[CANCER RESEARCH 26 Part 1, 1547-1560, July 1966]

Systematic Oscillations in Metabolic Activityin Rat Liver and in HepatomasI. Morris Hepatoma No. 77931

VAN R. POTTER, RONALD A. GEBERT, HENRY C. PITOT, CARL PERAINO,2 CARLOS LÕMAR, JR.,S. LESHER,3AND HAROLD P. MORRIS4

McArdle Laboratory, Medical Center, University of Wisconsin, Madison, Wisconsin

Summary

Environmentally induced changes in rat liver were used to determine the capability of a minimal deviation hepatoma (Morrishepatoma 7793) to respond to regulatory influences. Hepatoma-bearing rats were divided among 20 experimental groups testingall possible combinations of 5 levels of dietary protein and 4different times in the 24-hr day. Casein was fed at levels of 0, 12,30, 60, and 90 %, with glucose as the other variable. Lighting was6 A.M.to 6 P.M.,and animals were killed at 06:00, 12:00, 18:00,and 24:00. Only 2 rats were used in each group, but each groupwas reenforced by either 4 or 3 other adjacent groups in the experimental pattern. The hepatoma was 1 of the most slowlygrowing hepatomas available, and the animals were killed 149days after transplantation, when the hepatomas weighed between3 and 6 gm in most cases. The animals were adapted to the dietfor 44 days before they were killed. Incorporation of thymidineinto DNA showed marked cycling in rate with a maximum at6 A.M.and minimum at 6 P.M.Thymidine kinase also showed dailycycling in activity. A striking generalization could be made in thecase of all of the enzymes studied, which included Ferine dehy-drase, ornithine transaminase, tyrosine transaminase, and glu-cose-6-phosphate dehydrogenase. It was noted that at dietaryprotein levels that depressed enzyme activity in host liver, enzymeactivities in the hepatomas attained values 10-100 times higherthan those of livers in the same animals. Since the enhanced enzyme activities in the hepatomas were in the range of the highestvalues that could be produced in liver under conditions optimalfor liver, the phenomenon appears to be some kind of a "feedback deletion" the mechanism of which is as yet unspecified.

1This study was supported in part by Departmental Grant CA-07125 and Training Grant CRTY-5002 from the National CancerInstitute, USPHS. This publication is in honor of the 70th birthday of Dr. Jacob Furth. Cf. Cancer Res., 26: No. 3, 1966.

2 Present address: Biological Division, Argonne National Labo

ratory.3The autoradiographic measurements were performed at the

Argonne National Laboratory in the laboratory of S. L.1The hepatoma-bearing rats were produced at the National

Cancer Institute in the laboratory of H. M. and shipped toMadison.

Received for publication September 13, 1965; revised February11, 1966.

Introduction

Jacob Furth has made many contributions to the study oftumor-host relations. In his Clowes lecture (13) he pointed outthat, in addition to the frequently studied effects of tumors ontheir host, it is perhaps even more instructive to consider theeffects of the host on the tumors. Whereas the dogma was frequently assumed that tumors are autonomous, Furth emphasized that there are various degrees of autonomy and that in thedevelopment of tumors, hormones may play a determining role(Reference 13, p. 27). He developed a "stable transplantable,highly hormone-dependent mammary tumor which grew only infemales [rats] highly stimulated with MtH [mammotropic hormone]." Comparing these mammary tumors with the "minimaldeviation" hepatomas studied in our laboratories (37) he pointed

out that the mammary tumors referred to above are even closerto the normal cell of origin because they are not able to grow innormal hosts unless the mammotropic hormonal level is greatlyelevated. Summarizing, he stated, "Thus, the same mammary

tumor can be acute and kill the host rapidly, take a slow courseof several months, or can be dormant altogether, depending onthe mammotropin level of the blood" (Reference 13, p. 26).

Following some of the guidelines established by Furth, we haveendeavored to use the minimal deviation hepatomas in comparison with normal liver to study biochemical control mechanismsand have shown that numerous defects occur in the hepatomas inthe regulation of various inducible enzymes, some behaving as ifthey were noninducible and others as if they were derepressed(33, 37), i.e., a "simulated" derepression.5

Pitot suggested (29) that the defective controls of enzyme synthesis characteristic of the hepatomas studied might all be secondary to a more basic molecular lesion in control mechanismsthat need not necessarily involve DNA synthesis and cell division. Potter emphasized (37) a concept of multiple defects in

5 It is not considered appropriate to use the terms "repression"and "derepression" without qualification since we have not estab

lished whether the phenomena represent enzyme synthesis or enzyme activation. However, the qualified term "simulated derepression" has been chosen because it is much more likely that

an increase in a tumor enzyme under conditions that give a decreasein the enzyme activity in the corresponding normal tissue is analogousto derepression in microorganism than to induction or activationof enzymes (Cf. "Discussion").

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control mechanisms in minimal deviation hepatomas with theavailable hepatomas representing various possible combinationsof essential and nonessential changes and regarded defects in thecontrols for DNA synthesis and cell division as among the essential changes for the conversion of normal cells to malignant cells.

Casting about for a systematic way to study the controlledvariation in rates of DNA synthesis in rat liver in comparisonwith hepatomas, we were impressed with the demonstrations of"circadian" rhythms in the in vivolabeling of DNA and RNA in

normal and regenerating rodent liver as shown by Halberg andBaraum and their «workers (2,15). Moreover, it had been shownby Blumenfeld in 1943 (3) that the mito tic count in sections ofinduced epidermoid cancers in mice was high and constant whenobserved at 4-hr intervals for 24 hr, whereas normal mouse skinshowed a pronounced maximum at noon and a minimum at midnight (approximately 250 and 50 mitoses/500 fields, respectively).Since the minimal deviation hepatomas all grow very slowly, itseemed likely that the enzymes connected with DNA synthesismight be under partial control in every case but that the degreeof control might vary with the growth rate.

In the work to be described, the basic parameter was the rateof incorporation of Tdr6 into the DNA of Morris hepatoma 7793

during 60 min following a single injection. Similar measurementswere made on the livers of the same animals (host liver). In workto be presented subsequently, a variety of other hepatomas werealso studied, and samples of normal liver (from nontumorousrats) were included. Autoradiographic determination of the %of cells with labeled nuclei was also carried out. This parameterwas used in preference to mitotic counts to determine whether therate of DNA labeling as measured biochemically correlated withvariations in the number of cells engaged in DNA synthesis atthe time intervals studied, and this was found to be the case.

In addition to the studies based upon DNA labeling in vivo, anumber of enzyme assays were carried out. These included thymi-dine kinase, which is the 1st enzyme in the sequence required forthymidine incorporation into DNA, and a number of enzymes ofamino acid and carbohydrate metabolism. Liver glycogen wasalso determined, and this assay was very useful in confirming theperiodicity of the host metabolism.

Although the basis of the study was to test the response of theminimal deviation hepatomas to the natural daily rhythm of thenormal liver, it was decided also to study in the same experimentthe responses to variations in the protein level of the diet. Thisdecision complicated the research as well as its presentation, butit is believed that the results provide new insight into the problem of comparing tumor tissues with normal tissues.

The results show that the rate of DNA labeling in a minimaldeviation hepatoma (Morris No. 7793) varied markedly with thetime of day and in most instances followed the pattern for normalor regenerating liver [in sharp contrast to the earlier results basedon the mitotic index and a much more malignant tumor (3)].However, the level of thymidine incorporation was about 10times greater in the hepatomas than in the corresponding host

8The following abbreviations are used: Tdr, tritium-labeledthymidine; PCA, perchloric acid; dAMP, deoxyadeiiosine mono-phosphate; ATP, adenosine triphosphate; Tris, tris (hydroxy-methyl) aminomethane; DEAE, diethylaminoethyl; TPN, tri-phosphopyridine nucleotide.

livers. In the case of the other parameters, marked differences between host liver and hepatoma were also seen and could be generalized as showing a "simulated derepression"5 of hepatoma

enzymes under conditions that resulted in minimal or decreasinglevels of host liver enzymes. One of the most significant conclusions is that daily fluctuations in metabolism may need to beconsidered when comparisons are made between hepatomas andtheir tissue of origin. Such comparisons may benefit from a rangeof environmental conditions that can test the adaptive responsesof the hepatomas. The present report shows that when this approach was used the hepatoma always showed a systematic response to changes in the hormonal and/or nutritional state of thehost.

Materials and Methods

All experiments were carried out with Morris hepatoma 7793carried by transplantation into Buffalo rats at the National Cancer Institute. The average time between transplantations forthis hepatoma has been 4.9 months (41). On arrival in Madison,the animals were shifted to diets containing various amounts ofcasein made up as previously described (33) except that the dietscontaining 0, 12, 30, and 60% protein were prepared and pelletedby General Biochemicals, Inc. (Chagrin Falls, Ohio).

The lighting of the windowless animal rooms was automaticallyregulated to provide an "equinoctal" schedule of 12 hr of light

and 12 hr of darkness, corresponding to natural lighting from 6A.M.to 6 P.M.at the equinoxes. The lights were fluorescent typeand provided 110 foot candles at average cage levels.

The normal nocturnal feeding habits of the rat were reenforcedin this experiment by removing their food when the lights cameon and replacing it just before the lights were turned off. Thisprocedure prevented the rats from eating in the daytime whenunavoidable human activity in the room interrupted the animals'

normal period of sleep and may have made the results more uniform than otherwise. Animals were killed at 6 A.M., 12 noon, 6P.M.,and 12 midnight.

It should be emphasized that the animals were maintained onthe rigorous lighting schedule and the indicated diet for 44 daysbefore killing. In an earlier report we demonstrated markedchanges between 0, 4, and 7 days on a high protein diet (33), butit has not been rigorously established that longer periods ofadaptation represent a plateau for all functions measured. Nevertheless, it seems safe to assume that the period of rapid inductivechange has been passed and that the changes seen are due to adaily rhythm. For this reason, the 6:00 A.M.averages are plottedat both the beginning and the end of the time sequences. In thisway, the trend from 6 A.M.to 6 P.M.is shown by 3 points, and thetrend from 6 P.M.to 6 A.M.is shown by 3 points.

DNA LABELINGIN vivo. One hr before the animals were to bekilled each rat received 5.97 nmoles of tritiated thymidine s.c.(thymidine-methyPH, with a specific activity of 6.7 c/mmole,purchased from New England Nuclear Corporation, Boston,Mass.) in 1.0 ml of distilled water.

PREPARATION OF HOMOGENATES AND SUPERNATANT FRACTIONS.

One hr after the injection of the radioactive precursor each animal was killed by cervical dislocation. The liver and the tumorswere removed and immediately dropped into ice-cold buffersolution for rapid cooling. Connective tissue and necrotic tumor

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Systematic Metabolic Oscillations in Liver and Hepatomas. I

were carefully dissected away from the viable neoplastia tissue. Asample of liver, of approximately 600 mg (wet weight), was takenfor the determination of glycogen. The rest of the liver and thetumor were homogenized in 4 volumes of 0.2 Mpotassium phosphate buffer, pH 8.0, containing ethylene diamine tetraacetate(10~3M) and j3-mercaptoethanol (10~3M), by means of the UltraTurrax homogenize!- (Janke and Kunkel KG., Staufen, Breisgau,

Germany). The homogenates were centrifuged in a Spinco modelL2 preparative ultracentrifuge at 104,000 X s for 3 hr. The clearsupernatant» were stored at —20°Cand used for the differentenzymatic assays. The residual pellet was frozen at —20°Cand

subsequently used for the isolation of DNA.RADIOACTIVITY MEASUREMENT IN ISOLATED DNA. DNA was

isolated from the homogenate according to the method of Hechtand Potter (16). The precipitated DNA was resuspended in 0.5 NPC A, hydrolyzed by being heated at 100°Cfor 15 min, and ali-

quots of the clear supernatant were assayed in duplicate for DNAcontent by the Burton modification of the diphenylamine reaction (8) with dAMP used as a standard. For liquid scintillationcounting, 0.2-ml aliquots of samples were added to plastic vialsto which were then added 10 ml of scintillator solution (naphthalene, 80gm;2,5-diphenyloxazole, 5gm; a-naphthylphenyloxazole,50 mg; dissolved in 1 liter of a mixture of 5 parts xylene, 5 partsdioxane, and 3 parts ethanol) (20). The sample radioactivitywas measured with a 3-channel liquid scintillation spectrometer(Automatic Tri-Carb liquid scintillation spectrometer, PackardInstrument Co., La Grange, Illinois). The data were correctedfor quenching by the channels ratio method.

ASSAYFORTHYMIDINEJUNASE.Thymidine kinase was assayedby a modification of the method of Ivéset al. (18). The standardassay mixture contained the following components: thymidine-2-14C,0.10 HIM(10 mc/mmole); ATP, 9.0 HIM;MgCl2, 9.0 ÕÕIM;

potassium 3-phosphoglyeerate, 7.5 HIM;Tris-HCl, pH 7.8, 0.04 M;KC1, 0.031 M; and 0.4 ml of the supernatant enzyme mixture, togive a total volume of 1.0 ml. The reaction was run at 37°Cfor 40

min; aliquots were withdrawn every 10 min, placed into centrifuge tubes, and heated to 100°Cfor 2 min. The denatured protein

was removed by centrifugation, and a 25-¿ilportion of supernatant was applied to strips of DEAE-cellulose paper (WhatmanNo. DE20). The thymidine and thymine were eluted from thepaper with distilled water for 4 hr, the strips dried overnight, andthe origin containing the anionic phosphates was cut out, immersed in a scintillation solution, and counted in a Packard Tri-Carb liquid scintillation counter (12, 40). From these data andfrom the total counts obtained from a noneluted strip it is possible to obtain an initial reaction rate.

DETERMINATIONOFLIVERGLYCOGEN.Glycogen was determinedby use of the an throne reagent according to the method of Roeand co-workers (9, 39).

ORNITHINE TRANSAMINASE AND AUTOMATED ENZYME ASSAYS.

Ornithine transaminase was assayed by the method of Perainoand Pitot (26). Tyrosine transaminase, serine dehydrase, andglucose-6-phosphate dehydrogenase were assayed automaticallywith a combination unit previously described (34). The methodsof assay were as follows: tyrosine transaminase by the methods ofLin and Knox (21) as modified by Pitot, Priess, and Poirier (to bepublished); serine dehydrase by the method of Holzer (17) asmodified for automation (34). Glucose-6-phosphate dehydrogenase was déterminée!by Assay No. 1 of Bottomley et al. (5) withthe following solutions in the automated unit (34) :

Reservoir: 370 mg KC1, 105 mg magnesium acetate, 61 mgnicotinamide, 184 mg glucose-6-phosphate (potassium salt),made up to 100 ml volume with 0.10 M Tris buffer, pH 8.0; 40mg TPN added just prior to use.

TABLE 1WEIGHTDATAANDPLAN OF EXPERIMENTFORHBPATOMA7793°

TIMEOFDAY06:0012:0018:0024:00Av.±

S.E.DIETARY

PROTEIN'0%Body

wt.(gm)136140125127126100116140126±13.4Liver(gm)4.614.393.563.683.612.343.044.083.66±0.73Tumor(gm)0.611.704.332.872.813.153.362.272.63±1.1212%Bodywt.(gm)208193176204188200210168172191±15.9Liver(gm)6.876.316.265.955.456.006.114.544.565.78±0.81Tumor(gm)1.598.292.082.642.083.612.454.622.373.30±2.030%Bodywt.(gm)227202218210202221192230187209±15.5Liver(gm)6.216.536.946.757.036.155.506.694.806.28±0.73Tumor(gm)4.230.8310.306.454.204.719.826.305.885.85±2.960%Body

wt.(gm)209195211217220201194200205±9.6Liver(gm)6.876.697.067.337.205.375.406.376.53±0.77Tumor(gm)2.871.873.119.375.245.829.062.004.91±3.090%Body

wt.(gm)207213198204216180196203202±11.2Liver(gm)7.588.496.797.076.655.466.306.716.88±0.90Tumor(gm)0.903.805.272.376.832.925.351.823.65±0.2

* The hepatomas were inoculated on May 13, 1964, received in Madison on September 14, 1964,and placed on experimental diets onarrival. The animals were killed on October 28, 1964. This was Generation 9 and the hosts were females. Two of the groups that werekilled at 6:00 A.M.had an extra animal per group.

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Sample tubes contained 0.05 ml 2090 Sj, 2.0 ml 0.1 M Trisbuffer, pH 8.0, containing 10 mg TPN/100 ml buffer.

The sample time was 1 min at the X 4 speed and total cycletime was 7 min.

Results

Table 1 records the body weight, liver weight, and tumorweight for each individual rat among the various groups and alsoshows the average values as a function of the protein content ofthe diet. It is clear that the protein level of the diet has a significant effect on all 3 measurements with tumor weight slightlyhigher at 30% and 60% protein than at the other levels. Variations in these parameters due to time of day are probably smallcompared to the individual variations, and thus in Table 1 allof the data from animals on a given level of protein were averaged. In all other measurements, the averages and individualvalues are shown as a function of time of day as well as of protein.

In general this experiment represents a very mild host effectbecause the hepatomas grew very slowly and were small in size,averaging from 2.63 to 5.85 gm in the various dietary groups(Table 1). There were only 5 hepatomas out of 42 which weighedover 7 gm, and these were evenly distributed with 1 in the 12%protein group, 2 in the 30% protein group, and 2 in the 60% protein group. The largest tumor weighed only 10.3 gm, and it wascarried in the 4th largest rat in the series, suggesting that grossdisturbances in host metabolism hail not yet occurred, even after149 days of hepatoma bearing.

Measurements of DNA labeling, glycogen content, and enzyme activity show marked variations according to time of day.The data can be visualized as a graph in 3 dimensions, in whichthe average value from every pair of rats falls on a curve between 2 other pairs of measurements in the time dimension, whichis cyclic, and between 2 additional pairs of measurements in the

HOST LIVER (DARK SYMBOL)HEPATOMA 7793 (LIGHT SYMBOL)

HOST LIVER (DARK SYMBOL)HEPATOMA 7793 (LIGHT SYMBOL)

ORNITHINE TRANSAMINASECHART 1

CHARTS 1-3. The contrasting activities of ornithine transaminase, tyrosine transaminase, and serine dehydrase in Morris hepatoma 7793 and in livers of the tumor-bearing rats, as shown in3-dimensional graphs in which the vertical axis is used to show enzyme activity and the horizontal axes are dietary protein and timeof day. The dark bars represent host liver, and the light bars represent hepatoma values.

TYROSINE TRANSAMINASECHART 2

HOST LIVER (DARK SYMBOL)HEPATOMA 7793 (LIGHT SYMBOL)

SERINE DEHYDRASECHAUT 3

protein dimension for groups receiving 12, 30, or 60% protein(12 groups). In the case of animals receiving 0 or 90% protein,the data fall at the ends of the curves. Although supported by 2other pairs of measurements in the time dimension they are supported by only 1 other pair of measurements in the protein dimension (8 groups).

These relationships are illustrated in Charts 1, 2, and 3, whichare 3-dimensional representations of the findings for ornithinetransaminase, tyrosine transaminase, and serine dehydrase. Although useful in showing the general results for these 3 enzymes,Charts 1, 2, and 3 do not yield the detailed information that canbe obtained from a standard 2-dimensional graph, and it is therefor desirable to examine the data plotted in 2-dimensional chartswith each measurement 1st as a function of time of day and thenas a function of the protein content of the diet. In each case thedata for each individual rat is shown as well as the average foreach group of 2 or 3 rats. Although larger numbers of animals pergroup would have increased the significance of the averages, inspection of the curves to follow shows how each average is re-enforced by data from 6 or 8 other animals.

THYMIUINEINCORPORATIONINTODNA. In Chart 4, A-E, thedaily change in the rate of DNA labeling in the hepatoma is re-markablv consistent. Similar data were obtained with several

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Systematic Metabolic Oscillations in Liver and Hepatomas. I

THYMIDINE—DNAi PROTEIN DIET

THYMIDINE—DNAPROTEIN DIET

THYMIDINE—DNA30^ PROTEIN DIET

THYMIDINE—-DNA_60y. PROTEIN DIET

DARKPERIOD

THYMIDINE—DNA90% PROTEIN DIET

0600 1200 1800 2400 0600 1200 1800 2400 0600 1200 1800 2400 0600 1200 1800 2400 0600 1200 1800 2400 0600TIME OF DAY

THYMIDINE— DNAAT 2400 HOURS

60 900 12 30•7.PROTEIN IN DIET

CHART4, A-I. The incorporation of labeled thymidine into DNA of Morris hepatoma 7793,and into DNA of host liver during 60 minin vivo as a function of time of day and of dietary protein. In Chart 4, A-E, the individual charts are for rats receiving 0, 12, 30, 00, and90% protein in the diet, respectively, and the abscissa in each case is the time of day. In Chart 4, F-I, the individual charts are forrats killed at 00:00, 12:00, 18:00, and 24:00, respectively, and the abscissa in each case is the level of protein in the diet. The averagesat the end of the dark period in Charts A-E are repetitions of the averages at the beginning of the day period to show the trends forboth the light period and the dark period. The curves that are thus extended are shown by dashed lines. The solid circles representhepatoma data, and the open circles represent host liver. When such points coincide they are shown half black, and when they fall onan average, a point is placed in the center of the circle.

Each open or closed circle represents an individual rat, except when data from 2 or 3 rats fall on the same point. Such points areindicated by 2 or 3 small bars respectively on the appropriate symbol.

other strains of minimal deviation hepatoma. The rate is onlyslightly influenced by the protein content of the diet (Chart 4,F-I), and the 5 curves showing the daily oscillation in rate couldobviously be superimposed to obtain a S.D. calculated with 10 ormore rats at each time point. It can be seen that in every part ofthe chart the rate at 6 A.M.is about 10-fold higher than at 6 P.M.with the intermediate points falling on an approximately straightline between the maximum and the minimum values.

In comparing the hepatoma values with host liver it is clearfrom Chart 4, A-E, that the livers incoqiorated thymidine to anextent that was about 0.1 as great as the hepatoma rate. It appears from other studies that host liver in animals bearing otherminimal deviation hepatomas (e.g., Morris 5123) was similarlylow, and in fact lower than normal liver from non-tumor-bearinganimals.

The daily oscillation in DNA labeling by thymidine could conceivably reflect a variety of phenomena, and it would be mostdifficult to predict growth rate from an attempted integration of

rates that vary as much as 10-fold in 24 hr. There is no assurancethat labeling by thymidine reflects growth rate, nor is it known towhat extent competing pathways of thymidylic synthesis maydominate the over-all synthetic rate, or to what extent pyrimi-dine pools may vary during 24 hr. However, what can be done isto see what % of cells are actually labeled during the 60-min exposure to thymidine. This has been done by means of the auto-radiographic teehnie in a large series of other hepatomas to bepresented later wherein it was found that the hepatoma datasuch as are present in Chart 4, A-E, were paralleled by verylarge oscillations in % of cells labeled. Thus it may be inferredthat the oscillations must reflect the effect of factors acting in individual hepatoma cells and not a simple oscillation of circulating thymidine levels acting on all cells.

THYMIDINEKJNASE.In Chart 5, A-I, it may be seen that thethymidine kinase activity of the hepatomas was strongly influenced by both the time of day and the protein content of thediet, while the activity in host liver was not very responsive to

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900

THYMIDINE KINASE ACTIVITYJ?H PROTEIN DIET

THYMIDINE KINASE ACTIVITY127. PROTEIN DIET

THYMIDINE KINASE ACTIVITY3QV. PROTEIN DIET

•'iiai

THYMIDINE KINASE ACTIVITY60% PROTEIN DIET

THYMIDINE KINASE ACTIVITY90rt PROTEIN DIET

UJ 0600 1200 1800 2400 O600 1200 1800 2400 0600 1200 1800 2400 0600 1200 1800 2400 0600 I2OO ISÓO 2400 060O< TIME OF DAY

i THY KINASE ACTAT 0600 HOURS

THY KINASE ACTAT gOO HOURS

THY KINASE ACTAT I8OO HOURS

THY KINASE ACTAT 240O HOURS

O 12 3O 60 900 12 30 60 90012 30 60% PROTEIN IN DIET

900 12 30 60 90

CHART5, A-I. The activity of thymidine kinase in Morris hepatoma 7793and in host liver as a function of time of day and of dietaryprotein. Separate charts and symbols as in Chart 4.

GLYCOGEN IN HOST LIVER

9.0

0600 1200 1800 2400TIME OF DAY

0600

CHAHT6. Glycogen levels in host liver of animals bearing Morrishepatoma 7793as a function of time of day and of dietary protein.When protein was decreased in the diet the weight was made upwith glucose. Glycogen in hepatomas was too low to measure; henceno solid symbols are shown on the chart.

either factor. The hepatomas clearly showed a maximum at12:00 noon and a minimum at 6 P.M.which was very noticeableand consistent at all levels of protein above 0%. At the time ofpeak activity the protein effect was very marked (Chart 5G),rising from a value of about 170 to a value of about 750, whilehost livers in general showed an activity of less than 100 at alllevels of protein with closer agreement between individual samples.

The strong effect of dietary protein on hepatoma thymidinekinase (Chart 5, F-I) and the relatively weak effect on hepatomaDNA labeling in vivo (Chart 4, F-I) again raise doubt as towhether the thymidine kinase is rate limiting or is proportionalto DNA labeling. Nevertheless there is clearly a correlation inthat both parameters are lower in host liver than in hepatoma.

GLYCOGKN.The glycogen data for the host livers are shown inChart 6. The host livers contained glyeogen in inverse proportionto the amount of dietary protein and in proportion to the dietaryglucose at all time points studied (Chart 6). The hepatomas contained too little glycogen to be measured, and therefore noamount is shown. The maximum glycogen values in host liverswere all seen at 6:00 A.M.at the end of the feeding period, andranged from 3.1% on the 90% protein diet to about 8.7% on the0% protein diet. The 6:00 A.M.values tended to be maintaineduntil noon even though the food had been removed from thecages, and then dropped rapidly to a minimal value at 6 P.M.ormidnight. It was noted that the midnight value showed a sharpincrease only in the case of the 0% protein diet, and there was a

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Systematic Metabolic Oscillations in Liver and Hepatornas. I

marked lag in the glycogen response to feeding in the case of allthe other diets. These data on glycogen are for host livers in ratsbearing Morris hepatoma 7793, and do not necessarily depict theresponse of normal liver or host liver in rats bearing other tumorsor larger tumors.

The glycogen data demonstrate that the rats were systematically responding to both the protein content of the diet and thetime of day. This demonstration involves an in vivo parameterthat is not subject to the controversial interpretations that maybe placed upon measurements of enzyme activity, and hence maybe helpful in guiding the interpretation of the remaining enzymedata, which will now be presented. It should also be mentionedthat convincing data are in the literature regarding the fluctuations in rat plasma corticosterone, which apparently rises to amaximum during the late hours of daylight (14, 23), possiblytriggered by diminishing carbohydrate reserves.

GLUCOSE-6-PHOSPHATE DEHYDROGENASE. Chart 7, A-E, shoWS

the rather interesting result that the host liver exhibited essentially no daily variation on the 0% protein diet or on the 60 and90% protein diets, but showed marked variation on 12% protein (445-755, Chart IB) and on 30% protein (665-1140, Chart1C), which are in the optimum range for this enzyme except forthe 6:00 A.M. points (Chart 7F). The maximum activity wasreached at 30% protein, and the values declined smoothly fromthe maximum as the protein content of the diet was increased to60 and 90% (Chart 7, G-7). These data do not stand alone. Inthe acute experiments by Potter and Ono (38), it was shown thatthe synthesis of liver glucose-6-phosphate dehydrogenase aftera 3-day fast was optimum at 30% protein with lower values at20, 40, and 60% protein and almost zero at 0 or 90% protein (Fig.4 in Reference 38). Moreover, in the study by Ono et al. Reference 25, Charts 3a, 30, and 5), a marked synthesis of this enzymewas seen in 4-6 days after male or female rats were shifted to 30or 90% protein without previous fasting.

In the Morris hepatoma 5123 there was an increase in glucose-6-phosphate dehydrogenase when the diet was changed fromPurina Chow to a high protein diet for 1 week (25) but no increase when the animals were previously fasted for 3 days (38).In the present study, we are not dealing with a "metabolictransition" (38) that occurs during a few days when animals are

shifted from 1 diet to another. Rather we are dealing with themetabolic transition that occurs every 12 hr in animals thathave been on various diets for 44 days.

The 7793 hepatomas did not exhibit a clear pattern of dailyvariation in glucose-6-phosphate dehydrogenase (Chart 7, A-E)but in Chart 7, F-I, which shows enzyme levels as a function ofdietary protein, it is seen that the hepatoma parallels the hostliver quite closely up to 30% protein, but whereas host livervalues break above 30% protein at 12:00, 18:00, and 24:00, thehepatoma values continue to rise to a high plateau value at 90%protein. It is as if a repression function that set in for host liverwas inoperative in the case of the hepatoma. This phenomenonwill be recalled when it is seen in another circumstance to bereported below.

The assay for glucose-6-phosphate dehydrogenase was notcorrected for the further reaction of the product. Further workwith the automated system will be required to learn whether theinitial rates of TPN reduction are mainly due to glucose-6-phosphate dehydrogenase per se or whether Assay No. 2 (5) willbe preferable.

SERINEDEHYDRASE.This enzyme in the rat appears to be thesame as threonine dehydrase (24) and although both substrateswere used in earlier studies (33, 38) it was considered sufficientto employ serine alone in the present work. The previous studiesdid not include measurements at different times of day but didshow a linear increase in normal rat liver from very low levels tomuch higher levels at 4 and 7 days after shifting to a 91 % protein diet. Various studies have shown an increase in the enzvmeduring fasting, e.g. Pilot (27) and Potter and Ono (Reference 38,Fig. 15). It was also shown that hepatoma 5123 contained veryhigh levels of the enzyme and produced a marked reduction inthe enzyme activity in host livers (33). Hepatoma 7793 wasshown to have moderately high levels of threonine dehydrase ona chow diet, which gave low values in host liver, and to havesimilar high values after 1 week on 91 % protein (4).

The effect of the protein content of the diet on host liver serinedehydrase is strikingly revealed both in Chart 8, A-E, and Chart8, F-I. There appears to be a linear increase in enzyme after the30% protein level is reached, which is in contrast to data (27)with normal liver showing a linear increase above 12% proteinin the diet. This comparison suggests that the tumors depressserine dehydrase activity in host livers by a rather small fixedamount, but further experiments with paired controls will beneeded. Chart 8, A-E, shows that the time of day had very littleeffect on the serine dehydrase activity of host liver, althoughsome of the shifts at 60% and 90% protein may be significant.

'When we turn to the hepatoma data in Chart 8, A-I, the re

sults show very high levels of activity even at low levels of protein in the diet (in comparison with host liver or normal liver)while at the same time revealing marked shifts in activity atdifferent times of day. The tendency to show 2 peaks of activity,at noon and midnight respectively, seems to constitute a clearpattern, while the occurrence of a minimum value at 6:00 A.M.is unquestionable. The total impression gained from Chart 8,A-I, is one in which the hepatoma is responsive to the positive

effects of protein both in the long time adaptation basis and thedaily positive effects of carbohydrate depletion or high proteinintake, but is indifferent to the negative or repressive effects ofdietary carbohydrate, which have been clearly demonstrated innormal liver in short-time experiments bv Pitot and Peraino(31, 32).

TYROSINETRANSAMINASE.The value of the multivariable approach to the comparison of liver and hepatoma is especiallywell illustrated in Chart 9, A-7. The strong linear response ofthis enzyme to dietary protein in host liver with increments beginning between 0 and 12% protein (in contrast to serine dehydrase, see above, Chart 8, F-I) would have been missed if themidnight samplings had been omitted (Chart 87). Unpublishedstudies have repeatedly shown this marked peak at midnight innormal liver, comparable to the peaks in host liver at high levelsof protein (Chart 9, D and Tí).

The midnight peaks in host liver are paralleled by peaks inthe hepatoma, but only at the high levels of protein (Chart 9,D and E). In strong contrast are the results at 0 or 12% protein,wherein the hepatomas develop a new maximum of their own at6:00 A.M., which is the normal time for a falling level of thisenzyme in liver. The shape of the hepatoma curves in Chart 9,A and B, may be compared with their counterparts in Chart 8,A and B, which show reciprocal trends for the daily cycles ofserine dehydrase compared with tyrosine transaminase.

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Potter, hebert, Pilot, Peraino, Lámar,Lesher, and Morris

l40Or0 K PROTEIN DIETLIGHT

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1800 0600 1600

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0600 1800 0600TIME OF DAY

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60—900 12 30 60~~900 12 30 60 900 12 30 60 90

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PROTEIN IN DIETCHART7, A-I. The activity of glucose-6-phosphate dehydrogenase in Morris hepatoma 7793 and in host liver as a function of time of

day and of'dietary protein. Separate charts and symbols as in Chart 4.

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Systematic Metabolic Oscillations in Liver and Hepatomas. I

3500

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2500

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V. PROTEIN IN DIET

CHART8, A-I. The activity of serine dehydrase in Morris hepatoma 7793and in host liver as a function of time of day and of dietaryprotein. Separate charts and symbols as in Chart 4.

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Potter, Gebert, Pilot, Peraino, Lámar,Lesher, and Morris

07. PROTEIN DIET 12% PROTEIN DIPT30^ PROTEIN DIET &OK PROTEIN DIET 907. PROTEIN DIET

400-

ceco 1800 0600

AT 0600 HOURS AT IZOQHOURS

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AT 1800 HOURS

1800

AT 2400 HOURS

0600 1800 0600

0 12 30 60 900 12 30 60 900 12 30 60 900 12 30* PROTEIN IN DIET

CHART9, A-I. The activity of tyrosine transaminase in Morris hepatoma 7793 and in host liver as a function of time of day and of

dietary protein. Separate charts and symbols as in Chart 4.

The striking differences in metabolic responsiveness betweenthe hepatoma and liver would obviously have been missed if thecomparisons had been limited to a "normal" diet such as the30% protein with samplings during a "normal" working day

(Chart 9£).The inclusion of the 12% protein diet revealed a clear maxi

mum in enzyme activity in the hepatoma that was more markedat 6:00 A.M. and decreased progressively as the day advanced(Chart 9, F-H) only to be obliterated at midnight as the highprotein maximum was reached (Chart 97). The peak in tyrosinetransaminase at 12% protein and the resulting marked deviationfrom the linear increase with dietary protein in normal and hostliver was specific for tyrosine transaminase, as can be seen bycomparison with serine dehydrase and ornithine transaminase,each of which had their own specific responses to dietary protein(see below).

ORNITHINETRANSAMINASE.This enzyme occurs in rat livermitochondria (26) and probably has a slower turnover than the

preceding 2 enzymes (31, 32). Like tyrosine transaminase, it isvery sensitive to dietary protein and increases linearly from 0%protein to 90% (Chart 10, F-T). Unlike tyrosine transaminase itdoes not fall to low levels at noon and 6 P.M., but like tyrosinetransaminase the most marked change during the 24-hr cycle isat midnight (Chart 10, A-E). However, whereas tyrosine transaminase at high protein levels increased at midnight (Chart 9,D and E), the level of ornithine transaminase decreased in hostliver (Chart 10, D and E). This enzyme is completely unresponsive to steroid hormones (32), whereas tyrosine transaminase ishighly responsive to the corticosteroids (19).

In general the ornithine transaminase values in the hepatomawere fairly steady during the 24-hr cycle, and the most noteworthy phenomenon is the tendency for the hepatoma curves inChart 10, F-I, to cut across the host liver values which are linearand proportional to dietary protein, and average out to a peakat 30% protein, in contrast to the tyrosine transaminase valueswhich peaked at 12% protein.

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Systematic Metabolic Oscillations in Liver and Hepatomas. I

0% PROTEIN DIET iati PROTEIN DIET 30% PROTEIN DIET 60% PROTEIN DIET •PROTEIN DIETi F LIGHT I DARKS PERK» PERKXHOST LIVER

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CHAUT10, A-I. The activity of ornithine transaminase in Morris hepatoma 7793and in host liver as a function of time of day andof dietary protein. Separate charts and symbols as in Chart 4.

Comparisons among Charts 6-10 make it quite clear thatover-all changes in % protein in the soluble supernatant fractionper gm of wet weight could not possibly have accounted for theresults, since in many cases the enzymes changed in oppositedirections.

Discussion

The Morris hepatoma No. 7793 is one of the slowly growing-

minimal deviation hepatomas and was developed with the same

carcinogen that was used to induce the 5123 series. The growthrate expressed as average transplantation times has recentlybeen tabulated with other minimal deviation hepatomas andrapidly growing hepatomas by Shonk et al. (41), who studiedthe activity of 11 glycolytic enzymes. These workers reportedthe transplantation time for this hepatoma as 4.9 months, whichis much slower than the Morris 5123 hepatomas A, C, and D,which were all about 2.1 months. Only the Morris 7787 hepatomagrew more slowly and was reported as 9.8 months.

Despite the growth rates mentioned, the 7793 hepatomas ap-

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Potter, Gebert, Pilot, Peraino, Lámar,Lesher, and Morris

peared to be closer to normal liver than the 7787 on the basisof the 11 glycolytic enzymes (41) although it was not claimedthat either of these hepatomas differed significantly from normalliver in the instances studied.

With respect to tryptophan pyrrolase, Cho et al. (10) foundthat hepatoma 7793 normally contained more of this enzymethan any other hepatoma studied and responded to both trypto-phan and cortisone to a greater degree. Values of 4.2 units werereached in each of the latter cases starting from 1.2 units, whilehost livers started at 2.7 and reached values of 27.0 and 13.4with tryptophan and cortisone injection, respectively. Pitot(28) has shown that in the 7793 hepatoma, as in the Reuber H 35hepatoma, the response to tryptophan was dependent on thepresence of sufficient levels of corticosteroids in the host. Ad-renalectomy of the tumor-bearing host abolished the substrateresponse of this enzyme in the tumors but not in the liver. Dyeret al. (11) also studied hepatoma 7793 and reported nearly normal values for tryptophan pyrrolase and good responses to bothtryptophan and cortisone. No data on this enzyme have beenpublished for hepatoma 7787, and although in the study justcited hepatoma 7794 and 7795 appeared to resemble 7793, thesetumors were not employed in the other studies (10, 41).

Recent studies by Wu et al. (45) have shown that 3 out of 3rapidly growing hepatomas and 2 out of 3 minimal deviationhepatomas contained less than 10 units of glutamine syntheta.sewhile the remaining minimal deviation hepatoma contained morethan was found in normal liver. Thus the Morris 7800 hepatomacontained 276.8 units while control livers contained only 202.4units of the enzyme. These findings are particularly interestingin relation to the present work with hepatoma 7793 because thegrowth rates (transplantation times) of the minimal deviationhepatomas were reported as 9.8, 4.9, 3.8, 3.1, and 2.1 monthsfor hepatomas numbered 7787, 7793, 7316A, 7800, and 5123Drespectively (41). Since Wu et al. studied only the last 3 hepatomas, it becomes of great interest to study the glutamine syn-thetase of hepatomas 7787 and 7793 and to determine the effectof the protein content of the diet and the 24-hr variation, if any,particularly in view of the earlier data by Wu (44) showing theeffect of dietary protein on this enzyme in normal liver. He foundlow values on 0% protein, rising to values around 200 at 25%protein with no further increase or a slight decrease at 75% protein. Such a pattern resembles the pattern for glucose-6-phos-phate dehydrogenase (Chart 7) rather than the patterns for theserine, tyrosine, or ornithine enzymes (Charts 8-10), and thefact that hepatoma 7800 had higher than normal values raisesthe question of whether the glutamine synthetase in this tumorfollows a pattern of simulated derepression5 that seems to char

acterize hepatoma 7793 for the enzymes studied in the presentinstance.

If we examine the effect of dietary protein on the serine, tyro-sine, and ornithine enzymes (Charts 8-10) we see that in allcases the liver has a smooth linear response, modulated by the24-hr cycle, but in no case and at no time of day exhibiting areversal in the trend toward lower enzyme level at lower levelsof dietary protein. When we examine the hepatoma data we seea completely deviated but systematic pattern that includes thegeneralized result of high values at low levels of dietary proteinplus the individual features of a peak value for enzyme activityat some value other than the highest dietary protein as seen in

the host liver. Thus in the hepatoma, serine dehydrase showed apeak at 30-60% protein (Chart 8, F-I), tyrosine transaminaseshowed a peak at 12% protein (Chart 9, F-I), and ornithinetransaminase showed a peak at 30% protein (Chart 10, F-I).It was as if the host liver was repressed at low levels of proteinby the high glucose content of the diet (31, 32), while this repression failed in the hepatoma.

Equally impressive is the same phenomenon on the other endof the scale. In the case of glucose-6-phosphate dehydrogenase,some kind of a repression occurs the host liver at dietary proteinlevels above 30% (Chart 7, F-I), a phenomenon not seen in thecase of the amino acid enzymes studied. But now the hepatomarises to higher and higher values as the protein level is increased,again as if a failure in repression had occurred.

The phenomena of "simulated" derepression6 seen in Charts7-10 are quite in keeping with the data on DNA labeling and onthymidine kinase (Charts 4, 5) which can be interpreted as derepression at all levels of dietary protein, while in Chart 7 thederepression is at the high end of the protein scale and in Charts8-10 the derepression is at the low end of the protein scale. Itshould be noted that in Charts 9 and 10 the tumor values are alsodepressed at the high end of the protein scale in comparison withnormal liver. Whether these phenomena represent capabilitiesinherent in the hepatoma cells or whether they are artifactualand result from the absence of a portal circulation and a biledrainage system in the hepatomas cannot be easily proved, butin experiments with liver lobes in which the portal circulationwas ligated (5), the level of induced enzyme was lower than inthe nonligated lobes of the same animals. This finding suggeststhat the increased enzyme levels seen in Charts 5 and 7-10seems unlikely to be explained by the absence of input from theportal vein.

The generalized phenomenon of derepression or, in generalterms, feedback deletion, (36, 37) can only be studied in thecase of enzymes that are present in adequate amounts to bestudied. In the case of the minimal deviation hepatomas manyenzymes that characterize liver are present and seem to demonstrate something akin to derepression. However, some enzymessuch as tryptophan pyrrolase (30) and glutamine synthetase(45) appear to be deleted or present in extremely low amountsin some of the minimal deviation hepatomas and from all of therapidly growing hepatomas, and apparently cannot be dere-pressed in such hepatomas. Whether these facts should be interpreted along the lines of multiple mutations (36, 37) or in termsof a basic nonmutational event (29) cannot be decided at thistime, but further studies are clearly suggested by the presentdata. Moreover the question of how to attack the biochemistryof cancer is clearly raised, and it appears that the further studyof the phenomenon of "simulated" derepression in molecular

terms appropriate to the mammalian cell will be furthered by arealization that the phenomenon can only be studied in the caseof enzymes that are present and capable of responding to environmental change.

In previous discussions the term "feedback deletion" has been

used in its most general sense (36, 37) and in the present discussion the term "simulated derepression" has been used because

it appears that the activity (concentration?) of a considerablenumber of enzymes is greatly increased under conditions whichproduce a decreased activity (concentration?) in the correspond

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Systematic Metabolic Oscillations in Liver and H epatamos. I

ing normal tissues. Whether this increased activity is due to aderepression at the level of gene transcription, at the level of theenzyme-forming system, or at the level of preformed enzyme remains for further research. With conditions described for attaining simulated derepression, it should now be possible to learnmore about the molecular mechanisms of repression in mammalian liver, in the same sense that Loomis and Magasanik (22)envision the advantages that accrue from their development ofE. coli mutants that are catabolite repression negative (22), although there is no assurance that repression in the mammaloperates at the same level as in E. coli. Elsewhere the demonstration of the repression of serine dehydra.se and ornithine trans-aminase in normal liver by glucose has been described (31, 32)and evidence in favor of repression at the level of the enzyme-forming system was provided.7 The possible role of alteredtemplate, i.e., messenger RNA, stability has been discussed interms of the endoplasmic reticulum membranes (29), and sedimentation studies have demonstrated quantitative decreases inthe binding of the polysemes to the membrane in minimal deviation hepatomas as compared to normal liver (42, 43).

It seems possible that the increased glycolysis seen in advancedcancers is a manifestation of the same general phenomena reported in the present work; Burk (6) regards the increasedglycolysis as a result of a loss of hormonal regulation of gluco-kinase activity and has published many reports dealing with thisphenomenon. He has questioned the validity of the publicationsreporting that the glycolytic rate in certain minimal deviationhepatomas is not significantly different from normal liver and infact has claimed that a direct relationship exists between growthrate and anaerobic glycolysis, with the concomitant claim thatthere are no minimal deviation hepatomas that possess an anaerobic glycolysis that is not significantly greater than that ofnormal liver (7). This claim hinges on whether the glycolyticrate in certain minimal deviation hepatomas, particularly No.7787 and No. 7793, is in fact significantly different from normalliver, and in the data he presented the claim rested on the assignment of Q values below 0.5 to normal liver in comparisonwith a composite value for hepatomas 8624, 7794B, 7787, and7793 at around 0.7. The hepatoma values were all assigned agrowth rate of 10 months per generation despite the fact thatthe average rate for No. 7793 is reported to be 4.9 months, witha range of 4.1-6.2 months, while for No. 7787 it is reported as9.8 months, with a range of 5.9-12.6 months (41).

It appears that the prediction "that a tumor that is now minimal may be replaced by a tumor that represents a new minimum"

(35) has been substantiated by subsequent events. Hepatoma7793 now must be considered as the new minimal standard, andhepatoma 5123 is no longer the hepatoma that most resemblesliver. Further detailed studies are needed on hepatoma 7787.

The present report has emphasized the importance of recognizing and utilizing the existence of daily oscillations in metabolicactivity in the study of the biochemistry of cancer. We have

7Further studies with labeled amino acids and immunologietechnics have strongly indicated that true enzyme synthesis andits repression by glucose at the level of the enzyme-forming systemrather than activation or deactivation is involved in the case ofrat liver serine dehydrase (J. P. Jost and H. C. Pilot, FederationProc., in press).

avoided the use of the term "circadian rhythm" not because we

minimize its importance but because we have not attempted toprove that the observations are due to circadian rhythms. Ourstudies involved the reenforcement of the normal light and darkfeeding patterns by actually removing the food during the lightperiods, a procedure that has proved very helpful in standardizingthe metabolic control systems so that hepatomas could be compared with normal liver. H. A. Krebs (personal communication)has also found that fortuitous feeding by experimental rats during the day can lead to uncontrolled variation in metabolicstudies. Our studies emphasizing the need for better control ofenvironmental conditions leads to complete agreement with theconclusions of Andrews and Folk (1), who observed cyclic deviations as high as 80% in the Qo2of adrenal glands and commented"workers should be aware of the influence of circadian profile^when they measure metabolic quotients for isolated tissues."

As noted in the introduction, Jacob Furth remarked thattumor-host relations could be divided into effects of the tumoron the host and effects of the host on the tumor (13). The pre-entreport has dealt with the effects of the host on the tumor andhas utilized knowledge of the effect of the host and the host environment on its own liver in order to make comparisons betweenhepatoma and liver that otherwise would not have been possible.It is a pleasure to dedicate this report to Professor Furth, andwe feel that the subject matter is well within his sphere of influence and interest. Future work in this area will involve the interaction between the hormones of the host and the receptor systemsin the hepatornas, and here the systems described by Furth \\illsurely be helpful.

Ackiiowledgmeii t

The authors acknowledge the technical assistance of Mrs.Annabelle Cutler, Mrs. Melila Vedejs, Nancy Pries, and MargaretHohenstein. Charts 1-3 were made with the help of Dr. W. Wol-berg.

References

1. Andrews, R. V., and Folk, G. E., Jr. Circadian Metabolic Pat-lerns in Cultured Hamster Adrenal Glands. Comp. Biochem.Physiol., 11: 393-409, 19G4.

2. Barn urn, C. P., Jardetsky, C. D., and Halberg. F. NucleicAcid Synthesis in Regenerating Liver. Texas Kept. Biol. Med.,15: 134-47, 1957.

3. Blumenfeld, C. M. Studies of Normal and Abnormal MitoticActivity. II. The rate and the Periodicity of the Mitotic Activity of Experimental Epidermoid Cancer in Mice. Arch. Pathol,S6: 067-73, 1943.

4. Bottomley, R. II., Pilot, H. C., and Morris, H. P. MetabolicAdaptations in Hat Hepatomas. IV. Regulation of Threonineand Serine Dehydrase. Cancer Res., 2S:392-99, 1963.

5. Bottomley, R. H., Pitot, H. C., Potter, V. R., and Morris, H.P. Metabolic Adaptations in Rat Hepatomas. V. ReciprocalRelationship between Threonine Dehydrase and Glucose-6-Phosphate Dehydrogenase. Ibid., 23: 400-9, 1963.

6. Burk, D. Book Review. Cellular Control Mechanisms andCancer. Clin. Chem., //: 607, 19(15.

7. Burk, D., Woods, M., and Hunter, J. On the Cancer Metabolism of Minimal Deviation Hepatomas. Proc. Am. Assoc. Cancer Res., 6: 9, 1965.

8. Burton, K. A Study of the Conditions and Mechanism of the

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Potter, Gebert, Pilot, Peraino, Lámar,Lesher, and Morris

Diphenylamine Reaction for the Colorimetrie Estimation ofDeoxyribonucleic Acid. Biochem. J., 62: 315, 195(i.

9. Carrol, N. V., Longley, R. W., and Roe, J. H. The Determination of Glycogen in Liver and Muscle by use of Anthrone Reagent. J. Biol. Chem., 220: 583, 1055.

10. Cho, Y. S., Pitot, H. C., and Morris, H. P. Metabolic Adaptations in Rat Hepatomas. VI. Substrate-Hormone Relationships in Tryptophan Pyrrolase Induction. Cancer Res., 24-'52-59, 1964.

11. Dyer, H. M., Gullino, P. M., and Morris, II. P. TryptophanPyrrolase Activity in Transplanted "Minimal Deviation" Hepatomas. Ibid., 24: 97-104, 1964.

12. Furlong, N. B. A Rapid Assay for Nucleotide Kinases Using!4C- or 3H-labeled Nucleotides. Anal. Biochem., 5: 515, 1963.

13. Furth, J. Influence of Host Factors on the Growth of Neoplas-tic Cells, (second G. H. A. Clowes Memorial Lecture). CancerRes., 23: 21-34, 1963.

14. Glick, D., Ferguson, R. B., Greenberg, L. J., and Halberg F.Circadian Studies on Sticcinic Dehydrogenase, Panothenateand Biotin of Rodent Adrenal. Am. J. Physiol., 200: 811-14,

1901.15. Ilalberg, F., Barnum, C. P., Silber, R. H., and Bittner, J. J.

24-Hour Rhythms at Several Levels of Integration in Mice onDifferent Lighting Regimens. Proc. Soc. Exptl. Biol. Med.,97: 897-900, 1958.

16. Ilecht, L. I., and Potter, V. R. Nucleic Acid Metabolism inRegenerating Rat Liver. I. The Rate of Deoxyribonucleic AcidSynthesis in Vivo. Cancer Res., 16: 988, 1956.

17. Hölzer, H., Boll, M., and Gemiamo, C. The Biochemistry ofYeast Threonine Deaminase. Angew. Chem. (English Ed.), 8:101-7, 1964.

18. Ivés,D. H., Morse, P. A., Jr., and Potter, V. R. Feedback Inhibition of Thymidine Kinase by Thymidine Triphosphate. J.Biol. Chem., 238: 1467-74,1962.

19. Kenney, F. T., and Flora, R. M. Induction of Tyrosine a-Keto-glutnrate Transaminase in Rat Liver I. Hormonal Nature. J.Biol. Chem., 236: 2699-2702,1961.

20. Kinard, F. E. Liquid Scintillator for the Analysis of Tritiumin Water. Rev. Sci. Instr., 28: 293, 1957.

21. Lin, E. C. C., and Knox, W. E. Adaptation of the Rat LiverTyrosine a-Ketoglutarate Transaminase. Biochim. Biophys.Acta, «6:85-88,1957.

22. Loomis, W. F., Jr., and Magasanik, B. Genetic Controlof Catabolite Repression of the Lac Operon in Eschericia coli.Biochem. Biophys. Res. Commun., 20: 230-34, 1965.

23. McCarthy, J. L., Corley, R. C., and Zarrow, M. X. DiurnalRhythm in Plasma Corticosterone and Lack of DiurnalRhythm in Compound F-like Material in the Rat. Proc. Soc.Exptl. Biol. Med., 104: 787-89, 1960.

24. Nagabhushanam, A., and Greenberg, D. M. Isolation and Properties of a Homogeneous Preparation of Cystathionine Syn-thetase-L-Serine and L-Threonine Dehydrogenase. J. Biol.Chem., 240: 3002-9, 1965.

25. Ono, T., Potter, V. R., Pitot, H. C., and Morris, H. P. Metabolic Adaptations in Rat Hepatomas. III. Glucose-6-phos-

phate Dehydrogenase and Pyrimidine Reductases. CancerRes., 23: 385-91, 1963.

26. Peraino, C., and Pilot, II. C. Ornithine d Transaminase in the

Rat. I. Assay and Some General Properties. Biochim. Biophvs.Aeta, 73: 222-31, 1963.

27. Pitot, H. C. Studies on the Control of Protein Synthesis inNormal and Neoplastic Rat Liver. Ph.D. Dissertation, Tu-lane University, 1959.

28. . Substrate and Hormonal Interactions in the Regulation of Enzyme Levels in Rat Hepatomas. Advan. EnzymeRegulation, /.- 309-19, 1963.

29. - —. Altered Template Stability: The Molecular Mask ofMalignancy? Perspectives Biol. Med., 8: 50-70,1964.

30. Pitot, H. C., and Morris, H. P. Metabolic Adaptations in RatHepatomas. II. Tryptophan Pyrrolase and Tyrosine a-Keto-glutarate Transaminase. Cancer Res., 21: 1009-14, 1961.

31. Pitot, H. C., and Peraino, C., Carbohydrate Repression ofEnzyme Induction in Rat Liver. J. Biol. Chem., 238: PC1910-12, 1963.

32. . Studies on the Induction and Repression of Enzymesin Rat Liver. I. Induction of Threonine Dehydrase and Orni-thine-a-Transaminase by Oral Intubation of Casein Hydroly-sate. Ibid., 239: 1783-88, 1964.

33. Pitot, H. C., Potter, V. R., and Morris, H. P. Metabolic Adaptations in Rat Hepatomas. I. The Effect of Dietary Protein onSome Inducible Enzymes in Liver and Hepatoma 5123. CancerRes.«: 1001-8, 1961.

34. Pitot, H. C., and Pries, N. The Automated Assay of CompleteEnzyme Reaction Rates. 1. Methods and Results. Anal. Biochem., 9: 454-66, 1964.

35. Potter, V. R. Transplantable Animal Cancer, the PrimaryStandard. Guest Editorial. Cancer Res., 21: 1331-33, 1961.

36. . Biochemical Perspectives in Cancer Research (ClowesLecture). Ibid., 24: 1085-98, 1964.

37. . Biochemical Studies on Minimal Deviation Hepatomas.In: P. Emmelot and O. Mühlbock (eds.) Cellular ControlMechanisms and Cancer, pp. 190-210. Amsterdam: ElsevierPublishing Co., 1964.

38. Potter, V. R., and Ono, T. Enzyme Patterns in Rat Liver andMorris Hepatoma 5123 during Metabolic Transitions. ColdSpring Harbor Symp. Quant. Biol., XXVI: 355-62, 1961.

39. Roe, J. H., Bailey, J. M., Gray, R. R., and Robinson, J. N.Complete Removal of Glycogen from Tissues by Extractionwith Cold TCA Solution. J. Biol. Chem., 236:1244-46,1961.

40. Sherman, J. R. Rapid Assay Technique Utilizing RadioactiveSubstrate, Ion-Exchange Paper, and Liquid ScintillationCounting. Anal. Biochem., B: 548-54, 1963.

41. Shonk, C. E., Morris, H. P., and Boxer, G. E. Patterns of Gly-colytic Enzymes in Rat Liver and Hepatoma. Cancer Res., 25:671-77, 1965.

42. Webb, T. E., Blobel, G., and Potter, V. R. Polyribosomes inRat Tissues. I. A Study of in Vivo Patterns in Liver and Hepatomas. Ibid., 24: 1229-37, 1964.

43. . Polyribosomes in Rat Tissues. II. The PolyribosomeDistribution in the Minimal Deviation Hepatomas. Ibid.,m: 1219-24, 1965.

44. Wu, C. Glutamine Synthetase. IV. Its Formation in Rat Liverfollowing Partial Hepatectomy and during Repletion. Arch.Biophys., 106: 402-9, 1964.

45. Wu, C. Roberts, E. H., and Bauer, J. M. Enzymes Related toGlutamine Metabolism in Tumor-bearing Rats. Cancer Res.,25: 677-84, 1965.

1560 CANCER RESEARCH VOL. 26

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1966;26:1547-1560. Cancer Res   Van R. Potter, Ronald A. Gebert, Henry C. Pitot, et al.   Hepatomas: I. Morris Hepatoma No. 7793Systematic Oscillations in Metabolic Activity in Rat Liver and in

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