0@ CO â€˜ C') CO .2 E â€˜--Cl) 0 tO CO tO L() L() â€˜4 C') Câ€•J'4LC) C) â€˜4 â€˜4'4'4COC') C') 0. 0:2 Ev ZCS

QjC E .2@ C.) C@ @â€˜C,) Cl) CO Cl) NO 0@0 Cl) @ .@2 a) C.) C) 0 @@cl) .@:Cli@ Cli Cl) CS C,, U) E C) 0. 0. Cl) C.) o. 0.0. o@ â€¢@[email protected] @ Cl) ._: .@: â€˜4 Cl) .@: ._ U- .@: <@@-c@JC? E @Â° @fU)csjoâ‚¬@ CSâ€” C @ c%J,-. > > CO CSJ @ 0.@ â€˜ OÂ°CSJO)CO E Oe.E

496 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Creatine Kinase in Normal and Tumor Tissues tissues displayed only the BB form. Although early (13- to other enzymes (56). Two rat hepatomas, the 5123C and the 15-day) rat embryos had the BB form exclusively, by the S-19, and the 2 mouse ascites tumors exhibited only the BB 19th day of gestation there was in the whole embryo a isozyme. All the nest had some MM activity; however, the substantial MM activity as well as a low activity of the MB composition was extremely variable, ranging widely even in hybrid. These findings are also confirmatory of those of each single tumor type; and the isozyme composition bone Tunneretal. (49). no relationship to the growth rate on the degree of differen The MM form was the only one in mouse skeletal muscle tiation. Among the 4 hepatomas, 7288C, 3924A, 9618A, and also, and was the predominant form in mouse lung, to Novikoff, values for the MM isozyme ranged from 0 to 63% gethen with BB and traces of MB forms. The MM form was of the total. Two of the kidney tumors, MK-1 and MK-3, had also present as 20% of the total activity in mouse kidney. barely detectable traces of MM, whereas MK-2 had from 20 Most surprising was a high activity of the MM form in to 55% of the MM form. The mouse MCA sarcoma and the mouse liven; in marked contrast with the lack of this isozyme C-1300 neumoblastoma had high MM form activity, and this in rat liven, although the total activity was less than one-half form was oven 90% of the total in the mouse BW 10139 thatofratliver. rhabdomyosarcoma. Creatine Kinase and Adenylate Kinase Activities in The possibility was considered that the presence of MM Tumors.The data collectedin Table 2, Columns5 and 6, form in the tumors might reflect the entrainment of some establish the presence of substantial creatine kinase activity normal muscle fibers. This seems very unlikely, however. In in a wide variety of solid and ascites tumors of the rat and dissecting the tumors, special cane was taken to avoid the mouse. The values ranged much less widely than they do in inclusion of normal tissue; and all tumors were examined normal tissues, extending from a low of 3 units/g for the histologically to establish the absence of muscle fibers. mouse neuroblastoma to a high of 300 units/g for the MK-3 Moreover, solid tumors implanted i.p. in which inadvertent matkidney tumor. They were high also in 2 other matkidney inclusion of muscle is unlikely, and the Novikoff ascites tumors, in the poorly differentiated 3924A hepatoma, in the cells, had the same range of MM form as the s.c. on i.m. primary mammary tumors, and in the mouse Ehmlich and tumors. Sarcoma 37 ascites cells. Activity was also high in the Adenylate Kinase Activity in Tumors. Asidefrom very mouse rhabdomyosarcoma, although much lower than in low values for the 2 mouse ascites tumors, and one very muscle. high value for the MK-3 kidney tumor, all adenylate kinase Of the 6 transplantable rat hepatomas studied, activities activities fell within a relatively narrow range between 7 and ranged from 4 to 39 units/g. In contrast with our experience 43 units/g. Activities expressed pen 100 mg protein paral with other enzymes (56), there was no clear relationship leled the activities per g tissue. between the activities and tumor growth mate on degree of Creatine Kinase In Fetal and Neonatal Liver. To assess differentiation. Activity was highest in the fast growing, the significance of alterations of activity of cneatine kinase poorly differentiated 3924A and was low in the slow grow and its isozyme patterns in hepatomas, we examined late ing, well-differentiated 9618A hepatoma. The activities of fetal and neonatal rat liver, since marked alterations often the tumors when expressed pen 100 mg protein (Table 2, occur at this period (10). Table 3 reveals that creatine Column 6) paralleled the activities expressed pen g tissue. kinase was present at all stages, with total activities varying No marked differences were found whether solid tumors over a narrow range from 5 units/g in 19-day fetal liven to were implanted i.m., s.c., or i.p., although the numbers neonatal values between 7 and 12 units/g. These activities assayed were too few to establish statistical significance. were only slightly below those of adult liver, but there was Creatine Kinase IsozymeCompositionin Tumors. Iso a marked difference in isozyme pattern. Whereas we never zyme assays, as shown in Table 2, Columns 7 to 9, did not observed the MM isozyme in normal adult rat liver, all of the yield an ordered pattern as we had observed previously for neonatal livers had low but definite MM activity, ranging

Table 3 Creatinekinaseand adenylatekinaseactivities in fetal and neonatalrat liver Conditions and valuesarethe sameasthose given in Table 1. kinaseAdenylate kinaseA tivi composition c tyIsozyme (%of total)Units/gUnits/100

mg mgproteinMMMBBB19-dayfetalAgeofratCreatineUnits/g proteinUnits/100 9.1 19-dayfetal 4.4 6.8 98 6.3 9.7 1 day 7.6 9.6 2 98 13.8 17.6 lday 11.6 14.5 26 74 23.0 28.8 2day 7.5 10.5 14 Tn 86 13.6 19.1 3day 10.9 15.6 16 Tr 84 18.8 26.9 4day 8.5 11.0 9 Tr100 91 21.4 27.5 6day 8.8 10.3 6 94 23.3 27.1 2mos.5.926.5a 11.7 13.22 5Tn1' 957.4 23.511.5 Tr,trace.

FEBRUARY1979 497

from 2 to 26% of the total, and traces of MB hybrid were creatine kinase isozymes, we treated mixtures of the sepa also visible on starch gel electrophoresis. A typical elution rated MM and BB isozymes with 5 M urea, followed by curve of an Ultrogeb filtrate separated on DEAE-cellubose dialysis and then carried out electrophoresis on starch gel @ (Chant 3) clearly shows the presence of MM isozyme in a 3- as described in â€˜Materialsand Methods.' â€˜Controls were day neonatal rat liver. The data of Table 3 show that the MM run simultaneously, in the same way except for omission of form is high in the early neonatal period and then recedes urea. Results of such experiments collected in Table 5 gradually to a low level in the 6-day-old rat. make it clear that both the MM and BB forms of tumor Adenylate kinase activity, either per g liver or per 100 mg creatine kinase can hybridize with each other and also with protein in fetal liver, was about one-third to one-half that in the corresponding forms in normal muscle and brain; that neonatal rat liven, which ranged within the same magnitude the MM and BB forms in regenerating liver can also hybrid as adult matliver. ize; and that the MM, BB, and MB forms in all instances Creatine Kinase in RegeneratingRat Liver. Ratswere exhibit, respectively, identical electrophometic mobilities. partially hepatectomized, the portions that were removed These data provide additional evidence that the M and B (termed control liven)were assayed for total creatine kinase, subunits of creatine kinase are the same proteins in all and the isozyme composition was determined. At the stated tissues examined. intervals after hepatectomy, the regenerating livers were assayed for total activity and isozyme composition. Results DISCUSSION for 10 such pains of control and regenerating livers are displayed in Table 4. In the control livers, the total creatine The quantitative data on creatine kinase reported in this kinase activity ranged from 4.5 to 17.8 with a mean of paper demonstrate that this enzyme is present in a wide 9.2 Â±1.2 (SE.), whereas in the regenerating livers the range of normal tissues of the rat and mouse, that it activity ranged from 7.2 to 27.3 with a mean of 14.0 Â±2.1 survives the neoplastic transformation, and that in tumors units/g. These values were somewhat lower than the mean its isozyme composition may deviate from that of the tissue of 18 Â±3 units/g for adult rat liven shown in Table 1. of origin. However, the observed isozyme transitions differ Although the MM isozyme was not observed either by significantly from those observed for other enzymes, and DEAE-cellubose chromatography or by starch gel electro some findings require further exploration. phoresis in any of the 10 control livers, all of the early Relatively little firm quantitative data are available for regenerating livers, up to 48 hr after hepatectomy, had the comparison with our results on either normal or neoplastic MM form in proportions ranging from 4% to as high as 62% tissues. Our data for normal tissues are in the same range of the total. A typical example of the difference in isozyme of values as those reported by some investigators (3, 5, 44), pattern between a 41-hr regenerated liven and its control but in general they are considerably higher than are activi liver is shown in Charts 4 and 5. ties reported by others (15, 27, 33, 47). Considering the Adenybate kinase data are also available for the megenem many differences in conditions for enzyme extraction and ating and control livers, but because no significant differ methodologies of assay, it seems fruitless to try to reconcile ences were found, they were omitted from the table. The these differences. We feel that our data, determined after respective mean values Â±S.E. were: for regenerating livers, careful establishment of optimal assay conditions and me 21.4 Â±3.0 units/g and 26.5 Â±3.9 units/100 mg protein; for moval of the interfering adenylate kinase, give us reliable control livers, 16.8 Â±0.9 units/g and 19.1 Â±1.2 units/100 measures of maximal tissue creatine kinase activity. mg cytosol protein. Creatine Kinase in Tumors. The data in Table 2 leave no Hybrid Formation. It was surprising that despite the doubt that creatine kinase is retained in substantial activity presence of both MM and BB forms in some normal and in rat and mouse tumors. In general, the activities fell within neoplastic tissues, the hybrid MB form either was not a narrow range compared with that of normal tissues. Low evident onwas detectable only as a faint trace in electropho to intermediate values were found for rat hepatomas, mam netogmams.To further establish the identity of the separated mary and pituitary tumors, and all of the mouse tumors, but relatively high values were found for 3 rat kidney tumors, one of which, the MK-3, had activity exceeded only by skeletal muscle. In previous studies, the pattern of isozyme alteration in hepatomas always deviated toward the fetal or primordial form (38, 56). In conformity with this conception, the mouse Cl) BW 10139 nhabdomyosarcoma expressed some BB activity, z although the MM form, the sole form in skeletal muscle, D was preponderant in this tumor, and all of the tumors of C) 4 connective tissue origin exhibited substantial BB as well as MM activity, except for the highly anaplastic ascites San coma 37, which had only the BB form. When, however, the BB form was the sole isozyme in the tissue of origin, as in matlivenand kidney and matand mouse brain, the corresponding neoplasms expressed MM as well KCI CONcENTRATION,4m1FRACTIONS as BB activity. This surprising finding that in some tumors Chart 3. Creatine kinase isozyme composition of 3-day neonatal rat liver. ., creatine-Ppresent;0, creatine-Pabsentin assaysystem. isozyme composition may deviate toward the differentiated

498 CANCERRESEARCHVOL. 39

Table 4 Creatine kinase and adenylate kinase activities in regenerating rat liver Valuesare the sameas those given in Table 1. Each regeneratingliver had, as its own control, the tissue removedon hepatectomy.Creatine kinase Isozymecomposition

(Units/100 mg supernatant TimeBB22 after hepatectamy(hr) Units/g protein) MM MB 27.3 34.4 40 60 10041Control 17.8 22.2 21.4 25.2 62 38 Control10042 6.9 7.1 15.2 20.0 48 52 10043Control 10.9 12.4 17.8 24.6 34 66 Control10048 10.3 10.6 9.0 10.4 24 76 10068Control 5.3 5.4 8.0 8.9 100 Control10096 4.5 3.9 12.3 15.6 100 Control100Mean, 7.2 6.9 2.8Mean,regenerating 14.0 Â±2.1a 17.3 Â± 1.7a control 9.2 Â±1.2 10.2 Â±

Mean Â± SE.

Cl) Cl)

C C

UI U â€˜4 4

KCl concentration,4ml fractions KCI concentrotion,4m1fractions Chart5.Creatinekinaseisozymecompositionof41-hrrat regenerating Chart4.Creatinekinaseisozymecompositionofcontrolratliver,deter liver. Symbols are the same as for Chart 3. mined by DEAE-cellulose chromatography. Symbols are the same as for Chart 3. is active DNA synthesis accompanied by changes in the form is similar to the well-known ectopic production of activity of many enzymes (23, 55), there are also transient polypeptide hormones by many human tumors of nonen changes of isozyme composition in the same direction of docnine organs (18), where genes also are expressed for fetal expression found in hepatomas. Examples are produc proteins required for differentiated functions of tissue other tion of a-fetoprotein (1), increases in type Ill pyruvate kinase than the tissue of origin. Thus, in the misprognamming of (7) and types lb and III hexokinase (41 ), and the appearance protein synthesis in some tumors, the organ site specificity of aldolase A (38). It is especially noteworthy that the as well as temporal specificity of gene expression can be appearance of creatine kinase MM isozyme in hepatomas breached for enzymes as well as hormones. also has its parallel in the transient appearance of the MM Creatine Kinase Isozyme Transition in Regenerating isozyme in the regenerating liven. During the neonatal and PerinatalRat Liver. In regeneratingliver,wherethere period when many liven enzymes change markedly in activ

FEBRUARY1979 499

ity (10), theme was also observed an isozyme transition species and various tissues other than muscle and brain (6, toward the MM form. Thus, the unusual transition toward 20, 33). LePage (16) reported values for cmeatine-Pin several MM expression that occurs in hepatomas occurs also in primary and transplanted tumors ranging from 0.3 to 2.2 peninatal and regenerating liven. j.@mol/g.Because liver is the site of creatine synthesis, one Lackof MB ForminTissueswith MM and BB Isozymes. might expect an appreciable accumulation. However, me Experience with other obigomenic enzymes indicates that ported values are of the order of 1 or 2 jimol/g (6, 20, 33). hybridization should occur if different subunits are present The only recent data on tumor creatine-P were reported by in the same cell (13). It is puzzling therefore to note the Williamson et a!. (57) who found levels ranging from 0.19 to absence of all but traces of the hybrid MB form when both 1.34 @moI/gin3 kidney tumors. MM and BB forms were clearly present in the same tumor. In preliminary assays for creatine and creatine-P by a The back of MB isozyme remains unexplained, but several modified method based on the principles used by Lowry possibilities can be envisioned: in vivo, there may be some and Passonneau (21), we found values for creatine and intracellular factor which renders the MB form unstable on creatine-P, respectively, of the order of 1 @mol/g;however, that prevents the homodimers from dissociating once they a notable exception was the Novikoff ascites hepatoma are formed. Alternatively, the respective MM and BB forms which in 4 separate assays had a creatine content of 4.3 to may be present in different cells. The latter possibility would 7.8 and a creatine-P content of 0.23 to 1.45 @mol/ml of seem remote in tumors transplanted many times; however, packed cells. Such high levels warrant a further investiga evidence for heterogeneity does exist. Fidlen (8) has ne tion, and studies on the cmeatine and cmeatine-P levels in viewed an impressive body of evidence for heterogeneity of neoplasms are continuing. Whether or not the creatine subpopulations within transplantable tumors, and his labo creatine kinase system is significant in phosphate metabo ratomyhas established the presence within cultured mouse lism in tumors generally or in normal tissues other than tumor cell lines of preexisting clones with great variation in muscle or brain, the presence of creatine-P demonstrates their potential for invasion and metastasis in syngeneic that the system is functional in at least some tumors as well mice. This problem obviously requires further investigation. as in some normal tissues besides muscle and brain. Creatineand Creatlne-PinTumors.In muscleandbrain, the cneatine kinase serves an important function in that it is ACKNOWLEDGMENTS accompanied by high levels of total creatine of the order of 40 and 10 @.tmol/g,respectively (6), so that these concentra We acknowledge with thanks the aid of David Meranze in histological examination of tumors and the technical assistance of Albert Williams, tions of high-energy phosphate can be conserved as cmea Charity M. Jackson, and Louise Lawson. tine-P to maintain high ATP activities in the face of energy demand. Whether creatine kinase functions to any degree REFERENCES in this way in other tissues is problematical, since available data, although sparse, point to relatively low levels of 1. Abelev, G. I. a-Fetoprotein in Oncogenesis and Its Association with Malignant Tumors. Advan. Cancer Res., 14: 295-358, 1971. creatine generally. Values obtained by a variety of methods, 2. Adamson, E. Isoenzyme Transitions of Creatine Phosphokinase, Aldol some of which are old and probably unreliable, mangefrom ase and Phosphoglycerate Mutase in Differentiating Mouse Cells. J. Embryol. Exptl. Morphol., 35: 355-367, 1976. about 0.3 to 4 @moIof total creatine pen g for various 3. Armstrong, J. B., Lowden, J. A., and Sherman, A. L. Brain Enzyme of Creatine Kinase. J. Biol. Chem., 252: 3105â€”3111, 1977. 4. Aw, E. J., and Simons, P. J. Myogenic Cells Derived from Murine Table 5 Sarcoma Virus-Induced Mouse Tumors: DNA Synthesis and Effects of Hybridization of creatineandneoplastic kinase isozymes of normal Simian Virus 40 Infection. Exptl. Cell Res., 79: 231â€”235,1973. tissuesHybridization 5. Burger, A., Richterich, R., and Aebi, H. Die Heterogenitat der Kreatine Kinase. Biochem. Z., 339: 305-314, 1964. thetext, by 5 M urea was conducted as described in 6. Ennor, A. H., and Rosenberg, H. The Determination and Distribution of gelelectraphoresis.and the isozymecomposition was determined by starch Phosphocreatine in Animal Tissues. Biochem. J., 51: 606â€”610,1952. ureaomitted. Controls were run in the same manner with 7. Farina, F., Shatton, J. B., Morris, H. P., and Weinhouse, S. Isozymes of DEAE-cellubosechromatographyThe separated isozymes were obtained by Pyruvate Kinase in Liver and Hepatomas of the Rat. Cancer Res., 34: text.Width after filtration on Ultrogel as described in the 1439â€”1446,1974. of bands indicates intensity.Electrophoretic 8. Fidler, I. J., Gersten, D. M. , and Hart, I. R. The Biology of Cancer Invasion and Metastasis. Advan. Cancer Res., 28: 150-250, 1978. patternUntreated 9. Gosalvez, M., Perez-Garcia, J., and Weinhouse, S. Competition for ADP Between Pyruvate Kinase and Oxidative Phosphorylation as a Control Urea-treatedSource Mechanism in Glycolysis. European J. Biochem., 46: 133-140, 1974. 10. Greengard, 0. Enzymic Differentiation in Mammalian Tissues. Essays Biochem., 7: 159-205, 1971. BBNormal of isozyme MM MB BB MM MB 11. Gruenstein, M., Meranze, D. R., Thatcher, D., and Shimkin, M. B. INormalrat skeletalmuscle I Carcinogenic Effects of Intragastric 3-Methylcholanthrene and 7.12- rat brain I I Dimethylbenzanthracene in Wistar and Sprague Dawley Rats. J. NatI. Cancer Inst., 36: 483-502, 1966. Normalrat liver i I 12. Higgins, G. â€˜M.,andAnderson, R. M. Experimental Pathology of the 5-19 hepatoma IIIII Liver. Restoration of the Liver of the White Rat Following Partial Surgical 7288C hepatoma IIIII Removal. Arch. Pathol., 12: 186â€”202,1931. 3924Ahepatoma IIIII 13. Horecker, B. L. Biochemistry of Isozymes. In: C. L. Markert (ed), The @ Regeneratingrat liver â€¢ i I I Isozymes, Vol. 1, pp. 11-38. New York: Academic Press, Inc. , 1975. Normalratbrain rat muscle + normal 14. Jacobus, W. E., and Lehninger, A. L. Creatine Kinase of Rat Heart INormal IIII Mitochondria. J. Biol. Chem., 248: 4803-4810, 1973. rat muscle + S-19 hepa- I I 15. Jockers-Wretou, E., and Pfleiderer, G. Quantitation of Creatine Kinase lsoenzymes in Human Tissues and Sera by an Immunological Method. I5-19toma II Clin. Chim. Acta, 58: 223-232, 1975. imarhepatoma + MK-3 kidney tu- I i I I 16. LePage, G. A. Phosphorylated Intermediates in Tumor Glycolysis I. Analysis of Tumors. Cancer Res., 8: 193-196, 1948.

500 CANCER RESEARCH VOL. 39

17. Levine, A. J., Torosian, M., Sarokhan, A. J., and Teresky, A. K. Blachem Activity Taken as an Example.) Tsitalogiya, 12: 102-110, 1970. cal Criteria for the In Vitro Differentiation of Embryoid Bodies Produced 37. Sapica, V., Litwack, G., and Criss, W. E. Purification of Rat Liver by a Transplantable Teratoma of Mice. The Production of Acetylcholine Adenylate Kinase Isozyme II and Comparison with Isozyme Ill. Biochim. Esterase and Creatine Phosphokinase by Teratoma Cell. J. Cellular Biophys. Acta, 258: 436-445, 1972. Physiol., 84: 311-318, 1974. 38. Schapira, F. Isozymes and Cancer. Advan. Cancer Res., 18: 77-153, 18. Levine, R. J., and Metz, S. A. A Classification of Ectapic Hormone 1973. Producing Tumors. Ann. N. Y. Acad. Sci., 230: 533-546, 1974. 39. Schapira, F., Michaeu, C., and Junien, C. Foetal Enzyme Pattern of Two 19. Lienhard, G. E., and Secemski, I. I. P' ,P'-Di(adenosine-5')pentaphosphate, Alveolar Rhabdamysarcomas. Rev. Europeene Etudes Clin. Biol. , 17: a Patent Multisubstrate Inhibitor of Adenylate Kinase. J. Biol. Chem., 896-899, 1972. 248:1121-1123,1973. 40. Scrutton, M. C., and Utter, M. F. Regulation of Glycalysisand Glucone 20. Long, C. (ed), Biachemists Handbook, p. 780. New York: Nostrand ogenesis in Animal Tissues. Ann. Rev. Biochem. , 37: 249-302, 1968. ReinhaldCo., 1961. 41. Shatton, J. B., Morris, H. P., and Weinhouse, S. Kinetic, Electrophoretic, 21. Lowry, 0. H., and Passonneau, J. V. A Flexible System of Enzymatic and Chromatographic Studies an Glucose-ATP Phosphotransferases in Analysis. pp. 151-160. New York: Academic Press, Inc. 1972. Rat Hepatomas. Cancer Res., 29: 1161-1 172, 1969. 22. McGilvery, R. W., and Murray, T. W. Calculation of Equilibria of Phos 42. Sidransky, H. A Transplantable Rat Hepatocellular Carcinoma Induced phocreatine and Adenosine Phosphates during Utilization of High En by Ethianine. J. NatI. Cancer Inst., 28: 1425â€”1435,1962. ergy Phosphate by Muscle. J. Bial. Chem., 249: 5845-5850, 1974. 43. Simans, P. J., Tuffery, A. A., McCuIIy, D. J., and Aw, E. J. Cellular 23. Miura, Y. , and Fukui, N. Pleiotypic Responses of Regenerating Liver. Interactions in Vitro with Cells Derived from Tumors Induced by Murine Advan. Enzyme Regulation, 14: 393â€”406,1976. Sarcoma Virus (Harvey). J. NatI. Cancer Inst., 46: 1229-1235, 1971. 24. Morris, H. P. Induction and Some Characteristics of â€œMinimalDeviationâ€• 44. Smith, A. F. Separation ofTissue and Serum Creatine Kinase Isoenzymes and Other Transplantable Hepatomas. Recent Results Cancer Res., 44: on Polyacrylamide Gel Slabs. Clin. Chim. Acta, 39: 351-359, 1972. 103-114, 1974. 45. Soifer, D. (ed). The Biology of Cytaplasmic Microtubules. Ann. N. Y. 25. Murone, I., and Ogata, K. Studies on Creatine Kinase of Skeletal Muscle Acad. Sci., 253: 1975. and Brain with Special Reference to Subcellular Distribution and Iso 46. Tanzer, M. L., and Gilvarg, C. Creatine and Creatine Kinase Measure zymes. J . Biachem. Tokyo, 74: 41-48, 1973. ment. J. Biol. Chem., 234: 3201-3204, 1959. 26. Nada, L., Kuby, S., and Lardy, H. ATP-Creatine Transphosphorylase. 47. Traugatt, C. , and Massaro, E. An Electrophoretic Analysis of Rodent Methods Enzymol.. 2: 605-610, 1955. Creatine Phasphokinase Isozymes. Comp. Biachem. Physiol., 42B: 255- 27. Oliver, I. T. A Spectaphatametric Method for the Determination of 262, 1972. Creatine Phosphokinase and Myokinase. Biochem. J., 61: 116-122, 48. Turner, D. C., and Eppenberger, H. M. Developmental Changes in 1955. Creatine Kinase and Aldolase lsoenzymes in Their Possible Function in 28. Omenn, G. 5., and Hermadson, M-A. Human Phosphoglycerate Mutase: Association with Contractile Elements. Enzyme. 15: 224-238, 1973. Isozyme Marker for Muscle Differentiation and for Neaplasia. Isazymes. 49. Turner, D. C., Maier, V., and Eppenberger, H. M. Creatine Kinase and In: C. L. Markert (ed), Developmental Biology, New York: Academic Aldolase lsoenzyme Transitions in Cultures of Chick Muscle Cells. Press, Inc. , 1975. Develop. Biol., 37: 63-89, 1974. 29. Ostretsova, I. B. Changes in Creatine KinaseActivityin Hepatic Tumors. 50. Uriel, J. Cancer, Retrodifterentiatian, and the Myth of Faust. Cancer Vopr. Onkol., 16: 102-104, 1970. Res., 36: 4269â€”4275,1976. 30. Ostretsova, I. B., Kuznetsav, 0. K., and Zhudina, A. I. Activity of Creatine 51. Van Der Veen, K. J., and Willebrands, A. F. Isaenzymes of Creatine Kinase in the Cells Infected with Rous Virus. Vopr. Virusol., 18: 565â€”567, Phosphakinase in Tissue Extracts and in Normal and Pathological Sara. 1973. Clin. Chim. Acta, 13: 312-316, 1966. 31. Painter, R. G., Scheetz, M., and Singer, S. J. Detection and Ultrastruc 52. Waddell, W. J. A Simple Ultraviolet Spectraphotametric Method for the tural Localization of Human Smooth Muscle Myosin-Like Molecules in Determination of Protein. J. Lab. Clin. Med., 48: 311-314, 1956. Human Nan-Muscle Cells by Specific Antibodies. Proc. NatI. Acad. Sci., U.S., 72: 1359-1363, 1975. 53. Watts, D. C. Creatine Kinase, ATP-Creatine Phosphotransferase. In: P. 32. Prehn, R. T. Analysis of Antigenic Heterogeneity Within Individual 3- D. Bayer (ed), The Enzymes, Vol. 8, pp. 383-455, New York: Academic Methylcholanthrene-Induced Mouse Sarcomas. J. NatI. Cancer Inst., 45: Press, Inc. , 1973. 1039-1045, 1970. 54. Watts, D. C., Focant, B., Moreland, B. M., and Watts, R. L. Formation of 33. Rapin, A. M. C., and Burger, M. M. Tumor Cell Surfaces: General a Hybrid Enzyme between Echinoderm Arginine Kinase and Mammalian Alterations Detected by Agglutinins. Advan. Cancer Res., 20: 1-91 , 1974. Creatine Kinase. Nature New Bial., 237: 51-53, 1972. 34. Richterich, R., Wiesmann, U., and Cantz, B. Comparative Studies on 55. Weber, G., Prajda, N., and Jackson, R. C. Key Enzymes of IMP Metaba Creatine Kinase and its lsoenzymes. In: N. Van Thoac and J. Roche lism: Transformation- and Proliferation-Linked Alterations in Gene (eds.), Colloquium on Biochemical Evolution. Homologous Enzymes Expression. Advan. Enzyme Regulation, 14: 3-24, 1976. and Biochemical Evolution, pp. 243â€”253.NewYork: Gordon and Breach, 56. Weinhouse, 5. Glycalysis, Respiration, and Anomalous Gene Expression 1968. in Experimental Hepatomas: G. H. A. Clowes Memorial Lecture. Cancer 35. Rona,E., Nagy,A., Wollemann,M.,and Slawick,F. Localizationof Res., 32: 2007-2016, 1972. Various lsoenzymes in Different Cell Fractions of Brain Tumors. Neuro 57. Williamson, D. H., Krebs, H. A., Stubbs, M., Page, M. A., Morris, H. P., pathol. Polska, 10: 207-220, 1972. and Weber, G. Metabolism of Renal Tumarsin Situ and during Ischemia. 36. Salyamon, L. S., Ashmarin, I. P., Ostretsava, I. B., Lyzlova, S. N., Cancer Res., 30: 2049-2054, 1970. Pluzhnikova, G. F., and Vaskina, L. V. The Analysis of Tumor Transfor 58. Willingham, M. C., Ostlund, R., and Pastan, I. Myosin is a Component of mation of Tissues II. Biochemical Dedifferentiatian of Tissue During the Cell Surface of Cultured Cell. Proc. NatI. Acad. Sci. U. S., 71: 4144, Carcinogenesis (Changes of Creatine Kinase and Alkaline Phosphatase 1974.

FEBRUARY 1979 501

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1979 American Association for Cancer Research. Creatine Kinase Activity and Isozyme Composition in Normal Tissues and Neoplasms of Rats and Mice

Jennie B. Shatton, Harold P. Morris and Sidney Weinhouse

Cancer Res 1979;39:492-501.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/39/2_Part_1/492

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/39/2_Part_1/492. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.