glucose-6-phosphate utilization in hepatoma, regenerating ...g-6-p may be hydrolyzed by the enzyme...

Glucose-6-phosphate Utilization in Hepatoma, Regenerating

and Newborn Rat Liver, and in the Liver of Fedand Fasted Normal Rats*

GEORGEWEBERJANDANTONIOCANTERO^

(Montreal Cancer Institute, Research Laboratories, Notre Dame Hospital, Montreal, Canada)

Theoretical background.â€”The object of this

study is to find biochemical differences betweennormal liver and hepatoma to form a basis for arationally planned chemotherapeutic approach.The aim is to find chemical substances which willdestroy only neoplastic cells without significantlydamaging normal cells. The problem of cancer research from this point of view is that of selectivetoxicity.

The choice of tumor and control tissues.â€”It was

difficult to determine which tumor or spectrum oftumors to choose for the biochemical and pharmacological studies. It was decided to select for thebeginning of this investigation a single tumor forwhich suitable control tissues could be found. Thetumor used in the present phase of the studies isthe Novikoff hepatoma. For control tissue theliver of normal, fed rats was taken. As additionalcontrols, livers of fast-growing embryonic andnewborn animals were added. For rapidly growingadult tissue the livers of partially hepatectomizedanimals were used. Since rapidly growing transplanted tumors frequently cause anorexia andcachexia, as an additional control condition (33)the effect of fasting on G-6-P utilization in normalrat liver was also examined. By this arrangementa spectrum of normal resting, fast-growing embryonic, and rapidly growing adult tissues wasavailable to serve as multiple control for the fast-growing neoplastic tissue.

Carbohydrate metabolism and cancer.â€”The im

portance of carbohydrate metabolism in variousneoplastic tissues has been demonstrated. How-

*The following abbreviations are employed: G-6-P =glucose-6-phosphate; G-6-Pase = glucose-6-phosphatase; 6-PG = 6-phosphogluconic acid; DAB = 4-dimethylaminoazo-benzene; S'-Me-DAB = S'-methyl-4-dimethylammoazoben-zene; ADP = adenosine diphosphate.

t Senior Fellow of the Cancer Research Society.

ÃŒSupported by a Grant of the Cancer Research Society,Montreal. Grantee of the National Cancer Institute ofCanada.

Received for publication June 10, 1957.

ever, the main effect emphasized in early studieswas the increased glycolytic pathway, which doesnot give a complete picture of the carbohydratemetabolism changes in the neoplastic tissues.

The central role of G-6-P in carbohydrate metabolism.â€”Glucose circulates in the blood and can

not be metabolically utilized in this form. Theonly reaction glucose is known to undergo in theorganism is its phosphorylation resulting in theformation of G-6-P, which can then be utilized bythe cells of the organism. It is clear that G-6-Poccupies a key position in the carbohydrate metabolism. In the liver cells G-6-P may be channeled into four different metabolic pathways bythe activity of four enzymes: (a) G-6-Pase.â€”G-6-P may be hydrolyzed by the enzyme G-6-Pase,and thus glucose is made available for the othercells of the organism. (6) Phosphoglucomutase.â€”G-6-P may be routed into storage as glycogen,and phosphoglucomutase is the enzyme whichchannels it into this pathway, (c) Phosphohexose-isomerase.â€”G-6-P may be converted by this enzyme into F-6-P, and this way it may enter intothe glycolytic pathway, yielding energy for thespecialized functions of the cells, (d) G-6-P dehy-drogenase.â€”G-6-P may be directly oxidized bythis enzyme into 6-phosphogluconic acid. The roleof the hexosemonophosphate shunt in neoplastictissue is of particular interestâ€”as a metabolic

cycle alternative to the citric acid cycle for theaerobic metabolism of G-6-P, as a mechanismwhich may channel G-6-P into glycolysis or gly-

cogenesis, as a pathway for producing the pentosephosphate required in the formation of nucleicacids and coenzymes, and as a pathway to produce TPNH.

Systematic and comparative investigation of G-6-Putilization.â€”In the last 4 years in our laboratories

a systematic examination was conducted to elucidate the behavior of all four enzymes which utilizeG-6-P as a substrate. The present paper sum

marizes the results obtained with these enzymes

995

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996 Cancer Research

in normal, neoplastic, embryonic, newborn, andregenerating liver. The effect of fasting on nitrogen content, cellularity, G-6-Pase, phosphogluco-mutase, G-6-P dehydrogenase, and phosphohex-oseisomerase activities was also studied.

MATERIALS AND METHODSAnimals.â€”Male, adult Wistar rats weighing 180-220 gm.

were used in these experiments. Purina Fox Chow and waterwere available ad libitum. For regenerating liver studies theanimals were partially hepatectomized (66 per cent) underlight ether anesthesia, according to the method of Higgins andAnderson (16). Sham operations were performed at the sametime on rats of the same weight. At periods of 1, 8, 8, and 22days following the operation, four to eight partially hepatectomized and the same number of sham-operated rats weresacrificed. For the embryo studies, pregnant rats were sacrificed at 16, 19, and 21 days of gestation. The livers of 1-day-oldrats were also examined. The Novikoff hepatomas were transplanted intraperitoneally by the large inoculum method withthe injection of 50 million + 10 million cells. The Novikoffhepatomas were 7 or 8 days old when used. The fasted animalswere placed in individual cages with water available ad libitum.

Preparation of homogenates and supernatants.â€”The animals

were killed by a blow on the head and were decapitated andbled. The livers were quickly removed, blotted on filter paper,put in a beaker, and chilled on cracked ice for 5 minutes. Thetissues were minced with scissors, and 5 or 10 per cent homogenate was prepared, usually in 0.25 M ice-cold sucrose. Forspecial experiments homogenates were also made up in distilledwater or in isotonic KC1. The supernatant fluid was obtainedby centrifuging the tissue homogenates at 100,000 g for 30minutes at 0Â°C. A refrigerated Spinco Model L centrifuge

was used.Cellularity determination.â€”The number of nuclei/gm fresh

liver was counted by the technic of Price and Laird (20), asmodified by Allard et al. (8). The cellularity was expressed according to Ultman et al. (22) as number of nuclei per unit wetweight of tissue.

Biochemical procedures.â€”G-6-Pase and phosphohexoseisom-

erase were measured in the homogenate; phosphoglucomutaseand G-6-P dehydrogenase were assayed in the supernatantfluid. G-6-Pase was measured in /Â¿g.of phosphorus liberated,as described previously (27). Phosphohexoseisomerase activitywas measured according to Bruns and Hinsberg (6), as modified by Clock and McLean (10). One unit is the quantity ofenzyme which produces 1 /Â¿moleof F-6-P/min/gm wet weightof tissue at 87Â°C. Phosphoglucomutase activity was measured

by determining the decrease in acid-kbile phosphate after hydrolysis for 3 minutes in 5 N sulfuric acid at 100Â°C. (19). One

unit is the quantity of enzyme which catalyzes the disappearance of 1 mg. acid-labile phosphorus/10 min/gm wet weight oftissue at 87Â°C. G-6-P dehydrogenase activity was determined

by the method of Clock and McLean (9). The blank solutioncontained all reaction components except triphosphopyridinenucleotide. The reaction was started in the experimental cell byaddition of an appropriate amount of enzyme (supernatantfluid) to give a linear increase in optical density for at least5 minutes. The course of reaction was followed by observingthe increase in optical density at 340 rnju at 20-second intervalsusing the Beckman Quartz Spectrophotometer, Model DU,with glass cells No. 2097. One unit is the amount of enzymewhich reduces 0.01 ;IM of TPN/min/gm wet weight of tissueat 25Â°C. Enzymatic activities in these studies were expressed

per unit tissue (per wet weight, per nitrogen, and per averagecell), per total liver (total liver activity), and per 100 gm. body

weight. The micro-Kjeldahl procedure was employed fornitrogen determinations.

With all enzymes, preliminary kinetic studies were done tocheck pH optimum, substrate optimum, and proportionality ofenzymatic activity with amount added and with reactiontune. After these criteria were fulfilled the effect of fasting wasexamined. Studies have also been started to elucidate thehormonal control of these enzymatic activities. After sufficientknowledge had been obtained on the physiological behavior ofthese enzymes, the studies were started which are described inthe present paper.

RESULTSThe effect of fasting, regeneration, and neoplasia

on liver cellularity.â€”Chart 1 shows that fasting

increased normal liver cellularity by 34 per cent.The cellularity of the regenerating liver showedno significant changes from the correspondingsham-operated controls. The Novikoff hepatomacontained about 400 million cells/gm wet weight,which is 76 per cent higher than the number weobserved in the liver of normal fed rats. It wasdifficult to evaluate the cellularity of the embryonic and newborn liver because of the presence ofthe hemopoietic cells (28); therefore, these dataare not presented here.

Comparison of nitrogen content in supernatantfluid and homogenate of the liver of fed and fastedrats, of embryonic, regenerating, and newborn ratliver, and of Novikoff hepatoma.â€”Chart 2 shows

that the homogenate nitrogen content on a wetweight basis apparently increased in normal liverafter 24-hour fasting. There were no significantchanges in the regenerating liver, embryo liver,or liver of newborn rats ; however, it was markedlydecreased in the hepatoma.

The nitrogen content of the supernatant fluiddid not change significantly after 24-hour fastingor in regenerating liver. However, the nitrogencontent of the supernatant fluid decreased markedly in the Novikoff hepatoma and in the embryoliver and to a minor degree in the liver of newbornrats.

When the nitrogen content of supernatant fluidand homogenate was expressed per cell, the average liver cell of a 1-day fasted animal contained20 per cent less nitrogen than the average hepaticcell of a fed rat (31). An average cell of the Novikoff hepatoma contained only 41 per cent as muchnitrogen as a normal liver cell, and the supernatant fluid of the hepatoma contained only 29per cent as much nitrogen as the same fraction ofnormal liver cells (29).

Regenerating liver studies.â€”Chart 3 presents

data on the restoration rate of liver constituents inthree different experimental series. These experiments were performed during the course of thelast 2 years and during each series another en-



COMPARISON OF CELLULARITY

450 r-

(0ox 300h

a:<

luo

150

â€¢Â¡'X

N NF SI S3 R l R 3

CHART1.â€”Comparison of cellularity in: liver of fed adultrats (N); liver of 24-hour fasted adult rats (NF); 1-day sham-operated (SI); 8-day sham-operated (S3); 1-day regeneratingliver (Rl); 3-day regenerating liver (R3); Novikoff hepatoma

(H). Cellularity means the number of nuclei (in millions) pergm. of fresh tissue. The mean values represent data on six ormore animals.

COMPARISON OF NITROGEN CONTENT

E 2099E

g IOoce

SUPERNATANT

I 40I*

O20

HOMOGENATE

JNF SI S3 RI R3 NB E H

CHART2.â€”Comparison of nitrogen content in: liver of fedadult rats (N); liver of 24-hour fasted adult rats (NF); 1-daysham-operated (SI); 3-day sham-operated (S3); 1-day regenerating liver (Rl); 3-day regenerating liver (R3); liver of newborn rats (NB); liver of 19-day-old embryos (E); Novikoff

hepatoma (H). Nitrogen is expressed as mg nitrogen/gm freshtissue. The mean values represent data on six or more animals.The data on embryonic and newborn rats were obtained on sixor more pools of fifteen livers each.



998 Cancer Research

zymatic activity was followed. Chart 4 showsthat the restoration rate of liver weight, liver/body weight ratio, and total liver cellularity ranapproximately the same course in all three experiments. The restoration rate of supernatantnitrogen ran parallel with the restoration rate ofthe nitrogen content of the complete cell. Therestoration of liver constituents was the most rapid

at 1 and 3 days after partial hepatectomy, andat 3 days about 60-80 per cent of the liver constituents was restored. About 80-100 per cent ofthe liver constituents was restored at 8 days afterpartial hepatectomy, and a further slow restoration took place until 22 days.

The restoration rate of total liver enzymatic activities of G-6-Pase, G-6-P dehydrogenase, phos-

RESTORATION OF

LIVER WEIGHTI20r

biuocIMo.

ceOÂ»-tnuoc

RESTORATION OF

LIVER/BODY WEIGHT RATIO120 r

138 22

DAYS AFTER OPERATION

138 22


RESTORATION OF

TOTAL LIVER CELLULARITY120 r

z 100

oocIL!O.

KO

Uloc

80

60

20

RESTORATION OF

TOTAL LIVER NITROGEN120 r

100

138 22


40-

20

>HOMOGENATENITROGENÂ«ANO.SUPERNATANTNITROGENS

"38 22


CHART8.â€”Regenerating liver studies. The restoration rate tion of liver values is expressed in per cent, and the values ofof liver constituents and liver/body weight ratio after partial the sham-operated animals are taken as 100 per cent for eachhepatectomy in three different experimental series. The restora- interval.



WEBERANDCANTEROâ€”Studyof Enzymes Utilizing Glucose-6-Phosphatase 999

phohexoseisomerase, and phosphoglucomutase ispresented in Chart 4. The enzymatic activitiesare expressed as total liver wet weight activities,total liver specific activities, total liver cell activities, and total liver enzymatic activities per100 gm. body weight. Chart 4 shows that thetotal liver enzymatic activities of these carbohy-

RESTORATION OF TOTAL

LIVER WET WEIGHT ACTIVITIES120 r

UJU

ocluo.

KO

UJoc

drate enzymes are restored parallel with the restoration of liver constituents. The total liver enzymatic activities were almost completely restored by the 8th day after partial hepatectomy.At 22 days the total liver enzymatic activitiesreached control levels.

Results of enzyme studies as expressed per unit of


AVAILABLE ENZYMATIC ACTIVITIES

120 r

40

20

138 22


138 22


LUU

enluCL

OCOHcoblOC


LIVER CELL ACTIVITIES

120 r

100

60

60

40

20

hlOOClÃ¼o.

OCo>-W


LIVER SPECIFIC ACTIVITIES120 r

100

80

60

40

20

138 22


CHART4.â€”Regenerating liver studies. The restoration rateof total liver enzymatic activities after partial hepatectomy inthree different experimental series. The restoration of totalenzymatic activities is expressed in per cent, and the values ofthe sham-operated animals are taken as 100 per cent for each

138 22


time interval. X X = G-6-Pase; O O = G-6-P de-hydrogenase; â€¢ â€¢= phosphoglucomutase; A A =phosphohexoseisomerase. Total enzymatic activity means totalliver enzymatic activity/100 gm body weight.



lÃ¼Ã¼Ã¼ Cancer Research

tissue basis. â€”The behavior of the four enzymesinvolved in G-6-P utilization is summarized inTable 1. The enzymatic activities in this tableare expressed on a nitrogen basis. A comparisonof the enzymatic alterations in the Novikoff hepa-toma and in the liver of 1-day fasted normal ratsis shown in Chart 5. The enzymatic activities inthis chart are expressed on average cell basis.

Glucose-6-phosphatase studies. â€”The results of

TABLE 1

COMPARISONOF ENZYMESDIRECTLYINVOLVEDIN GLU-COSE-6-PHOSPHATEUTILIZATIONIN HEPATOMAAND

IN RESTINGANDRAPIDLYGROWINGLIVER(The effect of fasting is included as an addi

tional control condition.)

G-6-Pase

100144

90-100130

ENZYMESDIBECTLTINVOLVEDING-6-P UTILIZATION*

Phospho-gluco-mutase10084

1001S61*5

Phosplio-hexoseisom-erase10094100160160180G-6-Pdehydro

genase10011110010025-75500LIVER

TISSUESNormal fedNormal fasted fRegenerating tNewbornEmbryonicNeoplastic

* Enzymatic activity is expressed in per cent. The specificactivity of the liver of normal fed rats is taken as 100 per cent.The following data are the mean values and standard deviationsof the four enzymes in the liver of normal fed rats. G-6-Pase:87 Â±12 ng of P/mg nitrogen; phosphoglucomutase: 239 + 27units/mg nitrogen X102; phosphohexoseisomerase: 6.6 + 0.7units/mg nitrogen; G-6-P dehydrogenase: 5.9 + 2.3 units/mgnitrogen.

t Fasted for 1 day with water available ad libitum.ÃŽThe activities of the enzymes in the regenerating liver were

assayed at 1, 8, 8, and 22 days after operation.

G-6-Pase studies are summarized in Table 1. G-6-Pase activity was absent in the Novikoff hepa-toma, and it was almost completely absent in theprimary liver tumors produced by the feeding of4-dimethylaminoazobenzene (DAB). A similar decrease or absence of G-6-Pase activity was observed in embryonic liver (28). On the other hand,G-6-Pase activity was increased in the liver ofnewborn rats. The G-6-Pase activity per unit livertissue was not affected in the regenerating liver(28).

Recently, an investigation was carried out todetermine whether G-6-Pase activity could bedemonstrated under experimental conditions different from those described above. Since most ofthe G-6-Pase activity is concentrated in the micro-somal fraction (15, 25), the possibility arose thatin a homogenate prepared in isotonic sucrose thehepatoma microsomes were not broken and thatG-6-Pase could not be liberated or the substratecould not reach the enzyme. However, Novikoffhepatoma homogenates prepared in isotonic NaClor in distilled water also failed to show G-6-Paseactivity. Preparation of more concentrated (20 percent) or highly diluted (1 per cent) hepatomahomogenate also failed to show any G-6-Pase activity or the presence of an inhibitor. Further experiments showed that the absence of G-6-Paseactivity in neoplastic tissues is not due to thepresence of an inhibitor, since homogenates ofadenocarcinoma or hepatoma did not inhibit reaction mixtures containing normal liver. A 1-hourpreincubation of these tumor homogenates with

GLUCOSE-6-PHOSPHATE UTILIZATION*

i

FASTING

65%

108%G-6-P

80%

77%

NEOPLASIA

I 2o/0%<G-6-P -/% /u

00%300%

CHART5.â€”The rerouting of G-6-P utilization in Novikoffhepatoma. The absent G-6-Pase, markedly decreased phosphoglucomutase, maintained phosphohexoseisomerase, and highlyincreased G-6-P dehydrogenase are discussed in the text. Theeffect of 1-day fasting on the same enzymes in normal liver isshown for comparison. 1 = gIucose-6-phosphatase; 2 = phosphoglucomutase; 3 = G-6-P dehydrogenase; 4 = phosphohexoseisomerase.

* Enzymatic activity is expressed in per cent. The activityper cell (nucleus) of the liver of normal fed rats is taken as 100per cent. The following data are the mean values and standarddeviations of the four enzymes in the liver of normal fed rats.G-6-Pase: 10.9 + 1.4 jug of P/cell X 10Â«;phosphoglucomutase: 15.8 Â±1.7 units/cell X IO5; phosphohexoseisomerase:79.2 Â±10 units/cell X IO8; G-6-P dehydrogenase: 3.2 Â±1.2units/cell X 10'.

t Fasted for 1 day with water available ad libitum.




normal liver homogenate, prior to the enzymeassay, was also without any effect on the G-6-Paseactivity of the normal liver (27). Since fastinggreatly increases normal liver G-6-Pase activity(4, 26), rats bearing 6-day-old transplanted Novi-koff hepatoma were fasted for 24 hours and thensacrificed. The livers of these rats showed a 60per cent increase in G-6-Pase activity, but againno activity could be demonstrated in the intra-peritoneally transplanted hepatoma. Cortisone administration was shown to result in an increasedliver G-6-Pase activity (5, 18, 24, 25); however,injection of high doses of cortisone failed to causeany G-6-Pase activity in the Novikoff hepatoma.

G-G-P dehydrogenase studies.â€”Table 1 summarizes the results of G-6-P dehydrogenase studies.This enzyme was found to be highly increased inthe Novikoff hepatoma, and no similar increasewas shown in any of the control tissues studied(29). The embryonic liver showed decreased G-6-Pdehydrogenase, and the enzymatic level returnedto normal in the liver of newborn rat. The G-6-Pdehydrogenase activity as expressed per unit tissue was the same in the regenerating liver as inthe control livers (30).

The same difference between the G-6-P dehydrogenase activity of normal liver and Novikoffhepatoma was found when the homogenates wereprepared in 0.25 M sucrose or in isotonic KC1 orin distilled water.

Phosphoglucomutase studies.â€”The results of the

phosphoglucomutase studies are summarized inTable 1. Phosphoglucomutase activity was verymarkedly decreased in the Novikoff hepatoma,and no such change was found in the examinedcontrol tissues (30). Addition of glucose-l,6-di-phosphate did not increase the phosphoglucomutase activity of hepatoma supernatant. Addition ofADP was also without any effect. This enzyme wasincreased by 25-35 per cent in the embryonic and

newborn liver. The phosphoglucomutase activityper unit tissue was the same in the regeneratingliver as in the control liver (32).

Phosphohexoseisomerase studies.â€”The results of

phosphohexoseisomerase studies are summarizedin Table 1 and Chart 5. Phosphohexoseisomeraseactivity was markedly increased when expressedon a nitrogen basis. A similar type of increasewas found in newborn rat liver and in embryonicliver. When phosphohexoseisomerase activity wasexpressed on an average cell basis, the activitywas 80 per cent of that of the normal liver. Phosphohexoseisomerase activity of the regeneratingliver was in the same range as that of the sham-operated controls, 1, 3, 8, and 21 days after operation (22).

The effect of fasting on G-6-Pase, G-6-P dehydrogenase, phosphoglucomutase, and phosphohexoseisomerase.â€”The behavior of liver enzymes involved in G-6-P utilization after 1-day fasting issummarized in Table 1 and Chart 5. In Table 1,the enzymatic values are expressed as specific activities. Liver G-6-Pase specific activity was previously found (26) to show marked increase infasting. Recent studies showed that phosphoglucomutase, phosphohexoseisomerase, and G-6-P dehydrogenase activities did not change significantlyafter 1-day fasting when expressed on a nitrogenbasis. When the enzymatic activities were expressed on an average cell basis, it was shownthat G-6-Pase was maintained, but phosphoglucomutase decreased by 35 per cent; phosphohexoseisomerase decreased by 23 per cent, and G-6-Pdehydrogenase by 20 per cent (Chart 5). The decrease in phosphoglucomutase and phosphohexoseisomerase activities was statistically significant.A detailed study on the effect of fasting on theseenzymes will be published elsewhere (34).

DISCUSSIONChanges in the routing of G-6-P.â€”In the present

investigation an attempt was made to elucidateG-6-P utilization by studying the four enzymeswhich are immediately concerned with the utilization of this hexose monophosphate ester. Theresults of these studies show that in the Novikoffhepatoma those enzymes which channel G-6-Pinto storage (phosphoglucomutase) or release itinto the blood stream (G-6-Pase) are greatly diminished or absent. On the other hand, the enzymes which channel G-6-P into energy-liberatingpathway (phosphohexoseisomerase) and into nu-cleoprotein synthesis (G-6-P dehydrogenase) aremaintained or highly increased (see Chart 5).

The specificity of the changes in G-6-P utilizationof the hepatoma.â€”The question arises whether

these alterations from the normal liver are specific to neoplastic liver or whether they may simply reflect the type of changes which could takeplace in a rapidly growing tissue.

Comparison of hepatoma and embryonic liver.â€”The data summarized in this paper (Table 1)demonstrate some analogy between the Novikoffhepatoma and the embryonic and newborn liver.There is a similarity between the nitrogen contentof the embryonic and newborn liver and the hepatoma, all showing very low values. Among theexamined enzymes the G-6-Pase activity of theNovikoff hepatoma did have a parallel with theembryonic liver in showing no or only minimalactivity. However, the absence of G-6-Pase in theembryonic liver may be physiologically explained



1002 Cancer Research

by the fact that maternal blood supplies glucosefor the fetus. It is possible that G-6-Pase in theembryonic liver is not yet active, while this enzyme in the neoplastic liver is not active anymore. The fact that, in the liver of the newbornanimal, G-6-Pase is present and is very activeindependently of the availability of nourishmentfor the newborn,1 indicates the possibility that

this enzyme may be present in the embryonicliver in an inactive form. However, attempts toactivate the G-6-Pase in the neoplastic liver byfasting or cortisone administration were unsuccessful.

Another parallelism between the Novikoff hepa-toma and embryonic and newborn liver was shownin the behavior of phosphohexoseisomerase activity. However, it is not known whether the increased phosphohexoseisomerase activity in theembryonic and newborn liver was due to the pa-renchymal cells or to the hemopoietic cells presentin these tissues.

On the other hand, there was no parallelismbetween the hepatoma and embryonic or newbornliver as far as phosphoglucomutase and G-6-P de-hydrogenase activities were concerned.

It is important to draw attention to the difficulty of comparing the biochemistry of embryonicliver or the liver of newborn animals to neoplasticliver. An important source of error in studyingthe metabolism of the embryonic and newbornliver tissue is the presence of the different developmental forms of red and white blood cell series.Therefore, until we compare embryonic tissue withneoplastic tissue, the comparison is probably notvalid. It would be possible to make this comparisona valid one if we could compare neoplastic paren-chymal cells with the embryonic parenchymalcells. Two conclusions may be drawn from theseconsiderations : (a) The behavior of G-6-Pase parallels the behavior of various enzymes which areall lower in activity in both fetal liver and hepatoma than in normal or regenerating liver (28),and thus the G-6-Pase results seem to supportthe hypothesis that neoplasms generally may revert to a more primitive and less differentiatedmetabolism (12). The behavior of phosphohexoseisomerase also seems to support the concept ofa similar enzyme pattern in neoplastic and embryonic liver. However, the G-6-P dehydrogenaseand phosphoglucomutase data are against thisassumption. It is felt that this contradiction can-iiot be solved until a method is found to correctfor the presence of the developing blood elements.(6) Therefore, although the liver of embryonicand newborn animals is useful as a comparison,

1J Ashmore, personal communication.

it cannot be considered as a satisfactory controlfor neoplastic liver.

Comparison of hepatoma and regenerating liver.â€”

Perhaps a more satisfactory control tissue may befound in the regenerating liver. This tissue has arapid growth rate which, however, stops afterabout 2-3 weeks. Chart 3 demonstrates that the

restoration rate of liver constituents is remarkable steady and reproducible. The similarity inthe restoration rate is an especially close one atthe time of most rapid growth during the first 3days after partial hepatectomy.

The restoration of total liver G-6-Pase, phosphoglucomutase, phosphohexoseisomerase, andG-6-P dehydrogenase runs parallel to the regeneration of the liver parenchyma. G-6-Pase is amicrosomal enzyme (15, 25), and phosphoglucomutase (14), phosphohexoseisomerase, and G-6-P-dehydrogenase (9) are supernatant enzymes;therefore, it is of interest to note that the rate ofregeneration of these enzymes is in contrast tothe behavior of mitochondrial enzymes in regenerating liver. A summary of the behavior of somemitochondrial enzymes (13) shows that the increase of glutamic dehydrogenase, ATP-ase, suc-cinic oxidase, oxalacetic oxidase, DPN-cyto-chrome reductase, rhodanase, and amine oxidaselags behind that of the liver weight restorationafter partial hepatectomy.

Since the liver G-6-Pase, G-6-P dehydrogenase,phosphohexoseisomerase, and phosphoglucomutaseactivities of the hepatectomized and sham-operated animals did not differ significantly on a wetweight, nitrogen, or per cell basis, it is clear thatthe activity of these carbohydrate enzymes is notaffected in the adult fast-growing liver. Therefore,it may be concluded that, in the case of regenerating adult liver, it is possible to have rapid growthwithout a change in the activity of the four enzymes on whose behavior the metabolic routingof G-6-P depends.

Comparison of hepatoma and the liver of fastedanimals.â€”A comparison of various tissues (Chart

1) revealed that the cellularity of the Novikoffhepatoma was markedly increased (176 per cent).It is interesting to note that 24-hour fasting causeda similar, but less marked, increase in the cellularity of the normal liver (134 per cent), whichmay be owing to loss of water and glycogen fromthis tissue. The enzymatic changes in G-6-P utilization, however, were completely different infasting than in the Novikoff hepatoma. Therefore,the marked alterations in the G-6-P utilization ofthe Novikoff hepatoma could not be attributedto possible effects of anorexia and fasting. Furthermore, there were no signs of cachexia 7-8




days after transplantation of the Novikoff hepa-

toma.Comparison of enzymatic results with data ob

tained by isotope methods.â€”It is of importance tonote that the biochemical changes of the hepa-toma, reported in this paper, have been confirmedby other investigators using different methodsand approaches. The progressive decrease of liverG-6-Pase activity during DAB feeding was examined by Spain, using 3'-methyl-4-dimethylami-

noazobenzene. The control animals were pair-fedwith basal diet. A significant reduction in G-6-Paseactivity was observed on both wet and dry weightbasis in the 3'-Me-DAB-fed animals (21). This

may indicate that the decrease in liver G-6-Paseactivity is probably connected with precancerouschanges and may be independent of the carcinogenic agent used.

The absence of G-6-Pase activity in the hepa-toma was recently confirmed by Abraham, whofound no G-6-Pase present in the Hepatocarci-noma C954 of the C57L strain mouse.2 The absence of G-6-Pase activity in this tumor was alsoconfirmed by isotope methods. It was found thatin liver slices the specific activity of the administered glucose-C14 was depressed about five- to six

fold on incubation, indicating the biosynthesis ofunlabeled glucose, whereas in the case of hepatomaslices the initial and final specific activities werethe same. This experiment demonstrated thatthere was no glucose production by this tumor.2

Abraham also demonstrated that this mouse hepatoma synthesized no glycogen from glucose-1-C14, glucose-6-C14, or glucose-E-C14, while the nor

mal or host liver synthesized from 2 to 25 percent from these substrates. This finding is in agreement with the very markedly diminished phos-phoglucomutase activity in the Novikoff hepatoma.

The marked increase in hepatoma G-6-P de-hydrogenase activity is of interest in view of thevery low TPN content of the hepatomas (11).Such a discrepancy between the TPN contentand the G-6-P dehydrogenase activity of tumorswas noted by Glock and McLean (11). This phenomenon awaits further elucidation. The markedincrease in hepatoma G-6-P dehydrogenase activity, on the other hand, correlates well withreports on increased hexose monophosphate shuntpathway indicated by isotope studies. The dataof Abraham, Hill, and Chaikoff showed that theC1402 derived from glucose-1-C14 was about 3times that derived from glucose-6-C14 (2). A pre

dominance of the hexose monophosphate shunt

*S. Abraham, personal communication (to be published).

(25-58 per cent) in this tumor was also indicated

by recent observations on the lipogenetic abilityof this tumor (1). The preferential utilization ofglucose-1-C14 to C140a in the Hepatocarcinoma

C954 slices is in agreement with the increasedG-6-P dehydrogenase activity demonstrated inthe Novikoff hepatoma. Wenner and Weinhousealso observed a greater yield of glucose carbon 1than of carbon 6 in COa in various solid and ascitestumors (35). Campbell described that there was abetter utilization of glucose-E-C14 by rat hepatoma

slices for the synthesis of protein than there wasby liver tissue (7). The isotope data of Emmelotet al. also indicate an increased hexose monophos-phate shunt in T 26473-hepatoma (8). Kit observed that the intact lymphatic tumor cellsformed 2-5 times as much pentose from glucose

as did normal lymphatic cells (17). These isotopedata of Kit correlate well with the observationsof Villavicencio and Barron (23) that the activityof the enzymes of the hexose monophosphate shuntof lymphosarcoma cell extracts is several times,higher than the activity of the enzymes from extracts of normal lymphatic cells.

It should be emphasized that in the presentwork the activity levels or the maximum capacities of the enzymes were measured under optimal in vitro conditions. Therefore, we really knowonly what these enzymes could do in cell-freehomogenates or supernatants. The present dataalone do not necessarily mean that these enzymatic reactions actually take place in vivo andthat the quantitative relation of the pathways toeach other correspond to the enzymatic data obtained in these studies. However, it is reassuringto find that, when the same pathways of glucose-6-phosphate utilization were examined by isotope methods, results confirmed the enzymatic-

indications given in the present paper.Conclusions.â€”The rerouting of G-6-P metabo

lism in the neoplastic liver seems, therefore, aspecific one without parallel in the liver of normalor fasted animals or in embryonic, newborn, orregenerating liver. The neoplastic liver does notperform the characteristic carbohydrate metabolicfunctions of the normal liver to store glycogenand supply glucose for the extrahepatic cells ofthe organism. The neoplastic hepatoma cell, onthe other hand, utilizes most of its G-6-P to generate energy for the maintenance of the cell andto produce nucleoproteins for mitosis.

It is now necessary to consider the questionwhether the outlined specific enzymatic alterations in the Novikoff hepatoma are essentialchanges due to the nature of neoplasia or whetherthey may be only concomitant alterations due to



1004 Cancer Research

some nonessential and secondary change in thecancerous tissue.

It is possible that many of the biochemicalchanges described in cancer literature belong toa class which may be called "concomitant andnonessential changes." To determine the essen

tiality of a biochemical change in the neoplastictissue the following rule may be applied: Doesthe re-establishment of corresponding normal tissue values inhibit the growth of tumor?

Following this principle there are two possibilities in applying this approach to the changes observed in the G-6-P utilization in the Novikoffhepatoma: (a) Attempt to activate the enzymeswhich show a decrease in the hepatoma (G-6-Pase,phosphoglucomutase). (6) Attempt to inhibit theenzymes which show increased activities in thehepatoma (G-6-P dehydrogenase, phosphohexose-isomerase). Experiments based on this approachare in progress.

SUMMARYThe glucose-6-phosphate (G-6-P) utilization of

the Novikoff hepatoma was investigated by studying the four enzymes which are concerned withthe immediate utilization of G-6-P as substrate.For control tissues the following ones were used:normal resting liver, liver of embryonic and newborn animals, and regenerating liver. The effectof fasting was also examined to exclude the possible effects of anorexia or cachexia. Enzymaticactivities were expressed per wet weight, per mg.nitrogen, and per average cell.

1. The nitrogen content was markedly decreasedin the 'homogenate and supernatant fluid in theNovikoff hepatoma and in the embryonic liver.The cellularity was markedly increased in theNovikoff hepatoma (176 per cent) and in the liverof fasted normal rats (134 per cent), but it didnot change significantly in the regenerating liver.

2. Glucose-6-phosphatase (G-6-Pase) activitywas absent in the Novikoff hepatoma. There wasalso no or only minimal G-6-Pase activity in thefetal rat liver. On the other hand, in newborn ratliver the activity was 30-40 per cent higher thanin normal adult liver. In the regenerating liverthe G-6-Pase activity per unit was not decreased.

3. The phosphohexoseisomerase activity wasmarkedly increased in the hepatoma on a nitrogenbasis, but it was in the normal range when it wascalculated per average cell. Phosphohexoseisomerase activity (on nitrogen basis) was markedly increased in embryonic and newborn rat liver. Inthe regenerating liver this enzymatic activity didnot change.

4. The phosphoglucomutase activity of the

Novikoff hepatoma was decreased to 10 per centof normal values. This decrease may be one of thespecific biochemical changes in this tumor, because no such decrease was found in any of thecontrol tissues. The activity of this enzyme increased in embryonic and newborn liver, but itdid not change in regenerating liver.

5. Glucose-6-phosphate dehydrogenase activitywas markedly increased (500 per cent) in theNovikoff hepatoma. This may be another of thespecific changes occurring in the Novikoff hepatoma. This enzyme was decreased in embryonicliver, but it was in normal range in newborn andregenerating liver.

6. The significance and implications of the results of glucose-6-phosphate utilization studieswere discussed.

ACKNOWLEDG MENTSThe valuable technical assistance of Vilma Jansons and

Colette Ayotte is gratefully acknowledged.

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1957;17:995-1005. Cancer Res George Weber and Antonio Cantero RatsNewborn Rat Liver, and in the Liver of Fed and Fasted Normal Glucose-6-phosphate Utilization in Hepatoma, Regenerating and

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