(CANCER RESEARCH 36, 487-495, February 1976]

SUMMARY

Isozyme patterns of glycogen phosphomylase in the Morrisand Yoshida hepatomas were compared electrophoreticallyand immunochemically with those in rat tissues during development. A 3rd phosphomylase isozyme, observed previously in hepatomas and fetal tissues by isoelectnic focusingand by immunochemical titration, was also separated bypolyacrylamide disc gel electrophoresis. It was observedcommonly in various nat hepatomas, together with variableactivities of the liven type, depending on the degree ofdifferentiation. This isozyme is nearly the sole type in placenta and early embryo, and in liver and skeletal muscle it isreplaced during fetal development with the organ-specificliver and muscle type. In adult rat brain the fetal type isretainedat low levels,togetherwiththe muscle type.Inkidney, spleen, testis, uterus, lung, and stomach, the fetaltype is present together with the liver type. This isozyme inhepatomas and adult brain is identical with the fetal-type, asdetermined by Ouchtenlony double diffusion and activityinhibition tests. Thus, it is considered to be a prototypewhose appearance in hepatomas is one of many knownexamples of fetal protein expression in cancer.

In some poorly differentiated hepatomas, the liver-typeisozyme migrated slightly more slowly in polyacrylamide gelbut could not be distinguished from the liven isozyme immunochemically.

INTRODUCTION

During the course of studies on isozyme alterations ofhepatic “marker―enzymes in rat hepatomas ranging widelyin growth mateand degree of differentiation, it was found bySato et a!. (29, 30, 34) that there is a 3rd glycogen phosphorylase (EC 2.4.1 .1) isozyme in various hepatomas that differed kinetically, by isoelectnic focusing, and immunologically from the liver or skeletal muscle phosphorylase isozyme and was similar to an isozyme present in fetal liven,fetal skeletal muscle, or adult brain. In highly and well

1 This work was aided by Grants CA-10724 from the National CancerInstitute and Grants-in-Aid for Special Scientific Research on Cancer fromthe Ministry of Education in Japan. A preliminary report of this work wasgiven at the Third International Conference of Isozymes, Yale University, NewHaven, April 18 to 20, 1974, and the Colloquium on Carcino-fetal Proteins,Tokyo, November 20 to 24, 1974.

Received June 18, 1975; accepted October 22, 1975.

differentiated, slow-growing hepatomas, the 3rd isozymewas present as a minor component, with predominant livertype isozyme, whereas in poorly differentiated, rapid-growing hepatomas, the liver-type isozyme was largely replacedby the tumor or fetal type. In continuing this study, weexamined this phosphorylase isozyme in the Yoshida ascites hepatomas as well as in various Morris hepatomas,and we found that this type could be separated from theliven and muscle types by polyacrylamide disc gel electrophoresis. Using this method, we examined the distributionof this isozyme in normal fetal and adult rat tissues, itsappearance in hepatomas, and its loss on retention in rattissues during development. In this paper we report data onisozyme composition patterns of phosphorylases of mathepatomas and normal fetal and adult rat tissues by polyacrylamide disc gel electrophoresis. The results indicate thepresence of a fetal prototype in various hepatomas, which isidentical immunochemically with an isozyme in placentaand in adult brain, heart, and other tissues.

MATERIALS AND METHODS

Animals and Tumors. Normal male and pregnant femaleDonryu rats and Japanese albino rabbits were bred in ourmedical school farm and were fed commercial rat and rabbitfood (Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum.Normal male rats weighing 200 to 250 g were used forroutine experiments and for transplantation of the Yoshidaascites hepatomas.

The Yoshida ascites hepatomas (24) were propagated byi.p. implantation in our laboratory and were used within

7 to 8 days after inoculation. Morris hepatomas (19-22) weretransplanted im. in male Buffalo rats in Washington, D. C.;then they were shipped to Hirosaki and maintained as descnibed above until tumors reached 3 to 6 g wet weight,when they were used in the experiments.

Tissue Preparations. Procedures were the same as thosedescribed previously (30), except for the following slightmodifications. The homogenizing medium contained 63 mMTnis-HCI (pH 7.4), 25 mM mercaptoethanol, and 6.3 mMEDTA (pH 7.4). The supernatant obtained by centnifugationfor 10 mm at 5,000 x g was used for determination of thetotal phosphorylase activity (crude extract). For electrophoresis, immunochemical exami nations, on further purification of phosphorylase by column chromatography when thetissue contained no glycogen, crude extract was centri

FEBRUARY 1976 487

Isozyme Patterns of Glycogen Phosphorylase in Rat Tissuesand Transplantable Hepatomas1

Klyomi Sato, Kimihiko Satoh, Tsuyoshi Sato, Fusako Imai, and Harold P. MorrIs

Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Japan [K. S. , K. S. , T. S., F. I.]; and Department of Biochemistry, HowardUniversity School of Medicine, Washington, D. C. [H. P. M.]

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

K. Sato et a!.

fuged for 45 mm at 105,000 x g, and the supennatant wasfractionated by collecting by centnifugation the precipitateformed by treatment with ammonium sulfate between 30and 70% saturation at 0°and pH 7.4. When the tissue contamed glycogen, phosphorylases were sedimented with theglycogen by centnifugation at 105,000 x g for 45 mm (or at78,000 x g for60 mm). The precipitate containing phosphorylases was suspended in 10 mM Tnis-HCI (pH 7.4), 5 mMmercaptoethanol, and 2 mM EDTA (Solution B), and phosphorylases were solubilized by treatment with human salivary a-amylase and collected by ammonium sulfate precipitation between 30 and 70% saturation. These preparationswere kept at —20°without loss of phosphonylaseactivity for4 to 6 weeks.

Assay of Glycogen Phosphorylase. Glycogen phosphorylase was assayed in the direction of glycogen synthesis bymeasuring P1 liberated from glucose 1-phosphate determined by the method of Fiske and SubbaRow (12). Theassay mixture was the same as reported previously (30). Theactivity of the b form of the liver-type phosphorylase wasassayed in the presence of 0.5 M sodium sulfate togetherwith 1 mM AMP. Glycogen and protein were determined bythe same methods reported previously (30).

Chemicals. Glucose 1-phosphate, AMP, and glycogen(from mollusk) were obtained from Boehninger Mannheim,Mannheim, West Germany; other reagents were obtainedfrom Wako Pure Chemical Industries, Ltd., Tokyo, Japan;Freund's complete adjuvant was obtained from latron Laboratonies, Tokyo; and special agar was a product of Difco,Detroit,Mich.

Preparation of Enzymes for Immunological Studies. Ratskeletal muscle phosphorylase b was purified according tothe method of Sevilla and Fischer (36), and matliver phosphorylase was purified by the method of Appleman et a!. (4)or by the following method , by which thefetal-type phosphorylase of hepatoma AH 130, containing no detectable glycogen, was purified approximately 260-fold. The ammoniumsulfate precipitate from a 105,000 x g supemnatant comingdown between 30 and 70% saturation was dissolved in anddialyzed against Solution B; then protein-free rat liver glycogen purified according to the method of Bueding and Orrell(6) was added, and the phosphorylase absorbed to glycogen was collected as a pellet by centnifugation for 45 mm at105,000 x g. This glycogen pellet was suspended thoroughly with a Teflon-glass homogenizer in Solution B and,without treatment with a-amylase, was placed on a DEAEcellulose(Whatman DE-52 isthe best) column, equilibrated,and washed with Solution B. After the initial “breakthrough― of very small amounts of protein and largeamounts of glycogen separated from the phosphorylase, alinear gradient was made with equal amounts (100 ml) ofSolution B, without and with 0.4 M NaCI. A single sharppeak of phosphorylase activity was eluted at approximately0.1 M NaCI. This peak was collected and concentrated byammonium sulfatefractionation.Thisfraction,containingboth fetal and liver-like types was submitted to disc gelelectrophoresis; portions containing the fetal type of activity in 10 to 12 gels were cut out and collected, and they were

finally recovered by reelectrophoresis and elution from thegel. This procedure was also useful for purification of the

fetal-type phosphomylase in brain and liven. With liven, theglycogen pellet was suspended and applied directly to theDEAE-cellulose column without digestion by amylase, andthe purification by disc gel electrophomesis was not necessary.

Polyacrylamide Disc Gel Electrophoresis. The method ofTakeo and Nakamuna (38) was used with slight modifications. The glycogen concentration of the small-pore gel was0.005 instead of 0.01%, because the mobility of phosphorylase isozymes was increased thereby. Routinely, the 105,000x g supemnatant or ammonium sulfate fraction was dialyzed

against the medium of the lange-pore gel before applicationon gel. Electmophomesiswas carried out in a 6% gel at pH 8.9with a current of 2 ma/tube for 90 to 95 mm at 2'. Afterelectrophoresis, gels were washed with cold water, placedin 20 mM Tnis-maleate buffer (pH 6.1) for 30 mm to adjust thepH from 8.9 to 6.1 , and then incubated for 30 mm at 30°in a

mixture (2 ml) of 40 mM Tmis-maleate buffer (pH 6.1) and 40mM glucose 1-phosphate, with on without 3 mM AMP andwith or without 0.75 M sodium sulfate. The a forms sometimes showed multiple forms, apparently owing to aggregation on hybrids between the a and b forms, thus making itdifficult to identify each isozyme on disc electmophonesis.Therefore, the b form of each phosphorylase isozyme wasused in disc electrophonesis, after conversion, if necessary,in the crude extract.

Preparation of the Antibodies and Immunochemical Procedures. Preparation of antibodies and their use in inhibition tests of phosphonylase activity were described previously (30, 34). The fetal hepatoma isozyme was freshlypurified and it was injected on the same day in rabbitsbecause it was unstable.

Ouchterlony Double Diffusion Test. Agamplates 3 mmthick consisted of 1% agan, 2% NaCI, 0.1% sodium azide,and 20 mM Tris-HCI, pH 7.4. An ammonium sulfate-precipitated -y-globulin fraction of the antiserum was in the centerwell, and the crude extracts, ammonium sulfate fraction, orthe purified phosphonylase isozyme preparations were inthe peripheral wells. The ‘y-globulinfraction and enzymepreparations were dialyzed against a solution of 20 mM TnisHCI, pH 7.4, 5 mM mencaptoethanol, 2 mM EDTA, and 0.9%NaCI before immunological examinations.

RESULTS

Disc Gel Electrophoresis of Phosphorylase Isozymes. Inrat tissues, the 3 isozymes of phosphorylase b were clearlydistinguished by polyacrylamide disc gel electrophoresis atpH 8.9. As shown in Chart 1, the fetal type or prototype,purified from the 14-day whole embryo, migrates most rapidly, the muscle type migrates less rapidly, and the liver typemigrates least rapidly toward the anode. The muscle andfetal types required only AMP, and the liven type requiredboth AMP and sulfate, as expected from previous observations (30). The fetal-type activity dependent on AMP wassomewhat inhibited in the presence of sulfate, as seen inChart1 and Tables1 and 2.

lsozyme Composition of Normal Tissues on Disc GelElectrophoresis. Chart 2 shows phosphorylase isozyme

488 CANCER RESEARCH VOL. 36

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

TissuePhosphorylase

activity(units/gtissue)—AMP+AMP+AMP

+Na2SO4Fetal

tissuePlacenta (15—20-day) (3)°Wholeembryo (10-day)(2)Whole embryo (20-day) (2)Liver (21-day) (3)Brain (21-day) (2)Heart (21-day) (2)Skeletal muscle (21-day)(3)1

.28 ±1.781.851 .63 ±1.622.821.57±0.63b

0.68

0.762.77c

±1.802.45e2.353.40 ±0.182.56―

12.97c11 .97 ±5.062.31

±1.504,45c7.58c ±1.920.50

0.70Adult

tissueLiver (10)Kidney(9)Spleen (7)Testis (6)Uterus (4)Lung (7)Stomach (5)Intestine (6)Brain (8)Heart (9)Skeletal muscle (9)2.36

±1.19 ±1 .46 ±0.77 ±1 .39 ±1.91 ±1.79 ±1 .56 ±4.49 ±

11.50±2.01 ±0.79

0.470.860.490.591.300.860.860.573.060.744.25

±0.872.68c± 0.513.87c ±0.311 @37c±0.293.48―±0.68357―±0.563.90r ±1.202.71c ±0.478.30c ±0.58

41 .17c ±2.84172.6e ±20.019.38c

±2.11 ±3.22 ±0.64 ±2.76 ±2.59 ±2.15 ±1.61 ±5.93 ±

38.5 ±168.0 ±1.02

0.360.630.200.810.401.090.190.552.34

19.2

Phosphory!ase !sozymes in Hepatomas

LIVER TYPEMUSCLE TYPE

ORIGIN—―@

Chart 1. Polyacrylamide disc gel electrophoresis of phosphorylaseisozymes of rat muscle, liver, and fetus. Electrophoresis was carriedout at 2 maltube for 90 mm in 6% gel at pH 8.9 according to theprocedure of Takeo and Nakamura (38), slightly modified as in the text.The fetal type was partially purified from the 14-day-old whole embryoby amylase digestion and ammonium sulfate fractionation (30 to 7@saturation), and muscle and liver types were purified according toSevilla and Fischer (36) and Appleman et al. (4), respectively. Approximately 0.05 unit of each type was applied. The activities were detectedin the absenceand presenceof AMP (3 mM)without or with Na,S04(0.75 N), as indicated.

BPB—@ ____ ____

AMP — + — + +Na@SO4— — — — + — — +

Table 1Glycogen phosphorylase activities in fetal and adult tissues and hepatomas

Assay procedures are given in the text and activities are expressed in units/g tissue, a unit beingdefined as @molesP released from glucose 1-phosphate per mm in the direction of glycogensynthesis.

(1 Numbers in parentheses, number of tissue preparations from different rats. The same tissues

pooled from all fetuses in the same litter were used for a preparation. A mean value of duplicateassays was obtained on each preparation.

b Data are presented as mean or mean ± SE. (p = 0.05).

C These values were used as the total activities.

composition patterns of several fetal and adult mattissues.The placenta (15- to 20-day) has predominantly the fetaltype with occasionally a minor band of the liven-like type,the activity of which is variable in different preparations,perhaps owing to the cellular heterogeneity of this tissue.The early (10-day) whole embryo has only the fetal type,indicating that this is the prototype isozyme. In the late (20-day) whole embryo, both the muscle and liven types appear,together with the fetal type. In 21-day-old fetal liver andskeletal muscle, the fetal type is still present, together withpredominant liver and muscle types, respectively. The fetaltypes in the liven and skeletal muscle are still detectablewithin 2 weeks after birth but are not detectable in the adult

tissues.2 The fetal brain (21-day) contains almost entirely thefetal type, and this persists as the predominant type in theadult brain, together with a low activity of the muscle type ora hybrid between the fetal and muscle type. The fetal heart(21-day) has both the fetal and muscle-types, but the adultheart has predominantly the muscle type, together with alow fetal-type activity. These results differ from those ofDavis et a!. (9), who detected only the muscle type in the ratheart. The fetal type is widely retained in many adult tissues.

2 In some adult rat livers, especially those from pregnant rats, very weak

activities of the fetal type were detectable when total activities higher than 0.2unitwere used.

FEBRUARY 1976 489

FETALTYPE

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

HepatomasPhosphorylase

activity (unitslgtissue)—AMP+AMP+AMP

+Na2SO4Morris

hepatoma16(1)@7777(2)5123D (2)2.30

0.883.254.80

5,08b5.8cY'11.8―

4355.60Yoshida

ascites hepatomaAH 130 (7)AH7974(3)AH 13 (3)AH 66F (3)2.66

±1.82±0.93 ±0.78 ±0.2O@

0.420.240.386.37―

±0.16363―±0.412.85 ±0.103.07 ±0.414.90

±0.092.98 ±0.195@35―±0.106.46―±0.15

K. Sato et a!.

Table 2Glycogenphosphorylaseactivities in hepatomas

Assayproceduresare the sameas in Table 1.

aNumbersin parenthesesindicatenumberof preparations,on eachof whichduplicateassaysweredone.

b These values were used as the total activities.C Data are presented as mean or mean ± S.E. (p = 0.05).

LMF

a..

type, total phosphorylase activities are generally very low.This table shows also that total phosphonylase activities insuch tissues as the whole embryo (20-day), the fetal liver(21-day), and the adult liven depend strongly on the presence of sulfate in addition to AMP, indicating the presenceof the predominant liver-like isozyme. In other tissues, sulfate strongly inhibits activities dependent on AMP, owing toits inhibitory action on activities of the fetal type. In only afew tissues such as the liver, heart, and especially skeletalmuscle do the liven or muscle types increase markedly duning development, as reported previously (31, 32, 34).

Isozyme Composition of Hepatomas on Disc Gel Electrophoresis. Chart 3 shows disc electrophoneticzymograms ofphosphorylases of various hepatomas compared with fetaland adult normal and regenerating liver. Regenerating liver72 hr after partial hepatectomy has a weak fetal isozymeactivity that is not detectable in the normal liven. All hepatomas so far examined, including the Morris and Yoshidahepatomas [such as Novikoff hepatomas, as reported previously (30, 34)], contain the fetal type in common, althoughthe activities vary with the degree of dedifferentation andaccording to the characters of the strains. Almost all hepatomas also retain what appears to be the liver phosphorylaseas well as the fetal type, as demonstrated by its requirementfor both AMP and sulfate. Among the many individualstrains of the Yoshida ascites hepatoma, most, like AH 130and AH 7974, have very weak activities of the liver-like type;whereas other strains such as AH 66F and AH 13, which areexceptional in accumulating relatively large amounts of glycogen, contain predominantly the liver-like type and minoractivities of the fetal type. The Morris hepatomas 7777 and51230 have significant liver-like activities, although they arepoorly differentiated. On the other hand, the Morris hepatoma 16, a well-differentiated and slow-growing hepatoma,has a high liver-like activity together with a low fetal-typeactivity.

Table 2 shows phosphorylase activities in these hepatomas. The Morris hepatoma 16 and hepatomas AH 13 andAH 66F have activities dependent on the presence of sulfate, indicating the predominant presence of the liven-like

F.@ @sw.s@th(i5@sy) I

Eañywh@embryo(busy)

@ whole— @day)

L@

Brain

H@fl

Skeletalmuscle

Aduft @ss

@dn@

Ts@s(Uterus)

Lung

5tomach-a)

Bm@

Hun

@ muscle

Chart 2. Polyacrylamide disc gel electrophoretic patterns of phosphorylass isozymes in various normal, fetal, and adult rat tissues. Electrophoresiswas done under the same condition as in Chart 1. Fetal tissues were obtainedfrom 21-day-old fetuses except for placenta and whole embryos. The ammonium sulfate fractions of 105,000 X g supematants, after amylase digestionof 106,000 x g precipitates containing glycogen from such tissues as thewhole embryo, the fetal liver and heart, and the adult liver, were used, and0.02 to 0.06 unit of thetotal phosphorylase activities was applied to the gel.

Kidney, spleen, testis, uterus, lung, stomach, and intestinehave the fetal-type as well as a liver-like type, which has aslightly lower electrophoretic mobility than does the livenisozyme but is otherwise similar immunochemically andkinetically in requiring sulfate for activation. Brain and hearthave both the fetal and muscle type. As shown in Table 1,however, in both fetal and adult tissues having the fetal

K'

—

490 CANCER RESEARCH VOL. 36

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Fetus (14d)

Normal liver

@rr@?

::::4.@

@—-,

“‘@‘“ 1+

!__@___L@________________________@AH

66F

AH 13

Moms77n1

@DI

::..y_-

--+

@___A_ai_.___@.

FractionProtein (mg)Specificactiv

ity (units/mg)Yield (%)Punification,foldCrude

extract5,000x gsupernatant39780.036100.01.078,000

x gsupemnatant20740.05065.31.430—70%ammonium sulfatefraction11650.07252.82.0Glycogen

precipitate30.82.7653.476.7Eluatefrom DEAE-cellulose column chro 4.511 .131.1308matographyEluate

fromreelectrophoresisFetaltype(F)0.849.455.7262Liver-like

type (L')0.496.371 .8177

Phosphorylase !sozymes in Hepatomas

Na@S0@

Chart 3. Polyacrylamide disc gel electrophoretic patterns ofphosphorylases of various hepatomas. Electrophoresis was doneunder the same condition as in Chart I . Phosphorylases to eachhepatoma in ammonium sulfate fractions of 105,000 x g supernatants (0.02 to 0.04 unit/tube) were used. With 14-day-old fetuses,normal liver, hepatomas AH 66F, AH 13, and Morris 16, amylasedigestion, and then ammonium sulfate fractionation were carriedout with 105 000 x g precipitates containing glycogen. All activities were detected in the presence of AMP without or with sodiumsulfate, as indicated.

Purification of the phosphory!ase isozymes of hepatoma AH 130

Table 3

type, as already shown in disc gel electrophonetic patterns(Chart 3) and the immunotitration test (Chart 7). The fetaltype in hepatomas is unstable; its activity is easily lostduring storage at 2°and, therefore, may be underestimated.

Purification of the Fetal Type of Hepatoma AH 130. Previously, we purified the fetal type of the Novikoff asciteshepatoma (34), but the preparation was still contaminatedwith a small amount of the liven-like isozyme. To examine inmore detail the immunochemical properties of the fetal typein hepatomas, the fetal type of hepatoma AH 130 was punfied. The purification steps and the degree of purificationare summarized in Table 3. By DEAE-cellulose (DE-52) column chromatography, a single sharp peak of phosphonylase activity was obtained, as shown in Chart 4. Since thispeak, however, still contained both fetal and liver-like typeson disc gel electnophoresis Chart 5A, sections containingonly the fetal-type activity in 12 gels were cut, and the fetaltype was collected by reelectrophoresis (Chart SB). Thefetal-type isozyme was then purified 260-fold with a yield of6%. The fetal type of adult nat brain was similarly purified150-fold, free of the muscle type. The purified fetal type isunstable after separation from glycogen; it is inactivated by50% during 5 days of storage at 2°in Solution B at pH 7.4and more rapidly in the more alkaline buffer. Purificationprocedures by DEAE-cellulose column chromatographyand disc electrophoresis were therefore performed in 1 day;then injection was immediately given to a rabbit. Rabbit

‘VI0U,‘VI0

::@:

FRACTIONNUMBER

Chart 4. DEAE-cellulose chromatography of hepatoma AH 130 phosphorylase b. Glycogen suspension (28 ml)abserbing 87 units of AH 130 phosphorylases(b form) in 27.8mg protein wasplacedon a I .7 X 10-cmcolumnofthe resin equilibrated with the buffer solution (Solution B) containing 10mMTris-HCI (pH 7.4), 5 mM mercaptoethanol, and 1 mM EDTA, and eluted with alineargradientmadeby passingthrough 100ml of buffer without and with0.4 N NaCI. The flow rate was kept at 50 mLlhr and 3-mI fractions werecollected. •, absorbance at 280 nm; 0 , total phosphorylase b activity with 1mM AMP; ———,NaCI concentrations (M). The absorbance of the “breakthrough―fraction was due largely to glycogen turbidity.

FEBRUARY1976 491

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

K. Sato et a!.

antisera to the fetal types from AH 130 and rat brain wereprepared as described in the text.

Ouchterlony Double Diffusion Test. As shown in Chart6A, the Ouchterlony double diffusion test showed only 1precipitate line between the antibody to the fetal type of AH130 in the center well and the extract of AH 130, placenta,fetal liven and muscle, and brain, and the precipitate linesfused smoothly. It was further confirmed with the antibodyto the fetal type of adult rat brain that fetal types of AH 130

and brain were indistinguishable, as shown in Chart 6B.Activity Inhibition by Antibody. This test was carried out

for approximate quantitation of the activity of the fetal typein various normal rat tissues and hepatomas. As shown inChart 7A, the activity of the extract from AH 130 was inhibited linearly about 70% with increasing amounts of theantibody to the fetal type of AH 130, but it gave a biphasiccurve, and the noninhibited residual activity was absorbedby the antibody to liven isozyme, indicating that in this

hepatoma the fetal type is predominant and the liven-likeisozyme is minor. Hepatoma AH 13, in spite of being poorlydifferentiated, and the well-differentiated Morris hepatoma16 also gave a biphasic curve, but the lessen degree ofinhibition indicates the presence of only about 20% of thefetal-type isozyme. The predominant nonfetal type is appanently the liver-like type, judging from the electrophoneticpattern.Chart7,A and B, shows thattheadultliverandskeletal muscle contain no fetal type and also indicates thatthe liven and muscle types do not cross-react immunologically with the fetal type. The fetal liven and muscle (16 to 17days) have about 50% of the fetal type, and the residualactivity is the liver on muscle type, respectively, as given inour preliminary report (32). The adult matbrain has about80% of the fetal type and the residual activity is the muscletype, as shown by the disc gel electnophonesis. In contrast,the adult natheart has about 20% of the fetal type and 80% ofthe muscle type. In Chart 7, activity inhibition slopes of thevarious tissues are approximately the same, another indication, together with the electrophonetic patterns, that thefetal type in the hepatomas is identical with the predominant fetal isozyme.

Immunochemical Properties of the Liver-like Type in

AMPNa@SO+

+@— +

A+

+— +

B+

—+ +C

Chart 5. Separation of fetal and liver-like type phosphorylases of hepatoma AH 130 by polyacrylamide disc gel electrophoresis. A, a preparationobtained by DEAE-cellulose chromatography; B and C, the fetal type and theliver-like types, respectively, obtained from A by reelectrophoresis. Procedures are described in the text. The activity of each sample applied on gelwas 0.05 unit in A, and 0.02 unit in B and C, respectively. All activities weredetectedin thepresenceof AMP(3mM)withandwithoutNa@SO4(0.75M).Activities without AMP were not detectable, indicating that the preparationswere not contaminated with phosphorylase a.

,,.ø-@@

/

A BChart 6. Ouchterlony double diffusion tests of phosphorylases in hepatoma AH 130, rat fetal tissues, and adult brain against antibody to the fetal-type

isozyme of AH 130 or the brain. Procedures are described in the text. A, ammonium sulfate fractions of AH 130 (130), placenta (Pla), fetal liver (fL), and skeletalmuscle (fM) obtained from 17-day-old fetuses, and adult brain (Br) were in the peripheral wells, and the antibody (y-globulin fraction) to the fetal type of AH130 (130ab) was in the center well. B, ammonium sulfate fractions of the adult brain (Br) and AH 130 (130), antibodies to the fetal types of AH 130 (130ab) andof the brain (Br ab) were placed as indicated. The activity of each sample (about 1.2 mg protein) was about 0.12 unit.

492 CANCER RESEARCH VOL. 36

@fl

C

130•

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Phosphorylase !sozymes in Hepatomas

(B)

Adult heart@ V V—V--—--—@—--——

‘\@almusc@ (17d)

Adult brain

C

E>:

>

C-)

U)

>-

0IV.U)0IV.

754

Chart 7. Inhibition of phosphorylases of hepatomas and some ratfetal and adult tissues by antibody to the fetal type of hepatoma AH130. Procedures were described previously (30). The ammonium sulfate fraction (0.05 ml) of each tissue was used. Antibody (1 ml)inhibited about 2.5 units of the fetal type. Activities were assayed inthe presence of 1 mM AMP with 0.5 M Na@SO4in A and without it in B.A, 0, adult liver; •,fetal liver; & Morris hepatoma 16; A—A,hepatoma AH 130; A——A,AH 130 supernatant absorbed with antibody to liver isozyme (L-ab) at 2°for 3 hr; A, hepatoma AH 13. B, 0,adult muscle; •, fetal muscle (17-day); V, adult heart; V, adult brain.

5o@

25

0.03 0.050 0.01

ANTI-BODY TO FETAL TYPE OF AH 130. ml

Hepatomas. It issurprising that some strainsof the Yoshidaascites hepatoma contain considerable liven-like phosphorylase activity, because they grow rapidly and are regardedgenerally to be poorly differentiated. Indeed, most livermarker isozymes such as glucokinase, aldolase B, and pyruvate kinase L are lost (26). As shown in Chart 3, the liver-liketype phosphomylase predominates in the AH 66F and AH 13but is the minor type in the AH 130 hepatoma on discelectrohporesis. Chart 8 demonstrates that in Ouchtenlonydouble diffusion tests themewas only 1 precipitate line between the extracts of different hepatomas and the livenantibody present in the center well, and the precipitate linesfuse smoothly with each other. These results (together withresults of immunoelectmophoresis not shown) indicate thatthe adult liven isozyme and the liven-like type in hepatomasare quite similar immunochemically, despite the slightlydifferent mobilities in disc electrophonesis.

DISCUSSION

These results support and amplify previous reports ofSato et a!. (29, 30, 34) in showing that a 3rd phosphomylaseisozyme present commonly in various hepatomas is identical electrophonetically and immunochemically with an isozyme present predominantly in fetal tissues- This isozyme isa sole type in the early embryo and thus seems to be aprimordial prototype isozyme. During normal embryonaldevelopment, the prototypic isozyme decreases and ultimately disappears from liven and skeletal muscle in theadult stage as new isozyme species appear and increase inthese tissues. In contrast, in other tissues such as kidneyand brain, the prototype form is retained but is accompanied by variable activities of the liven or muscle isozyme.When differentiation is complete in the peninatal period, theisozyme pattern becomes ‘‘lockedin' ‘, and the general pattern of isozyme composition is rigidly fixed in adult tissues,although the liver-type isozymes are subject to variationunder hormonal influence. Differentiation during embryonic development must depend on rigidly controlled mechanisms of selective gene activation. However, in hepatomasthe rigidity of gene control is so loosened that not only arethe adult liven isozymes suppressed but the prototypic isozyme becomes expressed. As dediffenentiation proceeds

Chart 8. Ouchterlony double diffusion tests of phosphorylases in varioushepatomas against antibody to liver isozyme. Procedures are described inthe text. Lab, ‘y-globulinto liver isozyme; L, purified liver isozyme; 13,hepatoma AH 13; 5123D, Morris hepatoma 5123D; 130, hepatoma AH 130; 16,Morris hepatoma 16.

and growth rate increases, the hepatomas lose the livertype isozyme activity, and other isozymes that are normallylow or may be undetectable in the adult liven begin toappear. Furthermore the isozyme patterns of the poorlydifferentiated hepatomas resemble those of early fetal liver,where the adult isozyme is low on absent. Results frommany laboratories leave no doubt that this pattern of isozyme alteration is a general phenomenon in hepatomasobserved with other enzymes (8, 16, 26, 35, 41). Weinhouse(40, 41) proposed that the replacement of hepatic key isozymes under host dietary on hormonal control by otherisozymes not under host regulation might be an underlyingmolecular foundation for the lack of control of cell proliferation that characterizes the cancer cell.

Glycogen accumulation is an important hepatic functionand is highly regulated by hormones and diet through asynergistic cyclic activation and deactivation of glycogen

FEBRUARY1976 493

L)@ab@13@

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

K. Sato et a!.

synthetase and phosphonylase via phosphorylation and dephosphorylation of these enzymes catalyzed by kinases andphosphatases (23, 27). In such an enzyme cascade, cyclicAMP' plays a key role, and cyclic AMP is now strongly

implicated in certain characteristics of neoplasia. Glycogenlevels in hepatomas are generally very low (23), except for afew highly or well-differentiated Morris hepatomas or somepoorly differentiated hepatomas, such as the Novkoff ascites hepatoma and a few strains of Yoshida ascites hepatomas (e.g., AH 13 and AH 66F).

These alterations of isozyme expression toward a fetalpattern in hepatomas are remarkably similar to those oftumor antigens. In cancer, antigens specific to the adultdifferentiated cells of origin disappear, and tumor-specificor tumor-associated antigens appear, which, as with theisozymes, are also present in fetal tissue (1—3,5, 7, 11, 13,14, 17, 37). As pointed out in previous publications (26, 31,32, 35, 40, 41), the appearance of fetal isozymes in cancer issimilar not only to the alterations of antigen expression butalso in some respects to the ectopic production of polypeptide hormones (10, 15, 25) observed in human tissues ofnonendocrine origin. By whatever functional methods oneuses to identify proteins, there is an apparent disorder ofnormal differentiation in cancer (18, 39) involving an impairment of the normally rigid mechanism that determines thesite and temporal specificity of gene expression.

Thus far, we have not detected any hybrid types amongthe 3 different phosphorylase b isozymes, such as wasreported for rabbit heart by Daviseta!. (9), although preliminary data suggested that hybrid types between the fetal andmuscle type phosphorylase a are present in rat heart andbrain (32). We are now examining effects of pH and glycogen content in the gel on isozyme separation by disc gelelectrophoresis. Those hepatoma strains having the abilityto accumulate glycogen, even poorly differentiated hepatomas, have predominantly the liver-like type phosphorylase,although such poorly differentiated hepatomas have predominantly nonhepatic glycogen synthetase isozymes (28,33). These data suggest that the liven-like type phosphorylase may be responsible for the ability to store glycogen;however, the complexity ofthe system does not allow definitive conclusions.

ACKNOWLEDGMENTS

We sincerely thank Dr. Sidney Weinhouse for his continued encouragement and very helpful advice on preparing this report. Charts 1, 2, and 3 arereproduced through the courtesy of the New York Academy of Science.

REFERENCES

1 . Abelev, G. I. a-Fetoprotein in Ontogenesis and Its Association with Malignant Tumors. Advan. Cancer Res., 14: 295—358,1971.

2. Alexander, P. Foetal “Antigens―in Cancer. Nature, 235: 137—140,1972,3. Anderson, N. G., and Coggin, J. H., Jr. Models of Differentiation, Ret

rogression and Cancer. In: Proceedings of the First Conference onEmbryonic and Fetal Antigens of Cancer, pp. 7—37.Oak Ridge, Tenn.:Oak Ridge National Laboratory, 1971.

4. Appleman, M. M., Krebs, E. G., and Fischer, E. H. Purification andPropertiesof InactiveLiver Phosphorylase.Biochemistry,5: 2101—2107,1966.

5, Baldwin, R. W. Immunological Aspects of Chemical Carcinogenesis.Advan. Cancer Res., 18: 1—75,1973.

6. Buedlng, E., and Orrell, 5. A., Jr. SedimentationCoefficient Distributionsof Cold Water-extracted Glycogen of Fasciola Hepatica. J. Biol. Chem.,236: 2854—2857,1961.

7, Coggin, J. H., Jr., and Anderson, N. G. Cancer, Differentiation andEmbryonic Antigens: Some Central Problems. Advan. Cancer Res., 19:105—166,1974.

8. Cries, W. E. A Review of Isozymes in Cancer. Cancer Res., 31: 1523-.1542, 1971.

9. Davis, C. J., Schliselteld, L. H., Wolf, D. P., Leavitt, C. A., and Krebs, E.G. Interrelationshipsamong Glycogen PhosphorylaseIsozymes.J. Biol.Chem., 242: 4824—4833,1967.

10. Eliel, L. P. Non-Endocrine Secreting Neoplasms: Clinical Manifestations.Cancer Bull,, 20: 37—39,1968.

11. Fishman, W. H@,Inglis, N. R., and Green, S. Regan lsoenzyme: ACarcinoplacental Antigentm.Cancer Res., 31: 1054—1057,1971.

12. Fiske, C. H., and SubbaRow, Y. Phosphocreatine. J. Biol. Chem., 81:629—679,1929.

13. Gold, P. Embryonic Origin of Human Tumor-Specific Antigens. Progr.Exptl. Tumor Res., 14: 43—58,1971.

14. Gold, P., and Freedman, S. 0. Specific Carcinoembryonic Antigens ofthe Human Digestive Tract. J. Exptl. Med., 122: 467—481,1965.

15. Goodall, C. M. A Review: On Para-Endocrine Cancer Syndromes. Intern.J, Cancer, 4: 1—10,1969.

16. Knox, W. E. Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues.Basal: S. Karger AG, 1972.

17. Lengerova, L. Expression of Normal Histocompatibility Antigens in Tumor Cells. Advan. Cancer Res., 16: 235—271, 1972.

18. Matsushlma, T., Kawabe, 5., Shibuya, M., and Sugimura, T. AldolaseIsozymes in Rat Tumor Cells. Biochem. Biophys. Res. Commun. , 30:565—570,1968.

19. Morris, H. P. Some Growth Morphological and Biochemical Characteristics of Hepatoma 5123 and Other Transplantable Hepatomas. Progr.Exptl. Tumor Res., 3: 370—411, 1963.

20. Morris, H. P. Studies on the Development, Biochemistry and Biology ofExperimental Hepatomas. Advan. Cancer Res., 9: 227—302,1965.

21. Morris, H. P. Biological and Biochemical Characteristics of Transplantable Hepatomas. In: E. Grundmann (ed), Handbuch der allgemeinenPathologic, Vol. 6, pp. 277—334.Berlin, Heidelberg, New York: SpringerVerlag, 1975.

22. Morris, H. P., and Wagner, B. P. Induction and Transplantation of RatHepatomas with Different Growth Rate (Including Minimal DeviationHepatomas). In: H. Busch (ed), Methods in Cancer Research, Vol. 4, pp.125—152.New York: Academic Press, Inc., 1968.

23. Nigam, v. N., and Cantero, A. Polysaccharidesin Cancer. Advan. CancerRes., 16: 1—96,1972.

24, Odashima, S. Establishment of Ascites Hepatomas in the Rat, 1951—1962. NatI. Cancer Inst. Monograph, 16: 51—94,1964.

25. Omenn, G. S. Pathobiology of Ectopic Hormone Production by Neoplasms in Man. In: H. L. loachim (ed), Pathobiology Annual, vol. 3, pp.177—216.New York: Appleton-Century-Crofts, 1973.

26. Ono, T., and Weinhouse S. (eds.), Isozymes and Enzyme Regulation in

Cancer. Gann Monograph 13. Tokyo: University of Tokyo Press, 1972.27. Ryman, B. E., and Whelan, W. J. New Aspects of Glycogen Metabolism.

Advan. Enzymol., 34: 285—443,1971.28. Sato, K., Abe, N., and Tsuiki, S. Glycogen Synthetase of Rat Skeletal

Muscle and Hepatomas and Its Comparison with the Enzyme of the RatLiver. Biochim. Biophys. Acta, 268: 646—653,1972.

29. Sato, K., Morris, H. P., and Weinhouse, S. Phosphorylase:A New Isozyme in Rat Hepatic Tumors and Fetal Liver. Science, 178: 879—881,1972.

30. Sato, K., Morris, H. P., and Weinhouse, S. Characterization of GlycogenSynthetases and Phosphorylases in Transplantable Rat Hepatomas. Cancer Res., 33: 724—733,1973.

31. Sato, K. , Sato, T. , Morris, H. P., and Weinhouse, S. Isozyme Patterns ofGlycogen Phosphorylase in Rat Hepatomas. In: C. L. Markert (ed),Isozymes. Proceedings of the Third International Conference on Isozymes,YaleUniversity,NewHaven,April 18—20,1974.NewYork: Academic Press, Inc., 1975.

32. Sato, K., Sato, T., Morris, H. P., and Weinhouse, S. Carcino-FetalAlterations in Glycogen Phosphorylase Isozymes in Rat Hepatomas. Ann. N. Y.Acad. Sci., 259: 273—286,1975.

33. Sato, K., and Tsuiki, 5. Studies on the Mechanism of Glycogen Storagein AscitesHepatomas.CancerRes.,32: 1451—1455,1972.

34. Sato, K., and Weinhouse, S. Purification and Characterization of theNovikoff Hepatoma Glycogen Phosphorylase and Its Relations to a FetalForm. Arch. Biochem. Biophys., 159: 151—159,1973.

35. Schapira, F. Isozymes in Cancer. Advan. Cancer Res., 18: 76—153,1973.36. Sevilla, C. L., and Fishcer, E. H. The Purification and Properties of Rat

Muscle Glycogen Phosphorylase. Biochemistry,, 8: 2161—2171, 1969.37. Stonehill, E. H., and Bendich, A. Retrogenetic Expression. The Reap3 The abbreviation used is: cyclic AMP, cyclic adenosine 3':5'-phosphate.

494 CANCER RESEARCH VOL. 36

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

Phosphory!ase lsozymes in Hepatomas

pearance of Embryonal Antigens in Cancer Cells. Nature, 228: 370—371,1970.

38. Takeo, K., and Nakamura, S. Dissociation Constants of Glucan Phosphorylases of Rabbit Tissues Studied by Polyacrylamide Gel Disc Electrophoresis. Arch. Biochem. Biophys., 153: 1—7,1972.

39. Uriel, J. Transitory Liver Antigens and Primary Hepatoma in Man and Rat.

Pathol. Biol., 17: 877—884,1969.40. Weinhouse, S. Glycolysis, Respiration, and Anomalous Gene Expression

in ExperimentalHepatomas:G. H.A. ClowesMemorialLecture.CancerRes., 32: 2007—2016,1972.

41. Weinhouse, 5. Metabolism and Isozyme Alterations in Experimental Hepatomas. Federation Proc., 32: 2162—2167,1973.

495FEBRUARY 1976

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from

1976;36:487-495. Cancer Res   Kiyomi Sato, Kimihiko Satoh, Tsuyoshi Sato, et al.   and Transplantable HepatomasIsozyme Patterns of Glycogen Phosphorylase in Rat Tissues

  Updated version

  http://cancerres.aacrjournals.org/content/36/2_Part_1/487

Access the most recent version of this article at:

   

   

   

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

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/36/2_Part_1/487To request permission to re-use all or part of this article, use this link

Research. on February 2, 2020. © 1976 American Association for Cancercancerres.aacrjournals.org Downloaded from