The molecular correlation concept of neoplasia

THE MOLECULAR CORRELATION CONCEPT OF NEOPLASIA GEORGE WEBER and MICHAEL A. LEA Department of Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana

THE principal experimental material for the investigation of cancer in our laboratories is a spectrum of liver tumors of different growth rates. This spectrum provides the basis for an understanding of the neoplastic cell in terms of progressive alterations in the molecular pattern correlated with the growth rate of these tumors. ° -10) As a result of studies on the enzymology and intermediary metabolism of the spectrum we now can predict the type and extent of biochemical alterations which should be present in all rapidlygrowing cancer cells of the liver. The ability to predict is not necessarily a stage of full understanding, but it is a step in that direction. The ability to predict indicates that alterations near the core of neoplastic transformation have been uncovered and a pattern has emerged. A description of the molecular pattern of neoplasia in the liver cell offers the opportunity of using such information for design of chemotherapy and requires an extension of such knowledge to other types of cancers. How was this stage of information reached and what are the biochemical results on which a molecular pattern of cancer can be constructed for the cancerous cell? On such data the "molecular correlation concept" is built.

Mechanisms of Disease In medicine a disease process is understood when the etiology is clarified and the symptoms and signs can be correlated with the progress of the disease. In endeavoring an etiological or causative description or an understanding in terms of symptom grouping or syndromatic classification, the physician analyzes in terms of progression of disease. He considers the development of the extent of symptoms and signs and correlates this with the severity of the disease. The application of such concepts at the molecular level is now necessary for an understanding of the cancerous cell.

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GEORGE WEBER AND MICHAEL A. LEA

Causes of Cancer Cancer research probed intensively and identified successfully hundreds of agents which were capable of causing cancer in animals and in man. These cancer-causing agents, the carcinogens, can be classified into three groups: (I) physical agents (e.g. X-ray), (2) chemical agents (azo dyes, hydrocarbons, metals, etc.), and (3) biological agents (hormones, viruses). The extreme range in chemical structure of the different carcinogens caused concern and scientists hoped that one common final mechanism may unite the molecular action of the various carcinogens.

Search for the Final Common Path of Carcinogenesis However, the common mechanism of carcinogenesis postulated has so far escaped detection. The main problem may be identified as the lack of a proper system for the analysis of the progressive development of cancerous processes. Attempts to utilize the development of liver tumors during feeding of azo dyes reached nearest to the fulfillment of this hope. In the study of azo dye hepatocarcinogenesis the disappearance of certain proteins was observed in the hepatomas. This finding gave rise to the suggestion that loss of certain proteins might be the basis or even the cause of the uncontrolled growth of cancer. ~11) The possibility of a coincidental side effect, not related to cancer, was not raised although such phenomena are known in clinical oncology. Later it was suggested that the missing proteins might be enzymes¢12) and this hypothesis was subsequently changed to the suggestion that it may not be the enzymes but the controlling mechanisms of enzyme forming systems which are lost. °3,14) Yet the azo dye carcinogenesis studies have been beset by technical problems, unresolved or partly solved today. Difference and cyclic alterations in cell population, toxic conditions, endocrine imbalance, undernourishment and failure to chart and properly identify the change in the "'average liver cell" defied description of neoplastic transformation in biochemical terms. ¢6) The difficulty of repeatability from laboratory to laboratory and even from experiment to experiment in the same laboratory frustrated and delayed investigation. However, such studies were eminently useful in defining and identifying the needs of a proper system which may give the information required to analyze and understand cancer at the molecular level.

The Requirement for an Adequate Biological System A biological system was needed which offered repeatability at will in any laboratory. A system was needed where the stages of neoplastic transformation were fixed, as if frozen in time, in order to examine the advance of the

THE MOLECULAR CORRELATION CONCEPT OF NEOPLASIA

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disease at each step of its progression. For elucidation of the progression of the biochemical alterations, therefore, there was need for a spectrum of cancer tissues of stable cell fines, each of them representing a further stage of the neoplastic disease in ~lls. In other words, a spectrum of cancer cells was necessary which represents predominantly one of the key properties of ncoplasia: tumors of different growth rates.

Spectrum of Hepatomas of Different Growth Rates The suitable biological system became available with the production of a spectrum of hepatomas of different growth rates by Dr. H. P. Morris of the National Cancer Institute.tl 5,16) The fiver tumors were caused by chemical carcinogenesis and the arising hepatomas were maintained through serial transplantation in inbred strains of rats. It is important that these were tumors of the same cell t ~ n c e r of the liver parenchymal cells; they were all hepatomas, but the various lines exhibited different rates of growth. The individual tumor lines represented stable lines of neoplastic cells and thus offered transplantability and repeatable experiments. The transplantation and distribution of all tumors by Dr. Morris prevented the variations of tumor lines from laboratory to laboratory.

The Molecular Correlation Concept We assumed that the different growth rates of the various lines may be taken as indicative of the progression of the cancerous process, t l - lo) It was also assumed that rate of growth in these tumors is indicative of the severity of the disease, tl-l°) The severity of the disease in cancer signifies the "malignancy" of the process at the clinical level. However, when we wish to work at the molecular level of the disease we must choose a parameter we can measure with accuracy. We chose growth rate because this can be followed and measured, whereas other facets of "malignancy", such as invasiveness, earliness or extensiveness of metastasis, etc., are difficult to quantitate.(1-~°) With this concept in mind we began to search for a correlation of molecular symptoms and signs with the progress of the cancer disease. For most diseases in subclinical cases few symptoms or signs are recognizable. In mild cases certain of the symptoms and signs are present, but some of them may be still undetectable. In more advanced conditions most of the symptoms and signs become manifest and their presence can be readily recognized or elicited by the trained observer. In the full-blown cases all the symptoms and signs are present and the lesions are completely developed. This type of evaluation in terms of syndromes and progressive emergence of the different critical

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GEORGE WEBERAND MICHAELA. LEA

components of a pathological condition can be easily discerned in cases of pneumonia or various neurological or psychiatric conditions but it applies to most other diseases and we attempted to apply it to an understanding of cancer at the molecular level. (1- lO)

Symptoms and Signs at the Molecular Level Taking the growth rates of the various tumor lines, as established by measurements of the tumor weights, estimating the time required for transplantation of viable tumors (generation time), and finally, the time required for the various tumors to kill the host, the different hepatomas were classified according to growth rate, that is to say, slow, medium or rapidly-growing tumors. The slow-growing tumors killed the host in 3-12 months, the mediumgrowth rate hepatomas required one to one and a half months and the rapidlygrowing liver tumors 0.25-0.75 months. (15,1~) The various biochemical parameters, the behavior of over-all metabolic pathways and the activities of key rate-limiting enzymes were measured and their correlation with the growth rate of the tumor lines was examined.° - lo) The systematic investigation revealed that the metabolic alterations can be classified into 3 groups according to their relation to growth rate of the hepatomas: (i) parameters which correlate positively or negatively with the growth rate, (ii) parameters which are increased or decreased in all or almost all tumors, (iii) parameters which show no correlation with growth rate. (s) As a result of systematic work carried out in these laboratories, it is now possible to summarize the data obtained, in a meaningful pattern, for the metabolism of cancerous liver cells. In an attempt to give a picture as complete as possible for the metabolic trends we also included the relevant results of other investigators. Although most other workers did not look at the hepatoma spectrum with our framework of expectation to see the cancerous process in terms of a progressive emergence of key symptoms and signs, when their data were reassembled in tables set up according to hepatoma growth rates, a picture in line with our general conclusions emerged. The results indicate that in the cancer cells there are progressive alterations in a number of metabolic pathways and enzyme activities which reveal a pattern at the molecular level. The sum of evidence now is extensive enough to be integrated into the phenotypic description of the cell in liver cancer. It is hoped that such a documentation of the progressively altered metabolic pattern with the advance of growth rate will provide a framework for both analysis and chemotherapy of the cancer cell. It appears to be high time that inquiry is to be directed, not to rearranging old facts and explanations, but rather to the discovery of new patterns of understanding and synthesis.

THE MOLECULAR CORRELATION CONCEPT OF NEOPLASIA

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PATTERN OF CARBOHYDRATE METABOLISM IN HEPATOMAS The two prevailing views in 1959 suggested that (a) cancer means high glycolysis, high glycolysis being present in all cancers, (b) many different kinds of enzyme lesions are found in hepatomas, giving no recognizable picture. In contrast, we suggested in 1959 that a pattern of intermediary metabolic alterations emerges in tumors and is clearly discernible in the N K hepatoma. (17) In the very rapidly-growing NK* hepatoma the anabolic pathway in carbohydrate metabolism was interrupted by enzyme lesions, wi;,~reas the catabolic processes were increased. In nucleic acid metabolism the catabolic pathways were interfered with by enzyme lesions, whereas the synthetic pathways were increased. It was suggested that the loss o f strategic enzyme systems in carbohydrate metabolism might have resulted in the stimulation of the catabolic pathway, whereas the absence of enzymes along the catabolic pathway of nucleic acid metabolism might stimulate the synthesis of these building blocks. (17) Such views preceded our current concepts on functional genie units governing the synthesis of key giuconeogenic and glycolytie enzymes with the same type of arrangement envisaged for other metabolic pathways. (1°) We also emphasized that " . . . since various liver tumors differ in their histological structure, cellular population, biological behavior, and growth rate, it is not unexpected that the biochemical differences will be present in varying qualitative or quantitative extent. Such a concept agrees well with common medical experience of finding many variations of the same disease from subclinical through mild or severe manifestations to the rarely encountered, full-blown case in which all symptoms and signs are present to their maximum development. "(17) We also made the prediction that "a careful comparison of the metabolism, histology, and biological behavior may bring a new understanding of the role of biochemical alterations in the pathological behavior of various liver tumors". (17) With the identification of a meaningful pattern in the N K hepatoma and the prediction that when a number of liver tumors were investigated the pattern of intermediary metabolism would show a gradation in relation to the biological behavior of the tumors, hepatoma biochemistry was brought into the framework of current medical thinking regarding the correlation of symptoms and signs with the progress of the disease. In the seven years since our concepts were published the experimental results contributed to filling in the pattern along the lines of our suggestion. The salient features o f this * The following abbreviations are used in the text, tables and figures: N K = Novikoff; G-6-Pase = glucose-6-phosphata~; G-6-P dehydrogenase = glucose-6-phosphatedehydrogenase; FDPase = fructose-l,6-diphosphatase; PEP carboxykinase = phosphoenolpyruvate carboxykinase; PFK = phosphofructokinase; AA = amino acid; dCMP = deoxy~/tidylate; DNA = deoxyribonucleic acid; dUMP = deoxym'idylate; P = protein; KNA = ribonucleic acid; TMP = deoxythymidylate,

120

GEORGE WEBER AND MICHAEL A. LEA

metabolic pattern were summarized in a review when work was far enough advanced on a slow-growing tumor, the 5123, to show clearly that along with slow growth rate there appeared an attenuation of the symptoms and signs at the molecular level for a number of key parameters. (6) The biochemical results could only be interpreted in the framework suggested by us. The analysis of a number of hepatomas in between the slow and the most rapidlygrowing ones provided the missing information to complete the pattern of metabolism in this spectrum of liver tumors of different growth rates. A further adaptation of clinical concepts to molecular aspects of disease enabled us to classify the symptoms and signs at the molecular level into the TAnI~ 1. Pattern of Carbohydrate Metabolism in Hepatomas of Different Growth Rates The results are grouped from the point of view of definite trends which fit into one of the three categories. Correlation with growth rate Increased Glycolysis:

Low or high in all hepatomas

No correlation with growth rate

Increase in lactate production

Increase in the Key Shunt Enzyme:

Bifunctional Enzyme Activities:

Increase in Key Glycolytic Enzymes:

Increase in G-6-P dehydrogenase

Hexokinase Phosphofructokinase Pyruvate kinase

Decrease in Glycogen Metabolism:

Phosphohexoseisornerase Lactate dehydrogenase Phosphoglycerate kinase 3-Phosphoglyceraldehyde dehydrogenase

Decrease in pyruvate conversion to glucose

Decrease in glycogen content Decrease in phosphoglucomutase

Decrease in Key Gluconeogenic Enzymes:

Decrease in Fructose Metabolism:

Non Rate.limiting Shunt Enzyme:

Glucose 6-phosphatas¢ Fructose 1,6-diphosphatase PEP carboxykinase Pyruvate carboxylase

Decrease in fructose uptake Decrease in fructose to CO2

6-Phosphogluconate dehydrogenase

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Decrease in Carbohydrate to Lipid:

Increase in C-1/C-6 oxidation of

Decrease in glucose to fatty acid

Decreased Gluconeogenesis:

glucose

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Glucokinase Fructokinase Decrease in Fructose Metabolism:

Decrease in fructose incorporation into glycogen through fructokinase reaction Decreased Responsiveness to Glucocorticoid:

Decrease in response of gluconeogenic enzymes Decrease in glycogenic response

Glycogen Enzyme:

Activation of phosphoglucomutase

Malic enzyme

THE MOLECULARCORRELATIONCONCEPTOF NEOPLASIA

121

three classes discussed in this paper and in previous articles where the biochemical alterations are grouped according to their relation to growth rate. (1-1°) A summary of the enzymatic and metabolic alterations in carbohydrate metabolism from this point of view is given in Tables 1 and 2. The salient features are now briefly summarized. In the review in 1961 it was pointed out that there was a decrease ofgluco= neogenesis and a rise of glycolysis as we move from slow= to rapidly-growing tumors. (6) This antagonistic behavior of the two opposing metabolic pathways was noted, confirming the identification of this phenomenon in 1959.(17) The pattern now emerges with extensive documentation (Table 2).

Gluconeogenesis: Decreases with the Increase in Hepatoma Growth Rate Evidence. We have shown in a number of publications that the key gluconeogenic enzymes decrease parallel with the increase in the growth rate of liver tumors. ° , 3,e-~o, 1%1a> This was tabulated previously in Tables and in a Chart showing the decrease of the two specific phosphatases as related |

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firmation of the results by other laboratories appears to complete this aspect of the evidence for the progressive failure of gluconeogenesis along with the growth rate.

Regulatory Response of Gluconeogenic Enzyme Activities and Glycogen Level W Glucocorticoid Stimulation: Decreases with the Growth Rate of Hepatomas The behavior of the gluconeogenic pathway in terms of response of gluconeogenic enzymes and glycogen deposition to steroid injection was also investigated. The injection of glucocorticoid hormone leads to a pronounced rise in normal liver in gluconeogenic enzyme activity and glycogen content, a2~ The steroid-induced enzyme increases and glycogen deposition can be blocked by inhibition of protein synthesis, t22, 23) When steroid was injected there was a minor response of the gluconeogenic enzymes and glycogen deposition in slow-growing tumors, but no change in the rapidly-growing onesj s) Thus the responsiveness of the enzyme forming systems for gluconeogenic enzymes and the behavior of the over-all pathway leading to glycogenesis gradually fail with the increase of tumor growth rate. tS~

Glycolysis: Increases with the Increase in Hepatoma Growth Rate Evidence. The first group of evidence disposed of the long-maintained suggestion that all cancer cells have an increased glycolysisJ4,24-26) Examination of the slow-growing hepatoma 5123 proved that lactate production from glucose in this tumor was very similar to that by slices of normal liver.t4} The rates of aerobic and anaerobic glycolysis in such slices of this slow-growing hepatoma were in the same range or lower than those of normal liver, t24-26) Keeping in mind our earlier findings that in the NK hepatoma the lactate production was four times greater than in normal tissue ~21~ we examined the intermediate hepatomas of the spectrum. These studies proved that there was a positive correlation between tumor growth rate and lactate production from glucose in the hepatomas. *~4) Weinhouse and his associates also observed that in the slowly-growing hepatomas the glycolytic rate was in normal range but it increased in the more rapidly-growing tumorsJ z4, 25} He and his group provided evidence that glucose phosphorylation was ratelimiting for glycolysis under the examined conditions. Furthermore, they found that the increase in growth rate of hepatomas is accompanied by an increase in glucose phosphorylation together with a decrease in the activity of the enzyme having a high Km for glucose (glucokinase) and an increase in * The correlation of glycolysis with growth rate was recently also recognizedby Burk and associates, yet their report in form of abstracts at a cancer meeting was presented two yearsafter publicationof our papers havingestablishedthis relationship{3,4~and subsequent to a number of our publications in which this concept was outlined.{S-10}

124

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the enzyme having a low Km for glucose (hexokinase).(29,3o) In those hepatomas in which the high Km glucokinase is absent the glucose phosphoryfating system will be saturated at normal blood glucose concentrations and changes in blood glucose levelwillnot affectthe glycolyticrate as may be the case for normal liver. 340 320 300 280 260 240 ~A 220 200 180 160 140 120 100 0

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The progressive decrease of gluconeogenesis alone may permit the preponderance of glycolysis because of the altered ratios of key rate-limiting enzymes governing the over-all balance of gluconeogenesis and glycolysis. However, since glycolysis increased with the growth rate and gluconeogenic enzymes were progressively decreased, it was assumed that some or all of the key glycolytic enzymes might increase parallel with the growth rate. Evidence supporting this prediction of ours is summarized in Table 2 and in Figs. 2 and 3. Table 2 gives our enzyme results showing that the activities of hexokinase, phosphofructokinase and pyruvate kinase dO indeed

THE MOLECULARCORRELATIONCONCEPTOF NEOPLASIA

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increase parallel with the growth rate of hepatomas. Figure 2 also demonstrates that there is a close correlation between the phosphofructokinase activity and the growth rate of the hepatomas/31) When the phosphofructokinase results given in Fig. 2 were plotted along with earlier data on lactate production(4) the dose correlation between the activity of this key glycolytic enzyme and glycolysis, both increasing together, parallel with 340 ;

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Fxo. 3. The close correlation between phosphofructokinaseactivity (assayed in supernatant fluid)(31)and lactate production (in slices).(4) Both these parameters characterizingglycolysisincrease parallel with the increase in hepatoma growth rates. hepatoma growth rate, is seen (Fig. 3). The results on the behavior of hexokinase confirm the report of Weinhouse and his associates, c29,3°) The data presented regarding the behavior of pyruvate kinase and phosphofructokinase are in line with and further extend the findings of Shonk et al. (~°~ The evidence summarized shows the correlation of the over-all metabolic pathway, lactate production, and the behavior of key glycolytic enzymes, increasing parallel with the increasing growth rate of the hepatomas. The Direct Oxidative Pathway: Increases Evidence. We reported that G-6-P dehydrogenase was increased in all hepatomas/a) except one, and these findings were confirmed.(2°, 3z) We also showed that there was a positive correlation of growth rate with the relative oxidation to COz of the C-1 and C-6 atoms of glucose. (2,4,21) These results are interpreted with caution since such ratios do not necessarily give an

126

GEORGE WEBER AND MICHAEL A. LEA

exact measure of the pentose phosphate pathway activity, but it may permit an estimate for an approximation only.~4~ The direct oxidative pathway may provide the pentose required for nucleic acid synthesis. The correlation of the C-l/C-6 oxidation of glucose with lactate production and tumor growth rate is shown in Fig. 4. 350

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Both lactate production and the oxidation of C-1/C-6 of glucose increase with the increase in hepatomagrowthrate.(4~

Glycogen Deposition: Decreases in All Hepatomas Evidence. Very low glycogen levels were found in the hepatomas examined.(3,21) We also discovered low phosphoglucomutase activities in the hepatomas which is in line with the low glycogen levels.°,3) These findings were confirmed.(2°) The progressive increase of the K= of phosphoglucomutase with increasing growth rate would also tend to diminish the action of this enzyme.°) The activation of phosphoglucomutase remained normal in all hepatomas. ¢9) It has already been mentioned that the steroid response of glycogenesis decreased with the growth rate; it was also shown that refeeding after a 3-day fast which caused rapid accumulation of glycogen in normal liver resulted in no response in the 5123 hepatoma. ¢33) The Bifunctional Enzymes: No Correlation with Growth Rate Evidence. Phosphohexoseisomerase, aldolase, 3-phosphoglyceraldehyde dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase showed no correlation with the proliferative rate of tumors. (1.3,17,~8,2°) These findings which have been repeatedly confirmed underline our suggestion that

THE MOLECULAR CORRELATION CONCEPT OF NF~PLASIA

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one should expect correlation with the behavior of over-all metabolic pathways chiefly when one studies the behavior of key, rate-limiting enzymes. In summary, carbohydrate metabolism is characterized by (1) a decrease in gluconeogenesis, (2) an increase in glycolysis and (3) an increase in the pentose phosphate pathway in correlation with the growth rate. (4) The glycogen metabolism is decreased and (5) the bifunctional enzymes show no correlation. (6) There was a decrease in the responsiveness of the gluconeogenic enzymes and in glycogen deposition along with the increasing growth rate. No steroid response at all occurred in the intermediate and rapidly-growing hepatomas. The same approach will be applied to the investigation of the other metabolic pathways.

PATTERN OF LIPID METABOLISM IN HEPATOMAS It is unfortunate that there is available comparatively less information for an understanding of the lipid metabolism in hepatomas than for other parameters. Therefore, in this case only a preliminary and fragmentary picture "|

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can be constructed on the basis of current information. The results appear to indicate that there is a decrease in biosynthesis and possibly a decrease in breakdown of lipids in hepatomas which can be correlated with the increase in growth rate. A brief summary of the evidence is given below and in Fig. 5.

128

GEORGE WEBER AND MICHAEL A. LEA

Decrease in Biosynthesis Correlating with the Growth Rate of Hepatomas Evidence. There was a low lipid content in a number of hepatomas in comparison with levels found in host livers. ~34) In line with the low lipid content of hepatomas a greatly diminished incorporation of 14C from labeled glucose into fatty acids by slices of hepatoma 5123-D ¢2~ and also of N K hepatoma¢21) was observed when compared with normal livers. In fact, the incorporation into fatty acids was about I0 per cent or less in these tumors. The activity of alpha-glycerophosphate dehydrogenase decreased parallel with the increase in the hepatoma growth rate. ¢2°) These results are consistent with a decreased lipid synthesis in hepatomas. Further evidence of the relatively diminished importance of lipid metabolic activity in hepatomas was provided by the work of Weinhouse and his group. ¢35) These investigators found that the oxidation of both long and short chain fatty acids to CO 2 and the formation of acetoacetate from these fatty acids was progressively decreased in hepatomas of increasing growth rate. The distribution of butyrate carbon 3 between acetoacetate carbons 1 and 3 was similar in normal liver and in hepatomas 5123-C, 7793, 7787, H-35, 5123-t.c., and 7288-C, suggesting that similar biochemical mechanisms were operative, although at a decreased rate, in the examined hepatomas. ¢36) Regulatory Response of Lipid Metabolism: Decreased Response in Hepatomas Evidence. Whereas ethionine injections cause a two- to threefold increase in the ether extractable lipid of host livers, hepatomas H-35 and 7316-A responded by a twofold increase and there was no response at all observed in hepatomas 5123-D, 7793, and in an ethionine-induced hepatoma. ~34) PATTERN OF PROTEIN AND AMINO ACID METABOLISM IN HEPATOMAS In the hepatomas there is an increase in the over-all synthetic pathway of proteins and a decrease in the catabolic pathway. As a result of this imbalance in the neoplastic cells protein biosynthesis predominates. The chief evidence for such a pattern is now documented. Further evidence and supporting facts may be examined in the original papers referred to in the discussion which follows. A summary of the enzymatic and metabolic alterations in protein and amino acid metabolism is given in Table 3 and in Fig. 8.

Biosynthetic Pathway: Increases with the Increase in Hepatoma Growth Rate Evidence. Systematic studies were carried out to examine protein synthesis in slices of normal liver and hepatomas by measuring the incorporation of

THE MOLECULAR CORRELATION CONCEPT OF NEOPLASIA

129

TAeLE 3. Pattern of Protein and Amino Acid Metabolism in Hepatomas of Different Growth Rates The results are grouped from the point of view of definite trends which fit into one of the 3 categories. Correlation with growth rate Increased Protein Synthesis:

Amino acid incorporation into protein (alanine, aspartate, glycine, serine, isoleucine, valine) Activity of the post-microsomal protein synthesizing system Ratio of total free amino acid to total protein content

Low or high in all hepatomas Increased in Most Tumors:

Free amino acid level

Decreased Amino Acid Catabolizing Enzymes:

Decreased in Most Tumors:

Tryptophan pyrrolase Serotonin deaminase 5-Hydroxytryptophan decarboxylase Threonine dehydrase Serine dehydrase Glutamate dehydrogenase Glutamate-oxaloacetate transaminase Ornithine transcarbamylase

Protein level Amino acid response to glucocorticoid administration

No correlation with growth rate Amino acid oxidation (alanine, aspartate, glycine, serine, isoleucine, valine)

t4C-labeled amino acids. (s) It was found that the incorporation o f all amino acids tested (alanine, aspartate, glycine, serine, isoleucine, valine) exhibited an increase which correlated with the growth rate of the tumors (Fig. 6). (5) A correlation was also observed between the amino acid incorporating capacity o f the post-microsomal fraction and hepatoma growth rate. °7) Thus the slow-growing tumor, 7787, had normal incorporating capacity, whereas the medium-growth rate tumor, 7288-C, had 185 per cent of control activities and the rapidly growing tumors, 3683 and 3924-A, exhibited incorporating activities of 200 per cent of normal control liver values. The ratio of free amino acid level to total protein content increased with the increase in t u m o r growth rate. tt°) When the ratio of amino acid to protein o f the normal control livers was taken as 100 per cent, the ratio was 170 and 190 per cent in the slow-growing 5123-D and in 7800; it was 260 per cent in 7288-C of intermediate growth rate; it was 280 and 240 per cent in the rapidlygrowing hepatomas, 3924-A and 3683. These results indicate that there is a preferential maintenance and a progressive increase in the free amino acid pool in relation to the decline in total nitrogen content.

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Catabolic Pathway: Decreases with the Increase in Hepatoma Growth Rate Evidence. A major function in normal liver is the breaking down of amino acids which are in excess of the requirements for protein biosynthesis. It appears that the enzymes involved in this function in general show a tendency to decrease in the neoplastic liver especially in those tumors with intermediate and rapid growth rate. The enzymes which bring about the initial steps of amino acid breakdown do not exhibit a uniform pattern in the slow-growing hepatoma3. Thus, serine dehydrase was decreased, but threonine dehydrase was elevated.(39) However, in the hepatomas of medium and rapid growth rate, these enzyme activities were considerably decreased. (3a- 40) The activity of tryptophan pyrrolase, another enzyme of amino acid catabolism, was lower than normal in slow-growing hepatomas and very low in the rapidly-growing tumors. (41,42) In addition to the catabolic pathway initiated by tryptophan pyrrolase, tryptophan may also be catabolized via 5-hydroxytryptophan and serotonin. Examination of the behavior of 5hydroxytryptophan decarboxylase and serotonin deaminase revealed that this pathway was in a similar order of magnitude in normal liver and in the slowlygrowing hepatoma 5123. However, the pathway was considerably depressed in rapidly-growing tumors. (43~

THE MOLECULARCORRELATIONCONCEPTOF NEOPLASIA

131

Similarly, glutamate-oxaloacetate transaminase~44,45) and glutamate dehydrogenase~19} had normal or high activities in the slow-growing hepatomas but the activities were much lower in the rapidly-growing tumors. The decline in these enzyme activities parallel with the growth rate of hepatomas is important because it is believed that the combined reactions catalyzed by these two enzymes are chiefly responsible for the deamination of amino acids in the liver. The tendency to decrease in amino acid batabolism, which was most prevalent in the rapidly-growing hepatomas, may be involved in the conservation of amino acids in face of a decline in total protein content. The resulting imbalance between an increase in protein biosynthesis and a decrease in amino acid catabolism favors the predominance of the biosynthetic pathway. Oxidation of Amino Acids: No Correlation with Growth Rate Evidence. Systematic investigations were carried out to examine the oxidation of amino acids to carbon dioxide, and it was found that this proeess showed no correlation with the growth rate of hepatomas, cs} This lack of relationship of amino acid oxidation to growth rate suggests that the protein synthesis from amino acids is more important than the level of catabolic enzymes and the oxidation of amino acids in determining the basic mechanisms involved in the growth rate of hepatomas. Regulatory Response of Amino Acid Level and of Enzymes of Amino Acid Catabolism: Decreased or Absent in All or Most Hepatomas Evidence. Since a marked rise in the amino acid level in normal liver is an early and well-defined result of glucocorticoid stimulation,~46,47) the behavior of this biochemical parameter was studied in liver tumors, It was shown that the free amino acid level failed to increase in all examined hepatomas after injections of triamcinolone in a high dose, which resulted in a twofold rise in normal livers ~1o} (Fig. 7). The failure of response to this action of glucocortieoid hormone is an interesting sign of loss of biological response. There appears to be a drastic change in the hormonal control of metabolic features in hepatomas and it may be that one of the important aspects of such a loss of control is this fixity in the amino acid level in all hepatomas. When high protein diet is fed to normal rats there follows an increase in threonine dehydrase and serine dehydrase activities. However," under the same conditions in the hepatomas there was no change in the activities of these enzymes.(38-4o) Injection of tryptophan results in an increase in tryptophan pyrrolase activity in normal liver; however, this response is absent or decreased in most hepatomas.C41)

132

GEORGE WEBER AND MICHAEL A. LEA

The evidence appears to suggest that the levels of free amino acid and amino acid catabolizing enzymes in hepatomas are not influenced by regulatory factors such as hormonal, nutritional and substrate influences which are able to produce profound adaptation in normal liver. a K -

AMINO

ACID

NITROGEN

I

Flo. 7. Comparison of steroid-induced behavior in free amino acid content in normal liverand in hepatomas of differentgrowth rates. Injection oftriamcinolone caused a twofold increase in the liver of all control animals; however, there was no increase in any of the hepatomas tested. This is an example of a complete loss of steroid responsiveness in all hcpatomas.¢10)

A summary of the metabolic pattern of protein and amino acid metabolism in hepatomas is shown in Fig. 8. The synthetic pathways in protein metabolism are predominant and increase with the increase in growth rate. At the same time the catabolism of tryptophan and other amino acids is decreased by a gradual decrease in the enzyme activities involved in amino acid catabolism. The oxidation of amino acids is not altered along a recognizable pattern. On the other hand, the free amino acid level in all hepatomas examined was completely unresponsive t o the influence of glucocorticoid stimulation which causes a marked rise in amino acid level of normal liver.

PATTERN OF NUCLEIC ACID METABOLISM IN HEPATOMAS The collected results indicate that there is a gradual increase in biosynthesis and a decrease in catabolism of nucleic acids which can be correlated with the

THE

MOLECULAR

CORRELATION

CONCEPT

OF NEOPLASIA

133

increase in growth rate. The progressive rise in the anabolic pathway of D N A is especially well supported by the data available. A summary of the enzymatic and metabolic alterations in nucleic acid metabolism in hepatomas o f different growth rates is given in Tables 4, 5 and in Fig. 10. PROTEIN

SYNTHESIS

NO ACID

POOL

DEAMINATION

l

®

O)

t ",i',,,® ~-~TO ACIDS

(~

TRYPTOPHAN BREAKDOWN

STEROID RESPONSE (~

Incorporation of the following amino acids: alaninej aspartate, glycine, serine, isoleuctne, valine.

~)

Oxidation of amino acids.

(~) Tryptoplutn pyrrolase, 5-hydroxytryptophan decarboxylase, serotonin deaminase.

(~ Serinedehydrase, glutamate transaminaee and dshydrogenase. (~) GlucocorUcold- induced elevation of tissue free amino acid level.

FIO. 8. The pattern of amino acid and protein metabolism in hepatomas of different growth rates. Attention is drawn to the increase in the biosynthetic pathway in contrast to the decrease in the catabolic pathway. Further explanation is given in the text.

Biosynthetic Pathway: Increases with the Increase of Hepatoma Growth Pate Evidence. Increased DN.4 synthesis. It has been shown that the D N A levels were in the same range in the livers of control normal animals, in partially hepatectomized rats and in host livers. In studies with the hepatomas it was found that the D N A level was in the normal range in the slowgrowing tumors (7800, 5123-D). However, it was increased to 177 per cent in the more rapidly-growing hepatoma 7288-C; and it was markedly increased to 250 and 211 per cent, respectively, in the 2 most rapidly-growing tumors (3924-A, 3683). Thus, the D N A content shows a positive correlation with the growth rate in hepatomas (Table 5). <4s> The biosynthesis of D N A was studied by following the incorporation o f labeled precursors into DNA. Although incorporation of thymidine-2-x4C into D N A was low in normal liver, it was markedly elevated even in the slowgrowing tumors (7800, 839 per cent; 5123-D, 1596 per cent). Thymidine incorporation was further increased in the more rapidly-growing 7288-C

134

GEORGE WEBER AND MICHAEL A. LEA

TAeL~4. Pattern of Nucleic Acid Metabolism in Hepatomas of Different Growth Rates The results are grouped from the point of view of definite trends which fit into one of the three categories.

Correlation with growth rate Increased DNA Synthesis:

Increase in DNA content Increase in thymidine incorporation into DNA Increase in formate into DNA Increase in adenine into DNA Increase in thymidylate synthetase Increase in deoxycytidylate deaminase Decrease in DNA Catabolism:

Decrease in thymine degradation to CO2

Low or high in all hepatomas

No correlation with growth rate

Increase in adenine incorporation into RNA

RNA amount Thymidylate phosphatase Dcaminases for deoxyadenosine, deoxyguanosine, deoxyadenylate Uracil incorporation into RNA

Decrease in Nucleic Acid Catabolic Enzymes:

Decrease in thymine reductase Decrease in uracil reductase Decreased Regulatory Response:

No increase in RNA amount after steroid injection

Increased RNA Synthesis:

Increase in formate into RNA Increase in aspartate transcarhamylase Decrease in RNA Catabolism:

Decrease in xanthine oxidase Decrease in uricase

Decrease in orotate incorporation into RNA

Decrease in RNA Metabolic Response to Stimulation by Glucocorticoid:

Decrease in precursor incorporation into total tumor RNA after steroid injection (2131 per cent), and it was the highest in the most rapidly-growing tumors (3924-A, 6403 per cent; 3683, 3378 per cent). Thus, D N A production (thymidine incorporation into D N A ) and growth rate (as estimated by generation time) show a good correlation (Table 5). ~48) This correlation appears to be close enough to suggest that thymidine incorporation into D N A might be used as a measure o f growth rate in bepatomas. In the synthesis of purines there are two formylation steps which were examined in hepatomas by measuring the incorporation of formate into RNA. A good correlation was observed between the extent of formate incorporation in rive and the growth rate of hepatomas by Wheeler et al./49~ and it was confirmed by Weber et al. ~1°~ The incorporation of 14C labeled formate into D N A also correlated well with the growth rate of hepatomas. There was a lower degree of correlation for the incorporation of adenine-8-14C into D N A , whereas the incorporation into R N A was high in all the tumors examined, c49)

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136

GEORGE WEBER AND MICHAEL A. LEA

In the case of DNA synthesis, studies with pyrimidine compounds in addition to those with purines support the picture of increased synthesis in line with increased growth rate. This is evidenced by the behavior of deoxycytidylate deaminase, thymidylate synthetase, and thymidine kinase which is shown in Table 5. The activities of the three anabolic enzymes for DNA synthesis increase with the increase in hepatoma growth rate. (5°- 54) It was also shown that the in vitro conversion of labeled thymidine to thymidine phosphate was greater in the more rapidly-growing hepatomas which is in line with the behavior of DNA metabolic enzymes,t55, 56) 7000

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The correlation between thymidine incorporation into D N A and the increase in hepatoma growth rate.(48)

Correlation of DNA Production and Tumor Growth Rate

To compare DNA production (in terms of incorporation of thymidine into DNA) with the growth rate of liver tumors (in terms of estimates for the generation times of hepatomas) these data were brought together in Fig. 9. The data on thymidine incorporation into DNA were taken from Table 5 and the generation times were established previously by Dr. Morris. ° 5,16) With the increase in hepatoma growth rate there was an increase in thymidine incorporation into DNA. The incorporation values for 3683 were lower than expected and repeated experiments confirmed this observation. We suggested that this finding may be explained in part by the fact that hepatoma 3683 regularly showed extensive hemorrhage and necrosis throughout the tumors, making the selection of viable tissue extremely difficult. For all other tumors the correlation between incorporation rate and growth rate is close. (4a)

THE MOLECULARCORRELATIONCONCEPTOF NEOPLASIA

137

In analyzing the correlation of DNA biosynthesis with growth rate it is noteworthy that even in the slowest growing of the hepatomas examined, the 7800, thymidine incorporation into DNA was increased eightfold over that of normal liver. Other parameters which increase with the increasing growth rate, e.g. lactate production, ~4) C-1/C-6 oxidation of glucose, ~4)incorporation of amino acid into protein, ~5.37) and fructose into glycogen through hexokinase reaction, ~s~) are in normal range in the slow-growing hepatomas. In contrast, the incorporation of other precursors into DNA, such as formate and adenine, is in normal range or only slightly increased in the slowgrowing tumors. ~1°,49) It appears that the behavior of thymidine incorporation into DNA offers the first well-defined, repeatable, quantitative alteration present in slow-growing tumors to a marked extent and increasing progressively with the increase of cancer cell proliferation rate. ~4s)

Decreased DNA catabolism. A progressive decrease in activity was observed for certain enzymes which bring about nucleic acid breakdown (Table 5). Examples of these are the uracil and thymine reductases, enzymes which are rate limiting in the catabolism of the respective pyrimidines,tSa) This evidence for a reduced thymine catabolism was in accord with results of studies showing that the capacity of liver preparations to convert carbon 2 of thymine to carbon dioxide was present in the 5123 hepatoma but not in the more rapidly-growing tumors, tS°) Two enzymes which are involved in the catabolism of purines, xanthine oxidaset59) and uricase, t6°) in hepatoma 5123 have activities of 85 to 50 per cent of the normal liver. However, the activities decreased to traces or were altogether absent in the rapidly-growing hepatomas (Table 5). ~s9-61) Increased RNA synthesis and decreased RNA catabolism. The levels of total RNA in hepatomas show no correlation with growth rate. ~1°,6°) It may be worth mentioning that the relative proportions of different molecular species of RNA and the turnover rates should be more characteristic and relevant to the problems of neoplasia and these may prove to be different in the different tumors. At present an easily interpretable pattern has not emerged for the synthesis of pyrimidine precursors and their incorporation into RNA (Fig. 10). The need for caution is reinforced by data for the incorporation of uracil which is found to be increased in slowly-growing tumors, normal in those of intermediate growth rate, and decreased in the rapidly-growing tumors. ~1°) The positive evidence may be summarized by pointing out that there is an increase in incorporation of formate into RNA which correlates with the hepatoma growth rate t1°,49) and that the activity of aspartate transcarbamylase also gives a positive correlation. ~6~) However, the initial reaction in this sequence, carbamyl phosphate synthetase, was found to decrease with the increase in growth rate tSa) and incorporation of

138

GEORGE WEBER AND MICHAEL A. LEA NUCLEIC

ACID

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5

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FIG. 10. Pattern of nucleic acid metabolism in hepatomas of different growth rates. The thin arrows indicate normal reaction rates; the thick arrows denote an increase in the metabolic step or pathway. In contrast, the dotted arrows show a decrease in the metabolic step or pathway. When the arrow tapers it indicates that the reaction or the metabolic pathway increases (thick arrows) or decreases (dotted arrows) with the growth rate; e.g. thymidine into D N A , the step catalyzed by aspartate transearbamylase or thymine to CO2. When the arrows are straight it means that the reaction increased or decreased in all hepatomas; e.g. incorporation of orotat¢ or uracil reductase is decreased in all tumors. This Figure shows the over-all increase in the synthetic pathways and the decrease in the catabolic pathways of nucleic acid metabolism.

orotate into R N A was decreased in all hepatomas examined. (t°) (An increase of the specific activity of the injected orotate caused a proportionate increase in the incorporation, suggesting that the low tumor specific activity was not due to a sequestration of the labeled orotate by the liver.) The decrease in the activities of xanthine oxidase and uricase is also in line with a decrease in R N A catabolism. Additional evidence for a decreased formation of catabolic products is shown by examination of the fate of adenine and hypoxanthine. In the slow-growing tumors a normal amount of these products is formed, but product formation in the rapidly-growing tumors was markedly decreased. (49) METABOLIC

PATTERN OF HEPATOMAS GROWTH RATES

OF DIFFERENT

In an effort to summarize the pattern which emerges from the results presented in this paper, the following considerations are given.

THE MOLECULAR CORRELATION CONCEPT OF NEOPLABIA

139

The metabolic pattern of carbohydrate metabolism in normal liver is dominated by the fact that there is a channeling either in the direction of gluconeogenesis or glycolysis. An understanding of these phenomena may be reached by the functional genie unit concept. °°) The key glycolytic enzymes and the key gluconeogenic enzymes are assumed to be produced on two separate units of the genome, the bifunctional enzymes on a third unit. In the hepatomas the key gluconcogenic enzymes decrease in one block with the increasing growth rate, whereas the key glycolytic enzymes increase. The bifunctional enzymes are present in an excess and their activities show no correlation with the growth rate. Alterations in the genome resulted in a predominance of the functional genie unit governing the production of key glycolytic enzymes with a gradual decline in the ability to produce the key gluconeogenic enzymes. These alterations lead to an imbalance in the two antagonistic functions. The importance of the opposition of key gluconeogenic and glycolytic enzymes in diabetes and other conditions was emphasized in another paper in this volume. (62) As a result of these enzyme alterations in the hepatomas a progressive imbalance develops and a dominance of the glycolytic pathway emerges. The evidence reveals a similar imbalance with regard to the opposing synthetic and catabolic pathways in protein, lipid and nucleic acid metabolism. In an attempt to apply the "pathway antagonism concept" gained from our studies in carbohydrate metabolism to the other pathways the present cancer pattern studies are useful. The key rate-limiting reactions for the other metabolic pathways are not as clearly defined as are those for carbohydrate metabolism. However, from the data discussed we suggest that the cancer studies will assist us to pinpoint the rate-limiting reactions because they are the ones which tend to show a correlation with the growth rate.

Pathway Antagonism as Revealed in the Metabolic Pattern of Hepatomas of Different Growth Rates In Fig. 11 the behavior of metabolic pathways is summarized. In the normal liver there is a balance of the synthetic and catabolic pathways (upward and downward arrows). These represent the direction of the over-all metabolic pathways and the normal activities of the rate-limiting enzymes. The horizontal arrows refer to the bifunctional reactions, representing enzymes which are present in an excess. In the carbohydrate metabolism of hepatomas the synthetic pathway gradually decreases with the increasing growth rate, and the catabolic pathway is progressively increased whereas in lipid metabolism both are decreased. In contrast, in protein and nucleic acid metabolism the anabolic pathways become progressively predominant and the catabolic pathways decrease with the increasing tumor growth. (These trends are indicated by the tapering arrows.)

140

G E O R G E W E B E R A N D M I C H A E L A. LEA

O n e m a y raise the question w h e t h e r with all these b i o c h e m i c a l a n d enzym a t i c studies we have really progressed in o u r u n d e r s t a n d i n g o f the e c o n o m y o f the cancer cell m e t a b o l i s m .9 Such a critic m a y say t h a t " a l l this tells us is CAR mOI/YIIqATE

LIPID

A A "--'-~ P

RNA

DNA

0 0 CO O000rO / /

"~% \

I I*

S

!

t

REPATOMA8

",: ~t fl

OF DIFFERENT

REGENERATING

NORMAL

GROWTH

RATES

LIVER

LIVER

FlO. 11. Summary of metabolic pattern in hepatomas of different growth rates as compared to that of regenerating liver and normal liver in rat. Thin arrows indicate normal reaction rates and metabolic pathway activities. The heavy arrow, and also its thickness, indicates an" increase whereas that of the dotted arrow indicates a decrease in over-all metabolic pathway activity. The tapering of the arrows shows that the increase or decrease is a progressive one which correlates with the increase in the growth rate of the hepatomas; e.g., synthetic pathways of carbohydrate metabolism and the breakdown pathways of protein and nucleic acid metabolism decrease parallel with the increase in hepatoma growth rate. In contrast, the catabolic pathway of carbohydrate metabolism increases progressively, whereas the anabolic pathways in protein and nucleic acid metabolism increase gradually parallel with the increase in hepatoma growth rate. The metabolic parameters which do not correlate with the growth rate are shown as the bottom half circle for each metabolic pathway. This Figure also serves to picture the antagonistic behavior of anabolic and catabolic pathways. For further discussion see text. t h a t the c a n c e r cells lose their ability to p r o d u c e glucose a n d there is a loss o f s t o r a g e functions such as a c c u m u l a t i o n o f glycogen a n d fat. It w o u l d also be expected t h a t p r o t e i n a n d nucleic acid synthesis involved in cell m u l t i p l i c a t i o n s h o u l d increase." I n a n s w e r to such c o m m e n t the following c o n s i d e r a t i o n s

THE MOLECULAR CORRELATION CONCEPT OF NEOPLASIA

141

are relevant. One may well imagine, and indeed there are, cancers which do not lose certain functions, and may not have high mitotic rates. The important point appears to us to be that it is now possible to describe neoplastic disease of liver in terms of progressive alterations in the various biochemical parameters and to show correlation with the advance of the disease. It is good that this is not in contradiction with what one "expects" for the behavior of intermediary metabofism in tumors. However, previous to this paper it has never been demonstrated that such a pattern indeed does exist. The present recognition of such a pattern in hepatomas also makes it possible to design chemotherapy on the basis of these metabolic findings and it provides a basis for studying the action of effective and non=effective therapeutic approaches in terms of action or no attack on critical biochemical steps. The establishment of the present pattern makes it feasible to test experimentally the relevance to the neoplastic transformation of any or all of the biochemical alterations. If, for instance, the growth rate of a tumor may be markedly slowed down without interfering with glycolysis, this will change our views of the relevance of glycolysis to growth rate. On the other hand, a marked inhibition of giycolysis along with an inhibition of the growth rate would underline the critical significance of these metabolic pathways in determining the rate of proliferation in these cancer cells. The final and most important argument we would pose at this point is that the critic is entirely wrong in assuming that we should have expected this type of alterations and metabolic pattern in the neoplastic cells. That this expectation is wrong and that the neoplastic metabolic pattern is a specific one is shown by the fact that no similar alteration or pattern has ever been found in the rapidly-growing regenerating liver. In fact, it has been shown in the regenerating liver that there is no decrease in the activities of gluconeogenic enzymes ;¢~, ~a) there is no increase in lactate production. (~3) Although in regenerating liver there is an increase in the incorporation of thymidine into DNA, there is no increase in the level of DNA ¢4s) and it is important that there is no loss of the various nucleic acid catabolic enzymes. In other words, it is possible to have very rapid growth, in fact as rapid as that of the most rapidly-growing hepatomas in this series, without the synchronous loss of gluconeogenic enzymes or of enzymes of nucleic acid catabolism (Fig. 1I). These considerations underline the specificity of the metabolic pattern which emerges from the systematic study of the hepatomas of different growth rates as we identified it. It is clear that normal tissue cells are capable of rapid proliferation without complete loss of an extensive number of enzymes and metabolic pathways. Therefore, it is advisable that serious attention be paid to the metabolic pattern uncovered here and that it be followed up by testing its applicability to other types of tumors and its usefulness as a framework to design and test and interpret chemotherapeutic agents.

142

GEORGE WEBER AND MICHAEL A. LEA SUMMARY

We examined the intermediary metabolism and enzymology of a spectrum of liver tumors of different growth rates. The new results we presented along with those assembled from the literature indicate that a definite pattern of intermediary metabolism does exist. Special stress was laid on the identification of the biochemical parameters which correlate positively or negatively with the growth rate in the hepatomas. With this concept in mind a pattern was identified showing that the synthetic pathways of carbohydrate and lipid metabolism, and the breakdown pathways of protein and of nucleic acid metabolism decrease parallel with the increase in hepatoma growth rate. On the other hand, the catabolic pathway of carbohydrate metabolism increases progressively, whereas in protein and nucleic acid metabolism the anabolic pathways increase gradually parallel with the increase in hepatoma growth rate. An application of our concepts developed for carbohydrate metabolism interprets these findings as examples of antagonism between synthetic and breakdown pathways centering on opposing, key, one-way, rate-limiting reactions. The metabolic parameters and enzyme activities which do not correlate with the growth rate are usually identifiable as non-rate-limiting reactions or enzymes present in an excess. The metabolic pattern identified in the spectrum of liver tumors of different growth rates offers a framework for experimental testing and application of these concepts to other types of tumors and to chemotherapy. ACKNOWLEDGMENTS The research work outlined in this paper was supported by grants from the United States Public Health Service, National Cancer Inst. Grant No. CA-05034-07, the American Cancer Society, and the Damon Runyon Memorial Fund for Cancer Research, Inc. REFERENCES 1. G. W~SER,G. BAN~gJT~and H. P. MORRIS,Comparative biochemistry of hepatomas. I. Carbohydrate enzymes in Morris hepatoma 5123, Cancer Res. 21, 933-937 (1961). 2. G. WEBER,H. P. MORXIS,W. C. Low and J. ASHMOI~,Comparative biochemistry of hepatomas II. Isotope studies of carbohydrate metabolism in Morris hepatoma 5123, Cancer Res. 21, 1406-1411 (1961). 3. G. WEBERand H. P. M o ~ , Comparative biochemistry of hepatomas III. Carbohydrate enzymes in liver tumors of different growth rates, Cancer Res. 23, 987-994 (1963). 4. M.J. S ~ Y , J. Asm~ot~e,H. P. Molars and G. WEBER,Comparative biochemistry of hepatomas IV. Isotope studies of glucose and fructose metabolism in liver tumors of different growth rates, Cancer Res. 23, 995-1002 (1963).

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143

5. S.R. WAOLE,H. P. MORRISand O. W ~ , Comparative biochemistry of hepatomas V. Studies on amino acid incorporation in liver tumors of different growth rates, Cancer Res. 23, 1003-1007 (1963). 6. G. W ~ , Behavior of liver enzymes in hepatocarcinogenesis, Advances in Cancer Research 6, 403-494 (1961). 7. G. W ~ , Summary of informal discussions on basic concepts in neoplasia, Cancer Res. 23, 1491-1497 (1963). 8. G. W ~ , Behavior and regulation of enzyme systems in normal liver and in hepatomas of different growth rates, Advances in Enzyme Regulation 1, 321-340 (1963). 9. G. WenEg, M. C. I-Ie~mv, S. R. WAOt~ and D. S. WAOt~, Correlation of enzyme activities and metabolic pathways with growth rate of hepatomas, Advances in Enzyme Regulation 2, 335-346 (1964). 10. G. WEnER,R. L. S ~ q G ~ and S. K. SnrVASTAVA,Regulation of RNA metabolism and amino acid level in hepatomas of different growth rate, Advances in Enzyme Regulation 3, 369-387 (1965). 11. E.C. M ~ R and J. A. Mn~t~at, The presence and significance of bound aminoazo dyes in the livers of rats fed p-dimethylaminoazobenzene,Cancer Res. 7, 468--480(1947). 12. V.R. PcyrriR, The enzyme pattern in cancer tissues, Acta Unio Int. Contra Cancrum 6, 301-305 (1948). 13. V.R. POTteR, Biochemical perspectives in cancer research, Cancer Res. 24, 1085-1098 (1964). 14. H. C. PrroT, Altered template stability: The molecular mask of malignancy, Perspectives in Biology and Medicine 8, 50-70 (1964). 15. H. P. MORRIS, Some growth, morphological, and biochemical characteristics of hepatoma 5123 and other new transplantable hepatomas, Progress in Exptl. Tumor Research 3, 370-411 (1963), Karger, Basel. 16. H.P. MORRIS,Studies on the development, biochemistry and biology of experimental hepatomas, Advances in Cancer Research 9, 227-302 (1965). 17. G. WF.Be~Zand A. CAh'TegO, Fructose-l,6-diphosphatase and lactic dehydrogenase activity in hepatoma and in control human and animal tissues, Cancer Res. 19, 763-768 (1959). 18. G. W B m and A. CAh'~_.RO,Glucose-6-phosphate utilization in hepatoma, regenerating and newborn rat liver, and in the liver of fed and fasted normal rats, Cancer Res. 17, 995-1005 (1957). 19. H. C. PrroT, The comparative enzymology and cell origin of rat hepatomas II. Glutamic dehydrogenase, choline oxidase, and giucose-6-phosphatase, Cancer Res. 20, 1262-1268 (1960). 20. C.E. Stlo~rK, H. P. Monnls and G. E. Box~, Patterns of giycolytic enzymes in rat liver and hepatoma, Cancer Res. 25, 671-676 (1965). 21. J. ASHMORE,G. WEew and B. R. LANDAU,Isotope studies on the pathways of giucose6-phosphate metabolism in the Novikoff hepatoma, Cancer Res. 18, 974-979 (1958). 22. G. WeBeR, R. L. S I ~ d ~ , N. B. S T ~ a , E. A. Fm~R and M. A. Me~r~NDmK, Regulation of enzymes involved in gluconeogenesis, Advances in Enzyme Regulation 2, 1-38 (1964). 23. G. WEeER,R. L. Sn~G~J~and S. K. SVaVASTAVA,Action of giucocorticoid as inducer and insulinas suppressor of biosynthesis of hepatic 81uconeogenic enzymes, Advances in Enzyme Regulation 3, 43-75 (1965). 24. Y.C. Ln~, J. C. ELwOOD, A. ROSADO,H. P. MORRISand S. WEn~rlouse, Glucose metabolism in a low giycolyzing tumor, the Morris hepatoma 5123, Nature 195, 153-155 (1962). 25. J.C. ELWOOI),3(. C. LIN, V. J. CRISTOVALO,S. W ~ O U S E and H. P. MonJ~S, Glucose utilization in homogenates of the Morris hepatoma 5123 and related tumors, Cancer Res. 23, 906-913 (1963). 26. A.C. AJ~eR¢3 and H. P. MORRIS,Energy pathways of hepatoma no. 5123, Nature 191, 1314-1315 (1961). 27. D. BURX, M. WOODSand J. HUNTER,On the cancer metabolism of minimal deviation hepatomas, Proc. Am. Assoc. Cancer Res. 6, 9 (1965).

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