Oxidative Metabolism of Neoplastic Tissues

Oxidative Metabolism of Neoplastic Tissues SIDNEY WEINHOUSE The Lankenau Hospital Research Institute and The Institute f o r Cancer Research, Philadelphia, Pennsylvania Page 270 I. The Concepts of Warburg.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Glycolysisin ~ i v o. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 11. The Pasteur Effect.. . . ................................. 274 276 1. Respiratory Quotient Data of Dickens.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 111. Present Concept of Carbohydrate Oxidation. . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Catabolism of Glucose.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 2. Pyruvate Oxidatio .................................... 281 IV. p-Oxidation of Fatty .................................... 282 V. Mechanisms of Glycolysis in Tumors.. . . . . . . . . . . . . . . 283 VI. Electron Transport in Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 291 1. Cytochrome c and Cytochrome Oxidase.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Respiration in the Intact Tumor Cell.. .................... 4. Nature of Substrates for Respiration of Tumor Cells. . . . . . . . 5. Isotope Tracer Studies on Tumor Respiration-Glucose Oxidat 6. Oxidation of Fatty Acids in Neoplastic Tissues.. . . . . . . . . . . . . . . . . . . . . 305 7. Oxidation of Acetoacetate in Liver and Tumor Slices.. . . . . . . . . . . . . . . . 309 311 8. The Citric Acid Cycle.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 313 9. Isotopic Studies 10. The “Condensin e. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 VII. Oxidation in Tumor Homogenates.. . . ......................... 315 ......................... 315 1. Citric Acid Cycle Intermediates. . . 2. The DPN+ Effect.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 3. Effects of Phosphorylation and Dephosphorylation on Oxidation in 320 Tumor Homogenates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 4. Possible Causes of High Glycolysis in Tumors References. . . . .............................................. 323

The biochemist concerned with the cancer problem is guided and probably also motivated by the belief that the uncontrolled growth of the cancer cell has its origin in some metabolic or enzymatic peculiaritya point of departure from the normal cell-which might provide a rational basis for the control or annihilation of this disease. The invasive growth of cancer cells, depending as it does on a high synthetic capacity, has directed much attention t o the mechanisms by which energy is made available for anabolic processes, and since the main source of such energy is the oxidation of fats and carbohydrates, it is in this field that bio269

270

S IDNE Y WEINHOUSE

chemical exploration of the cancer cell has probed most deeply. Many attempts have been made t o formulate differences between normal and neoplastic tissues on the basis of differences in metabolism, particularly of glucose. Stimulated originally by the pioneering efforts of Warburg, a concept of tumor metabolism has arisen which maintains as a fundamental thesis that the neoplastic process is somehow associated with disturbances or peculiarities of oxidative metabolism. It is the object of this report to review the evidence for this concept and t o examine it against our present knowledge of intermediary cell metabolism.

I. THECOSCEPTSOF WARBURG Biochemical thought concerning oxidative metabolism of tumor cells has been dominated by Otto Warburg, whose pioneering work in the metabolism of cancer tissue extended over some eight years of intensive work up t o 1930 and has continued sporadically to the present time. So completely has Warburg’s approach to the subject captured the imagination of biochemists that no serious discussion of this problem is complete without some description of \Tarburg’s results and ideas. These may be found i n extenso in a collection of the researches of Warburg and his colleagues ( l ) , and in a lucid review and interpretation of Warburg’s work on tumor metabolism by Dean Burk (2). Warburg’s important contributions to the biochemistry of cancer stem from the development of techniques for measurement of gas exchanges, made possible by the manometric apparatus which bears his name. I-sing these techniques, he and his colleagues measured the consumption of oxygen and at the same time measured the production of lactic acid by these tissues, either in oxygen (aerobic glycolysis) or in nitrogen (anaerobic glycolysis). T ii t hesc studies Warburg discovered a metabolic characteristic of tumor tissues, which to this day represents perhaps their most outstanding hiochemical feature, namely, a high aerobic and anaerobic glycolysis. Dean Hurk has summarized the data of Warburg and other early workers in a series of tables which not o d y reveal the experimental results of the early investigations but also give an idea of the type of information upon which the various concepts were built. For a detailed description these should be consulted in the original. For the purpose of the present discussion, these data have beem drastically condensed and are presented in Table I. As shown in this table, lactic acid formation in tumor slices under nitrogen, averaging 25.6, is over three times as high as in slices of nongrowing normal tissues. Glycolysis persists in the absence of oxygen for long periods, for days in fact, without diminution in rate. The process has a pH optimum of i . 3 and a temperature optimum of 37°C. It occurs

271

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

in the presence of other sugars, such as mannose, fructose, and galactose, but not nearly so rapidly, and is optimal a t a glucose concentration of 0.2%, which is in the range of its physiological concentration in body fluids in the postabsorptive state. TABLE I Condensed Tumor Metabolism Data of Warburg and Others from the Review by Burk (2)

Respiration, Qo, Aerobic glycolysis, Q L O ~ Anaerobic glycolysis, Q L N ~ Absolute Pasteur Effect QLNZ

- &LO,

Normal, Nongrowinga

Malignantb

9 . 3 (3-21) 2 . 1 (0-10) 7 . 2 (2-19) 5 . 1 (1-16.5)

11.8 (5.3-19.8) 14.0 (4.7-24.6) 25.6 (14.0-34.8) 11.6 (6.3-17.8)

9 . 7 (4-14) 7 (0-15) 20 (13-28) 12 (4-19)

46

64

Per cent Pasteur Effect 78 (12-100) ~ O O ( Q L-~ ~Q L O ~ ) / Q L ~ ~ Meyerhof oxidation quotient 2 . 1 (0.2-4.5)

-

(23-70)

3 . 2 (1.4-4.3)

Growing"

(28-100)

4 . 1 (1.7-6.0)

( Q L ~ ~ &LO,) /%Qo,

The Q notation refers t o microliters of gas corresponding to the product in question consumed or produced per milligram dry weight of tissue per hour. Averages and ranges of 14 different tissues of various animals. These are meam and ranges of mean values obtained with 15 different tumor types, but they are not weighted means. Individual values on 7 tiesues of 3 tissue types: chicken embryo, and rat placenta and embryo. (1

,t

@

More striking is the difference between neoplastic and nonneoplastic tissue sliceq in the presence of oxygen. Aerobically, glycolysis is on the average seven times as high in tumor slices as in nongrowing tissues; in fact, aerobic glycolysis in tumor slices is about twice as high on the average as anaerobic glycolysis in normal tissue slices. Warburg pointed out (1) that such rates are equivalent t o a lactic acid production of as much as 12% of their dry weight per hour; under the same conditions red blood cells produce only 0.1%) and frog muscle 0.06% a t rest and 1.5% doing maximum work. 1. Glycolysis in Vivo That glycolysis is high in the intact tumor growing in situ in its host as well as in slices of the excised tissue was first demonstrated by Cori and Cori (3). They showed that tissues of .the fasting, tumor-bearing mouse had a low lactic acid content, ranging from about 0.01% to 0.1 % in tumors, liver, and muscle. On administration of glucose, the lactic acid content was considerably increased in the tumor but not in the liver. Later these investigators (4),in comparing the glucose and lactic acid content of the blood from the axillary veins of a chicken carrying a Rous

272

SIDNEY WEINHOUSE

sarcoma in one wing, found 23 mg. less glucose and 16 mg. more lactic acid per 100 ml. in the vein draining the tumor. A similar experiment with a human patient carrying an axillary tumor gave similar results. Warburg, Wind, and Negelein (5) also observed large differences in lactic acid and glucose content between arterial and venous blood of rats bearing the Jensen sarcoma; these findings again pointed t o a rapid utilization of glucose by the tumor, with production of lactic acid. Similar, though somewhat less direct, evidence for lactic acid production in cico by tumors has been advanced by Voegtlin et al. (6) and by Kahler and Robinson (7), who found that the intercellular p H of a rat hepatoma decreased in response to glucose administration, whereas the p H of liver did not change. Despite many subsequent in vitro studies of tumor metabolism, the author has not found any exceptions t o the high glycolysis of tumor tissues. This appears t o be a distinct metabolic feature of tumor cells, regardless of type or host. The force of this conclusion is blunted somewhat by the fact th at glycolysis is not a n exclusive feature of neoplastic cells. Apparently all cells glycolyze under certain conditions, more so in the absence of oxygen; and as seen in the last column of Table I, actively growing tissues have a considerably higher glycolysis than adult, nongrowing cells. As seen in Table I, the glycolysis pattern of embryonic tissue is not far different from th a t of neoplastic cells (extended discussions of glycolysis in various tissues will be found in reviews by Burk ( 2 ) arid Dickeris (8) and in the monographs by Greenstein (9), and Stern and Willheim (lo)). It can therefore be assumed that high glycolysis is a general phenomenon of growing cells, whether it is the physiological, orderly development of the embryo or the pathological invasive growth of the tumor. It thus appears, as Warburg pointed out, that whereas normal tissues display predominantly an oxidative type of metabolism, tumor slices predominantly ferment glucose. This difference can easily be seen by comparing data for normal tissue with those for malignant tissue in Table I. Since the oxidation of one molecule of glucose requires six molecules of oxygen, we can calculate that under aerobic conditions the normal tissues on the average osidize 9.3,'6 = 1.5 molecules of glucose while they glycolyze 2.1/2 = 1 molecule of glucose. The tumor slices, on the other hand, osidize 11.8/6 = 2.0 molecules of glucose, on the average, while they glycolyze 14.0/2 = 7 molecules of glucose. Thus, the ratio, glucose fermented/glucose oxidized is 0.7 for normal tissue and 3.5 for tumors. It is important t o note th at the high aerobic and anaerobic glycolysis of tumor tissues is not associated with any noticeable derangement in oxygen consumption, since the averages and ranges of oxygen consumption correspond closely in all three tissue types. This fact should be kept firmly

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

273

in mind, since, as we shall see, many ideas, even of present-day investigators in this field, have been predicated on a supposed quantitative impairment in the oxygen consumption of tumor slices. Warburg’s interpretations of his findings can perhaps be best expressed in his own words, taken from the preface of the English Edition of his book (1): “Aerobic glycolysis results if the respiration of growing cells is injured, whether by diminishing its extent or by interfering with the relationship which holds between respiration and fermentation (glycolysis). . . . Interference with the respiration in growing cells is, from the standpoint of the physiology of metabolism, the cause of tumors. If the respiration of a growing cell is disturbed, as a rule the cell dies. If it does not die, a tumor cell results. This is no theory, but a comprehensive summary of all the measurements a t present available.” This categorical statement by Warburg with its somewhat mystical connotations, and carrying the authority of a recognized leader in the field, directed the course of many years of subsequent biochemical research in tumor metabolism. In many instances the Warburg concept became distorted and misinterpreted by subsequent investigators, and though a great mass of additional information accumulated in the twenty odd years since Warburg’s book on tumor metabolism appeared, no decisive advances were recorded and the Warburg view generally prevailed, namely, that a disturbance in respiration was a characteristic feature of tumor metabolism. If the experimental observations used by Warburg in support of this point of view are examined critically, it is seen that they offer relatively little support t o his idea. Originally Warburg’s choice of tissue was the Flexner-Jobling carcinoma and various human tumors, all of which had low respiration. It was easy to see, therefore, how an apparent association of high aerobic and anaerobic glycolysis with a low oxygen consumption could lead t o the conclusion that these phenomena are causally interrelated. Subsequent studies with tumors (no less malignant) with high Q0,% led Warburg t o modify his original view that there is a quantitatively lower respiration in tumors. Though Warburg relinquished the idea that respiration may be quantitatively disturbed in tumor cells, he still insisted that there was a disturbance in the relationship between respiration and fermentation. In justification of this idea Warburg pointed t o the fact that in normal tissues the respiration is able t o abolish glycolysis; that is, in normal cells aerobic glycolysis is zero or close to zero, whereas in tumor cells the

274

SIDNEY WEINHOUSE

aerobic glycolysis is high. In his words (1, p. 327), ‘(Whether the respiration of the tumor cell is large or small, aerobic glycolysis is present in every case. The respiration is always disturbed, inasmuch as it is incapable of causing the disappearance of the fermentation (i.e. glycolysis).’J This insistence on a disturbance of respiration as being the cause of aerobic glycolysis is not only not justified by the high respiration of tumor cells; it is also inconsistent with Warburg’s own thoughts expressed elsewhere in this book (1, p. 139). But before discussing this matter further it will be necessary to digress to say a few words about the relationship between respiration and fermentation, which Warburg termed the Pasteur Effect. 11. THEPASTEUR EFFECT The fact that living cells carry out a slower rate of fermentation in the presence than in the absence of air stems from an observation originally made by Pasteur. His words (11, see p. 276) are: “Free oxygen imparts to yeast an increased vital activity.

...

If we supply yeast with a sufficient quantity of free oxygen for the necessities of life, nutrition and respiratory combustion, it ceases to be

a ferment, that is, the ratio between the weight of the plant developed and that of the sugar decomposed is similar in amount to that in the case of fungi. On the other hand, if we deprive the yeast of air entirely it will multiply just as if air were present, although with less activity, and under these circumstances its fermentative character will be most marked ;under these circumstances, moreover, we shall find the greatest disproportion, all other conditions being the same, between the weight of yeast formed and the weight of sugar decomposed . . . if free oxygen occurs in varying quantities, the ferment power of yeast may pass through all the degrees comprehended between the two extreme limits of which me have just spoken. It must be borne in mind that the equation of a fermentation varies essentially with the conditions under which that fermentation is accomplished, and that a statement of this equation is a problem no loss complicated than that of a living being.”

It was logical to assume, of course, that this effect was due merely to the removal of fermentation product or some intermediary thereof by oxidation. By measuring simultaneously oxygen consumption, glucose utilization, and fermentation product appearance (alcohol in the case of yeast, lactic acid in the case of muscle), Meyerhof (12, 13) demonstrated that such a theory was untenable. He observed that t
OXIDATNE METABOLISM OF NEOPLASTIC TISSUES

275

was merely to oxidize away the cleavage product. Since in the case of lactic acid formation three molecules of oxygen are required to oxidize a molecule of lactic acid, the decrease in lactic acid divided by onethird of the oxygen consumption should equal unity if the only effect of oxygen is to remove lactic acid by oxidation. However, Meyerhof found that the decrease ranged from three to six times the amount which could have been oxidized by the oxygen consumed. Put in terms of experimentally definable quantities.

This decrease in fermentation brought about by oxygen was termed by Warburg the ((Pasteur Effect” and the above ratio, the “Meyerhof Quotient.” Warburg noted that, by and large, tumor tissues had the same Meyerhof Quotient ” as normal tissues. In Warburg’s words (1, p. 139) : ((

“We determined the Meyerhof quotient for carcinoma tissue, lactic acid bacteria, embryonic tissue and a number of other glycolyzing tissues, and as a rule obtained the same mean values as Meyerhof. As a rule 1 mol. of breathed oxygen, just as in muscle, causes the disappearance of 1-2 mol. lactic acid. This result . . . proves that the influence of the respiration on the cleavage metabolism in the carcinoma-cell is normal. . . . Although in the tumor every oxygen molecule breathed is just as effective as in muscle-the Meyerhof Quotient is equal in the two cases-yet the respiration does not cause the glycolysis to disappear. The respiration of the carcinoma tissue is too small in comparison with its glycolytic power.” Nowhere in this statement is there any mention of a quantitatively disturbed respiration or any other respiratory disturbance, nor is there any mention of any disturbance in the relationship between respiration and glycolysis-in fact, the opposite is explicitly stated. It is difficult to recognize in this statement any similarity to the categorical dictum quoted earlier. It is true, of course, that because of the high aerobic glycolysis the decrease in glycolysis due to oxygen is lower percentagewise in tumors than in most normal tissues (Table I). For this reason a low Pasteur Effect has been mistakenly attributed to cancer cells. Examination of the data of Table I reveals that if we measure the Pasteur Effect, most simply and directly, as the differences between glycolysis in air and glycolysis in nitrogen (absolute Pasteur Effect, Table I), the values for tumor slices are on the average over twice as

276

SIDNEY WEINHOUSE

high as those for normal tissues. Here we must be on guard to avoid the equally wrong but opposite conclusion, namely, that the Pasteur Effect is greater in tumor tissues. The reason why the Pasteur Effect is smaller in most normal tissues is that it is limited by the low rate of anaerobic glycolysis. Obviously tissues such as kidney or liver, which have an anaerobic glycolysis of three, cannot have a greater absolute Pasteur Effect than three. However, rat brain cortex, which has an anaerobic glycolysis of 19, displays a Pasteur Effect of 16.5, which is in the range exhibited by tumor slices. For the same reasons it is obvious why the percentage Pasteur Effect gives an entirely erroneous impression of the magnitude of this quantity. From Table I it may be seen that whereas glycolysis in normal tissues is decreased 78% by oxygen, glycolysis in tumors is lowered only 46% on the average. Warburg stated: “The respiration is always disturbed, inasmuch as it is incapable of causing the disappearance of the fermentation.” It would have been more accurate to state that the anaerobic glycolysis of tumor slices is so high that a normal respiration and a normal Pasteur Effect are incapable of eliminating it. This latter statement places the emphasis on the high glycolysis of tumor slices rather than on the respiration or on the absolute Pasteur Effect, neither of which are quantitatively diminished in neoplastic cells. 1 . Respiratory Quotient Data of Dickens

In the various quantitative expressions employed by Warburg, Meyerhof, and others, the possibility of the metabolism of other foodstuffs was ignored, and it was tacitly assumed that under the conditions of the experiments with tissue slices, i.e., in the presence of glucose, only sugar was being metabolized. Dickens and Simer (14) recognized the possibility that disturbances in carbohydrate metabolism may not be manifested in quantitative changes in respiration, since it is possible that other foodstuffs such as fats, etc., might be undergoing catabolism and thus contribute t o the respiratory activity. Dickens and his colleagues accordingly developed methods which made it possible to measure glycolysis, oxygen consumption, and carbon dioxide production simultaneously in citro with tissue slices, and embarked on a study of the respiratory quotients (R.Q.) of various normal and neoplastic tissues. These studies revealed an interesting relationship between glycolysis and R.Q. Reference to Table 11, in which data are taken from the work of Dickens and Simer, reveals that normal tissues fall into two main groups. The first, containing all of the resting tissues except brain and retina, has a low R.Q., which is closer to the theoretical value of 0.7 for fat oxidation, and a low anaerobic glycolysis, indicating that these tissues predominantly

277

OXIDATIVE ‘METABOLISM O F N E O P L A S T I C T I S S U E S

oxidize fat despite the presence of glucose in the medium. The second group, which includes brain and retina in addition to growing tissues such as embryo and chorion, displays a high glycolysis and R.Q. values characteristic of the oxidation of carbohydrate. TABLE I1 Mean Values of R.Q. and Anaerobic Glycolysis of Normal and Neoplastic Tissuesa (14) Tissue

R.Q.

Liver Kidney Intestinal mucosa Submaxillary gland Spleen Testis Embryo Embryo, chicken Brain cortex Chorion

0.79 0.85 0.85 0.87

7

Rat Rat Chicken Mouse

0.89 0.94 1.04 1.00 0.99 1.02

8 8 8 18 19 32

Mouse Mouse Mouse Mouse Mouse Human

Retina

1.00

88

Human

a

Q c ~ ~Animal ~ z

3 3 4

Tissue

R.Q.

Qco,~~

Jensen sarcoma Slow sarcoma Rous sarcoma Spindle-cell tar tumor 173 Tar carcinoma 2146 Crocker sarcoma Sarcoma 37 S Spontaneous tumor I Spontaneous tumor I1 Papillary carcinoma of bladder Carcinoma of breast

0.82 0.92 0.93 0.91

34 18 30 21

0.87 22 0.89 22 0.86 27 0.91 20 0.87 (16)a 0.86 (3.4)b 0.84

(7.1)b

Of rat unless otherwise stated. Mixed tumors; much connective tiaaue.

Thus in normal tissues a low value for R.Q. is associated with low ability t o form lactate anaerobically. There appears to be a gradual transition to a type which displays high glycolysis and exclusive carbohydrate respiration. In these tissues carbohydrate can be recognized as the main fuel for respiration. In contrast with these data for normal tissues, the data for tumor slices reveal a third metabolic pattern; here a low R.Q. is associated with a high rate of glycolysis. All of the tumors studied displayed R.Q. values which are distinctly below the value characteristic of total carbohydrate oxidation, while giving high values for Q C o n N 2 . Dickens and Simer reasoned that in normal, resting cells carbohydrate metabolism is limited by a low rate of formation of the substrate for respiration, whereas in tumors carbohydrate oxidation is limited by a defective mechanism for oxidation of glycolysis products. These conclusions represent an important extension of Warburg’s ideas in that they agree fundamentally that there is a defective respiration in tumor cells, and they extend his ideas by “pinpointing” the defect in the oxidation of the glycolysis product. There are several reasons why this hypothesis may not be entirely acceptable. Dickens and Simer themselves point out that the R.Q. of

278

SIDSEY WEISHOUSE

tumor slices is increased in the presence of pyruvate t o values close to the theoretical for pyruvate oxidation. It is obvious that this finding is not in accord with a disturbance in oxidative metabolism of carbohydrate unless the assumption is made that the defect is between lactate and pyruvate, an assumption which seems rather unlikely. This theory has been criticized on other grounds. Boyland (15) showed th a t high rates of glycolysis and low respiratory quotients are not necessarily characteristic of tumors, and Berenblum et al. (16) pointed out th a t the tumors, whose metabolism had been studied by Dickens and Simer (14), were derived from tissues which have the same types of metabolism as their neoplastic counterparts, e.g., skin, intestinal epithelium, and fibroblasts. These investigators found (17), using a specially constructed microrespirometer, that Shope papilloma has a glycolytic and respiratory pattern very similar t o normal skin epithelium (see Table 111). TABLE I11 Mean Values for Oxygen IJptake, Aerobic and Anaerobic Glycolysis, and Respiratory Quotient of Normal Skin Epithelium and Shope Papilloma (17)”

~

Sormal skin cpithelium

Shope papilloma

0 9 0 6

0 45 0 3

1.3 1 25

0.7 0.6

a The PQ values represent cu. mm of gas per pg. of nucleic acid phosphorus content of tissue per hour, as distinguished from the usual Q values. which represent cu. mm. of gas per mg. dry weight of tissue per hour.

111.

PRESENT CONCEPT O F C.4RBOHYDR.4TE OXIDATION

hfter thus presenting the background of early information leading to the concept of an impaired oxidative metabolism in tumor cells, it will be our task t o try to interpret these findings in terms of our present knowledge of cellular metabolism. The terms glycolysis, respiration, and Pasteur Effect were used by Warburg t o describe phenomena which had relatively little meaning with respect to detailed reaction processes. At the time IVarburg was carrying out his pioneering studies of glycolysis in tumors, little was known of the reaction pathways by which glucose is converted t o lactic acid. The adenine and pyridine nucleotides were yet, undiscovered. Investigators at th at time knew little more than Pasteur concerning the processes by which carbon compounds became converted to carbon dioxide. It is not surprising, therefore, that Warburg and others regarded the Pasteur Effect in terms of what Warburg called a “Pasteur reaction,” or t hat investigators even much later sought what they called a “Pasteur Enzyme” (2). Though such a view no doubt seemed eminently reasonable a t the time, our present-day picture of the complicated multi-

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

279

step processes of glycolysis and respiration with their numerous crosslinkages through electron transport factors, and the interlinkages with fa t t y acid and amino acid metabolism, leaves no provision for a single reaction by which respiration (or oxygen) can influence glycolysis. It will probably be worth while a t this point to review briefly our present concepts of the intermediary metabolism of carbohydrate in animal cells. Though there still remains much to be learned before the complete picture of energy metabolism is brought into sharp focus, biochemistry has now reached a stage where a broad outline of intermediary metabolism is visible. Pathways can now be envisioned by which the major metabolic fuels, such as the carbohydrates and the fatty acids, are interconverted or are converted t o COz or t o the constituents of proteins and other cell components. I n many instances chemical equations for these reactions can be written with confidence that they are representative of occurrences in the intact cell; in addition, knowledge of electron transport and oxidative phosphorylation has increased to such an extent that we now have a t least a rudimentary idea of how oxidative energy production may be coupled with the processes of synthesis which underlie all growth phenomena. I . Catabolism of Glucose As illustrated in Fig. 1, glucose is activated by conversion to glucose&phosphate, from which two routes diverge for the further catabolism of the sugar. One of these processes comprises a series of isomerizations, transphosphorylations, and an oxidation and a reduction step, resulting in the conversion of one molecule of glucose t o two molecules of lactic acid. Glucose

1

Glucose-6-Phosphate

1

Fructose-6-Phosphate

Dihydroxyacetone Phosphate

6-Phosphogluconic Acid

1 1 Fructose-1,6-Diphosphate Ribulose-5-Phosphate J-1 J1

$ Glyceraldehyde-3-Phosphate

I

.L

3-Phosphoglyceric Acid

1 1 Phosphoenolpyruvic 1

2-Phosphoglyceric Acid

Lactic Acid +ri Pyruvi.: Acid

FIG 1.

Acid

+ Glycolaldehyde

280

SIDNEY WEINHOUSE

This is the so-called pathway of Embden and Meyerhof. The other mechanism branches off with the oxidation of glucose-6-phosphate to 6-phosphogluconic acid. The latter substance is then presumed to undergo decarboxylation and a further oxidation to yield a pentose-bphosphate, which is probably the ketopentose, ribulose-5-phophate (18, 19), but which ultimately yields ribose-5-phosphate. The further metabolism of this substance is not entirely certain, but from the recent studies of Racker (20) it would appear that it undergoes an ((aldolase” type of split to triose phosphate and a “diose,” possibly glycolaldehyde. This so-called ‘(oxidative ” or hexose monophosphate shunt ” mechanism of glucose breakdown has been studied primarily in microorganisms, and little information is yet available concerning its extent in animal tissues. According to Bloom, Stetten, and Stetten (21) the shunt pathway does not occur to an appreciable extent in the normal, intact rat. This conclusion was based on an isotope tracer procedure, involving measurement of the relative extents of conversion to COZof variously labeled glucoses, viz., glucose-1, and 6-CI4, and uniformly labeled glucose; and of the three C14-labeled lactates. Application of the same method to tissue slices revealed that the shunt was not operative in kidney and diaphragm but occurred t o a major extent in liver (22). Similar conclusions were drawn by Lewis et al. (23), using a more direct method based on comparison of the specific activity of lactates and acetoacetates arising when slices of rat tissues were incubated with glucose-l-Cl4 or uniformly labeled glucose. With the esception of liver, therefore, whose low rate of glucose catabolism is overshadowed by that in the musculature, the hexose monophosphate shunt does not appear to occupy a prominent quantitative position in glucose catabolism in the normal intact animal. It is generally assumed that the glycolysis which occurs anaerobically proceeds via the Embden-Neyerhof pathway. Anaerobic glycolysis via the EmbdenMeyerhof process results in the quantitative conversion of glucose to lactic acid, whereas the so-called shunt mechanism gives a t most one lactic acid molecule per molecule of glucose consumed. Negelein (24) found that the Flexner-Jobling carcinoma gave two molecules of lactic acid per molecule of glucose disappearing from the medium, indicating the major occurrence of the E.M. process in this tumor; and the isotopic tracer method of Lewis et al. (23) indicated that the shunt is not an important pathway in the mouse hepatoma 98/15. On the other hand, preliminary data of Lewis et al. (23) indicate that the shunt operates to the extent of 40% to 50% in the mouse sarcoma 37 and in an ascites form of the T.43 carcinoma. It would thus appear that the shunt mechanism is of quantitative significance a t least in some tumor tissues; however, the matter requires further investigation and extension to more tumor ((

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

281

types. The pathway of glucose breakdown via 6-phosphogluconate has been designated the " oxidative pathway "-the implication of course being that this mechanism may come into play only in presence of oxygen. The possibility that such a mechanism may account for the Pasteur Effect has been entertained (8). Such a mechanism might be favored in the presence of oxygen, since in contrast with the balanced oxidoreductions of ordinary glycolysis, the shunt requires dehydrogenations equivalent to the consumption of a mole of oxygen per mole of glucose, as shown in Eq. 1. CsHlzOe

+ Oz

4

C3H603

+ CzH,02 + COz + HzO

(1)

There is, however, no compelling reason why the shunt mechanism would be restricted to oxidative conditions. It is conceivable that in an intact cell these dehydrogenations may be coupled with other reductive processes, and it is therefore possible that the shunt reaction can also occur anaerobically, just as other oxidative processes may be coupled, through common coenzymes, with reductive reactions. 2. Pyruvate Oxidation

Whatever the mechanism by which it is formed, triose phosphate is oxidized to pyruvic acid, and the further oxidation of this substance results in the formation of carbon dioxide and an active acetyl derivative. Evidence of recent years has shed considerable light on the mechanism of this hitherto obscure reaction. In addition to the apoenzymes, at least four cofactors are apparently necessary : coenzyme A, diphosphopyridine nucleotide, diphosphothiamine, and a-lipoic (thioctic) acid. The over-all course of events is formulated by Korkes et al. (25) as in Eq. 2. Pyruvate

+ DPN+ + CoA + Acetyl CoA + DPNH + H+

(2)

According t o Gunsalus (26) the process of pyruvate decarboxylation to yield acetyl CoA in various microbial species can be represented in the following four equations.

+ DPT+ + S-R-S I CHaCO: S-R-S+ COASH HSRS- + DPN+ CHaCOCOOCHaCO :DPT

-+

+

-+

-+

+ COz + DPT+ CHaCOSCoA + HSRSS-R-S + DPNH

CHaC0:DPT CHaCO :S-R-S-

u

(3) (4)

(5) (6)

The first step, Eq. 3, is a straight, diphosphothiamine-catalyzed decarboxylation to COZ and an acetaldehyde-diphosphothiamine complex. I n the second step, Eq. 4,the acetaldehyde is transferred to a sulfur of

282

SIDNEY TVEINHOUSE

a-lipoic acid. This cyclic disulfide of 6,8-dimercapto-octanoic acid (desigundergoes a reductive cleavage to nated in the equation by S-R-S)

u

form acetyl lipoate, and in the third step, Eq. 5 , the acetJyl group is transferred t o the thiol group of coenzyme A, yielding acetyl SCoA and reduced lipoic acid. The latter is reoxidized via DPN+ t o continue the cycle of reactions. Further details of this process will be found in several recent reviews (27, 28).

IJ-. P-OXIDATIOXOF F a w Y ACIDS Present conceptions of fatty acid osidation envision this process as occurring by successive removal of units of acetyl CoA to complete degradation of the carbon chain. Summaries of recent findings with respect to the individual enzymatic steps will be found in reviews by Mahler (291, Lyncii (30), and JVeinhouse ( 2 7 ) . The initial step is the formation of an acyl V0-i ester, either by transacylation from a natural lipid such as a phospholipid to CoASH or by activation of the acid with Coh and adenosirietriphosphate (AXTI'),as shown in Eq. 7 . The succeeding steps shoivn i n Eqs. 8 to 11 are: a,p-dehydrogeiiation, by a flnvoprotein enzyme; hydration t o the 0-hydroxy-acid ; dehydrogenation to the corresponding 8-keto arid: atid finally, a "thiolytic" split of the P-keto acid t o one molecule of ncrtyl SC'oA and the COX ester of the next lower homologous fatty acid. The process continues to complete conversion of the fatty acid carbon chain t o acetyl SCoA.

+ Co.4SH + ATP RCH?CH?COSCoA + A3IY + inorganic pyrophosphate ( 7 ) IiCHiC'H-C'C)SCo.\ + F h D RCH=CHCOSCoA + FADH? (8) ItCki=CHC'OYCoh + € 1 2 0 RCHOHCH~COSCOA (9) RCHOHCEItCOOII + DPS+ RCOCH,COSCoA4 + DPKH + H + (10) I1COC'II,COSCoh + COASH * RCOSCo.4 + CH~COSCOA (11)

RCII,C'H-('OOH

----t

----t

+

The nest step in the catabolism of acetyl COX is its entry into the citric acid cycle by condensation with oxalacetate to yield citric acid plus coenzyme -1 (31). There then ensues the now well-established stepwise reaction sequence resulting in the complete oxidation of a molecule of acetate arid the regeneration of osalacetate to carry 011 the cycle. During the course of the complete oxidation of a molecule of glucose, 23 electrons are transferred t o oxygen, 12 for each triose molecule, and these come off in pairs of 2 a t the following stages: triose phosphate, pyruvate, isocitrate, a-ketoglutarate, succinate, and malate. Before reaching oxygen, the electrons pass through the pyridine and flavoprotein enzymes and probably all funnel through the cytochrome system.

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

283

From the foregoing discussion, it is possible to consider various overall influences which may be exerted on the intermediary formation of lactic acid. First, lactic acid will accumulate if the glycolytic reactions occur too rapidly for the pathways of carbon or electron transport t o dispose of pyruvic acid. The accumulation of pyruvate would then result in its competing with the flavoproteins for the electrons taken up by the pyridine nucleotides, and would result therefore in the accumulation of lactic acid. Similarly, any defect in carbon transport through the citric acid cycle or in one of the electron transport steps could also conceivably result in the reduction of pyruvate. Evidence bearing on these possibilities will be discussed in subsequent sections.

V. MECHANISMS OF GLYCOLYSIS IN TUMORS Ever since the delineation of the route of sugar catabolism in muscle by Embden and Meyerhof (13), wide interest has been manifested in the possible existence of other pathways, particularly one not involving the usual phosphorylated intermediates. For the earlier history of these studies the reader is referred t o the excellent review of Dorfman (32). No attempt will be made t o cover the voluminous literature related t o glycolysis in tumor cells. Only a few examples have been chosen which illustrate progress in our conceptions of the mechanisms of this process. Stimulated by Warburg’s findings, Barr, Ronzoni, and Glaser (33) observed that an extract of pancreas inhibited glycolysis in slices of the Rous sarcoma; and in contrast with the behavior of muscle, they observed in tumor tissues a diminished glycolytic activity on mincing, grinding, or freezing. They concluded that the characteristic phosphorylative glycolysis of normal tissues was not occurring in tumor tissues. This view was supported in experiments conducted by Scharles et al. (34). It was found that although glycolysis from glucose was completely inhibited by cell destruction in both normal and tumor tissues (a finding previously made also by Warburg), this process did occur in tissue extracts in the presence of hexose diphosphate. They found, however, that glycolysis was not affected by fluoride in tumors as it was in normal tissues; it was less susceptible t o inhibition by iodoacetate, and it did not require any “ coenzyme” (which a t that time meant adenosinetriphosphate) . At the same time a systematic study by Boyland and Boyland (35) revealed that some of these differences could be attributed to the fact that adenosinetriphosphate (ATP)was rapidly destroyed by frozen malignant tissues. They found that tumor extracts would glycolyze hexose diphosphate (but not glucose) if supplied with relatively high concentrations of ATP, and they also observed the presence of zymohexase (a mixture of aldolase and triose phosphate isomerase) in these

284

SIDNEY WEINHOUSE

tumor extracts. Though the rates of glycolysis from hexose diphosphate were only about one-fourth those from glucose in slices, and considerably lower than those in muscle extracts, in view of the losses of enzymes by extraction and by the action of adenylpyrophosphatase, these investigators saw no reason to assume that hexose diphosphate was not an intermediate of glycolysis in the intact cell, or that the mechanism of glycolysis differed in any significant manner in normal and neoplastic cells. Following upon the finding of Meyerhof and Ohlmeyer (36) that cozymase (DPX) was required for glycolysis in muscle, Boyland et al. (37) found that in extracts of frozen Crocker sarcoma 180 the addition of cozymase raised the levels of glycolysis to virtually that of slices. They also found that glycolysis from glucose, fructose, and glycogen occurred in these extracts and was enhanced by the addition of adenylic acid. The recent exhaustive studies of LePage and his colleagues have brought forward overwhelming support to the idea that, qualitatively, glycolysis is similar in tumor and other tissue types. LePage (38), in 1948, developed a medium in which suspended tissue homogenates could carry out glycolysis of hexose diphosphate at a high rate. The system contained phosphate, magnesium, and bicarbonate ions, ATP and Dl" as cofactors, hexose diphosphate as substrate, and nicotinamide as an inhibitor of DPN breakdown. The system contained fluoride ions to inhibit dephosphorylation reactions, and since this inhibited the further reactions of phosphoglycerate, pyruvate was added t,o couple the oxidation of triose phosphate to lactic acid production. In this system, homogenates of the Flexner-Jobling rat carcinoma actively glycolyzed hexose diphosphate (and glucose when hexose diphosphate was present in low concentration) and maintained organic phosphate a t a high level, indicating that phosphorylation was a t least as rapid as organic phosphate breakdown. Cnder optimal conditions lactic acid formation occurred at a rate of 10 micromoles per 30 mg. of tissue in 40 minutes, which corresponds to a Q (lartic) of over 50. No " Pasteur EfTect " was displayed by these homogenates, the rate of lactate formation being as high in air as in nitrogen. A further study by Novikoff, Potter, and LePage (39) extended these results to the Jensen and Walker 256 tumors. The authors concluded, on the basis of their detailed study, that the Embden-Meyerhof scheme of phosphorylative glycolysis operates in tumors. This conclusion was further substantiated by a detailed analysis, by LePage (40), of a large number of phosphorylated intermediates, and cofactors of the Embden-Meyerhof scheme. Table IV shows that all of the intermediates found in normal, resting tissues were present also in a number of primary and transplanted tumors.

TABLE IV Analyses of Glycolysis Intermediates and Factors of Normal and Neoplastic Tissues of Rat (40) (Values are in micromoles per 100 g. tissue.)

Components Lactic acid Glycogen” Acid-soluble phosphorus Inorganic phosphorus Organic phosphorus Phosphocreatine Adenylic acid Adenosine diphosphate Adenosine triphosphate Glucose-1-phosphate Glucose-6-phosphate Fructose-&phosphate Hexose diphosphate Phosphoglyceric acid “Coenzymes” Free pentose phosphate Per cent of organic phosphate accounted for 0

As herose.

Primary Primary Liver Mouse CarciCarci- noma Brain Muscle Liver Kidney Heart noma (rat) 141 531 2390 495 1895 311 151 27 179 61 185 30 6 98 17 42

188 3480 5070 748 4322 1630 155 59 542 80 250 33 7 140 17 22

230 28450 3040 417 2623 274 144 330 8 42 423 24 17 183 35 48

155 81 2530 497 2033 116 213 48 138 42 264 17 4 102 16 72

578 2460 3200 730 2470 219 329 65 105 175 249 53 7 209 25 50

833 232 1830 723 1108 88 97 12 93 47 335 11 5 167

80

81

70

68

65

95

590 468 2810 828 1983 0 278 133 54 100 555 34 21 116

9D

0

Human Flexner- Walker Mouse Breast Jobling 256 Ear Carci- Carci- Carcino- Jensen Carcinoma noma sarcoma Sarcoma noma 1458 1620 ,2030 382 1649 46 137 51 67 101 470 16 15 161

862 67 2650 1035 1615 92 131 49 106 104 278 7 5 119

824 65 2430 622 1808 116 171 25 152 130 454 14 6 148

637 43 2130 580 1550 78 183 46 142 106 393 17 5 98

704 56 2950 726 2224 94 251 135 161 156 500 37 11 165

7 M

E w

g

3 $

5s d

M

m 78

77

72

86

91

89

286

SIDNEY WEINHOUSE

LePage and Schneider (41) found, using the differential centrifugation technique, that the enzymes which carry out glycolysis, both in rabbit liver and Flexner-Jobling carcinoma cells, are concentrated in the soluble portions of the cytoplasm. Though the presence of other cell components, principally the small granules (microsomes), considerably enhances the glycolytic activity of the supernatant fraction, none of the other fractions by themselves were nearly as active as the supernatant alone (see Table V). TABLE V Glycolysis Obtained in 40 Minutes with 30 mg. of Tissue or Fraction Obtained Therefrom (41) Flesner-Jobling Carcinonia __.____

Tissue Fraction Homogenatc Nuclei Mitochondria hIicrosomes Supernatant fluid

Lactic acid produced S e t P bptake per flask per flask (micromoles) i.35 1.31

0 0.05 2.69

3.62 1.23 -0.13 - 0 . 76 2.07

Rabbit Liver -____.____~

Lactic acid producccl Net P uptake per flask per flask (micromoles) 6.26 0.79 0 0.17 3.30

0.23 0.26 0.05 -0.31 0.42

Some of the differences between tumor tissues and normal tissues in their metabolism of glucose ha\-e been brought out more clearly by LePage (-12) in a further study of glycolysis in whole homogenates. As shown in Table TI,all tissues glycolyzed rapidly in the basic medium containing hexose diphosphate, but only brain and Flexner-Jobling carcinoma displayed an increased glycolysis when glucose was added t o the medium. ,4s shown in Table VII, however, tissues which were apparently unable t o utilize glucose, such as diaphragm, kidney, and liver, were able t o utilize glucose-6- and fructose-6-phosphate. LePage therefore was able to pinpoint the limiting step in glucose utilization for glycolysis to the hesokinase reaction by which glucose and ATP react to give glucose-6phosphate. He suggested that this step remains under hormonal inhibition even i n the dissected, surviving tissue. However, attempts t o relieve this inhibition i n zdro or t o demonstrate hormonal effects on glycolysis were i n general not successful. A highly significant feature of LePage’s results is that potential rates of glycolysis of normal tissues are as great as, or greater than, those of tumor tissues, and th at the low rates of glycolysis displayed by normal tissue slices are not due to lack of enzymes for the reactions involved.

287

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

TABLE VI Anaerobic Glycolysis with Homogenates of Tissues from Normal Intact Rats (42) (40-Minute incubation with 30 mg. wet weight of tissue except in the case of diaphragm, where 15 mg. was used. Each figure represents the average of 15-20 experiments except those for heart and skeletal muscle, where only three experiments were available.)

Tissue

Basic Medium Minus Glucose

Basic Medium with Glucose

Lactic acid Net P production uptake (micromoles)

Lactic acid Net P production uptake (micromoles)

Brain Flexner-Jobling carcinoma Liver Kidney Diaphragm muscle Heart Skeletal muscle

0.45 0 -1.5 -2.5 -1.0 -1.9 -2.2

6.0 6.2 6.4 6.5 5.5 9.1 9.0

9.8 9.5 6.8 6.8 5.6 9.9 8.7

6.5 4.5 -1.3 -2.3 -0.65 -0.39 -5.3

TABLE VII Anaerobic Glycolysis with Glucose and Phosphorylated Sugars b y Homogenates of Rat Tissues (42) (40-Minute incubation in each case, with 30 mg. wet weight of tissue except in the case of diaphragm (20 mg.). Reaction mixture contained cofactors as listed in the basic medium.) (Values are in micromoles lactic acid.) Substrate Added in Addition t o Glucose None Hexose diphosphate Hexose diphospbate Glucose Hexose phosphate Glucose-6-phosphate Hexose diphosphate Fructose-6-phosphate

Micromoles

Brain

Diaphragm Kidney Liver

0 6 6 30 6 4.5 6 4.5

0.1 6.8

0.1 6.2

0.1 6.0

0.1 7.7

9.1

6.6

6.0

7.6

8.6

8.9

8.3

9.0

9.0

9.5

8.2

9.6

Experiments by Meyerhof and collaborators (43, 44) have in general confirmed the idea that over-all processes of lactic acid formation are a t least qualitatively similar in normal and neoplastic cells. Their data emphasize the probability that quantitative differences observed in homogenates, extracts, or other broken cell preparations are more likely due to differences in the intracellular distribution or to differences in

288

SIDNEY WEINHOUSE

breakdown rates of coenzymes or other factors than to actual differences in types or amounts of enzymes. In seeking an explanation for the fact that glycolysis from glucose occurred readily in extracts of brain, but not in homogenates, Meyerhof and Geliazkowa (13)found th a t most of the ATPase of brain cells was in the particles removed on low-speed centrifugation. Consequently the level of ATP could be maintained readily in extracts, from which these particles were removed, hut not in homogenates. Subsequently, Meyerhof and Wilson (44) found th a t most of the ATPase of tumor cells was in soluble form, not removed by centrifugation, and they reasoned th at the glycolytic inactivity of tumor extracts or homogenates was due t o excessively rapid breakdown of ATP. I n confirmation of this hypothesis they found that if ATPase was inhibited by addition of agents like octyl alcohol or toluene, or if yeast hexokinase was added, steady and high glycolysis from free glucose could be achieved in tumor extracts and homogenates. There is no evidence known which would indicate that the catabolism of glucose t o lactic acid occurs in tumors via channels not present in normal cells. From the foregoing data it is clear th a t careful, critical studies offer no suggestion of a nonphosphorylative pathway, but, on the contrary, point t o the Embden-Meyerhof pathway as the major mechanism. The possible occurrence of the oxidative hexose monophosphate shunt remains for further exploration; indeed preliminary data already discussed (23) make it probable that this process may be of considerable significance in the carbohydrate metabolism of tumor cells.

JrI. ELECTROX TRANSPORT IN TUMORS

.4 great deal of evidence has been advanced in favor of the plausible idea that the high aerobic glycolysis of tumor tissues might have its origin in a disturbance in the pathways by which electrons are transported to oxygen. Shortly after the role of the pyridine nucleotides in respiration was recognized, Euler and colleagues (45) compared a number of normal and neoplastic tissues of the rat and found the diphosphopyridine nucleotide (DPK+) and triphosphopyridine nucleotide ( T P Nf) content of the Jensen tumor to be atsabout the same level as in muscle or liver. An interesting observation of these authors was a markedly higher proportion of reduced DPN in the Jensen sarcoma. The ratio D P N H / D P N + was 8.5 and 6.2 in two assays on the tumor, whereas rat muscle had approximately equal amounts of each form, the ratio ranging from 0.56 t o 1.0 in three analyses cited. Somewhat later Kensler et al. (46) reported a lowering in the DPK+ content of rat liver from 1390 pg./g. fresh weight to 500 pg. in the “precancerous” liver after long-term feeding of butter yellow. T he tumors thus induced had a still much lower DP N f content,

289

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

namely, 150 pg./g. fresh weight. Bernheim and Felsovanyi (47) reported DPN+ plus TPN+ values of 71 pg./g. of Walker 256 tumor as compared with values of about 500 pg./g. for muscle, spleen, kidney, and liver. More recently, Fisher and Schlenk (48) reported results of analyses of several tumors for their content of the oxidized and reduced forms of DPN+. Their values for DPN+, as shown in Table VIII, ranged from 9 t o 165 pg./g. fresh tissue, whereas the DPNH values ranged from 21 t o 91 pg. In contrast with the previously cited study of Euler et al. (45), the content of reduced DPN was generally lower than that of the oxidized form. The authors concluded that neoplastic tissues have a lower total DPN content than normal tissues, but their comparison was based only on data for normal rat liver, whose DPN content was in the vicinity of 500 pg./g. tissue. TABLE VIII Content of Reduced and Oxidized Forms of DPN in Tumor Tissues (48)

Tissue Rat sarcoma AH R a t tumor (methylcholanthrene) Mouse breast carcinoma (C3H) R a t fetus

Nucleotide Content (pg./g. fresh tissue)

No. of Determinations

DPN+

DPNH

Total

8 2 2 2

49-165 9, 31 75, 123 41, 31

41-91 36, 21 42, 57 25, 25

98-240 49,45 124,189 61,56

For reasons to be discussed in a later section a comparison of normal and neoplastic tissues with respect to their content of DPN+ and DPNH was carried out in the author’s laboratory by L. Jedeikin. It was found that both DPN+ and DPNH are highly susceptible both to enzymatic and nonenzymatic destruction, and their recovery from tissues requires rigid control of experimental conditions. Using conditions for extraction which ensured a predictable recovery of both forms of the nucleotide, and a highly specific enzymatic method of assay, with crystalline alcohol dehydrogenase, a generally much lower content of total pyridine nucleotide was found in neoplastic than in normal mouse and rat tissues. In both tissue types, however, the same pattern of reduced and oxidized forms was observed, the latter being greatly preponderant. A summary of data secured thus far is given in Table IX. Carruthers and Suntzeff (49) developed a polarographic procedure for determination of total pyridine nucleotides (DPN+ and TPN+). Their values for normal tissues ranged from 110 pg./g. fresh tissue for mouse epidermis up to 540 pg. for mouse liver. Comparable values for a series

SIDKET IVEIWHOUSE

DPNH

338-592 206-435 334-477 102,204 162,184 344-370 530,630

13 7 6 2 2 3 2 57-372 137-264 41-272 80, 114 37) 44 16-38 27,38

290

DPX+ No. of Analyses Range

TABLE I S Content of DPS+and DPXH in Various TissuesD (Values are in micrograms per gram fresh tissue.)

No. of Analyses Range

13 6 6 2 2 4 2 2 2 2 2 35) 38 19,29 33, 51 37, 41 38 0

___ -

hlormal Tissues Liverb Kidney b Heartb Brain< Spleenc Muscle (skeletal)' Muscle (pigeon breast) 92,114 99,118 89, 121 99,111 206 297

Human Cancer Tissues

50 29 75 54 21 21 138 200

( %,)

Cancer/ Sormal

3.7 9.6 87.0 20.4 0.87 0.22 924 3.7

Rat Sormal Tissues

1.54 3.4 23.6 7.7 0.196 0.05 516 3.51

Rat Cancer Tissues

42 35 27 32 22 23 56 100

Cancer/ Normal ( %)

1 1

2 2 2 2 1 1

Unpublished date of Jedeikin and Weinhouse. Of rat, mouse, rabbit, pigeon. Of rat and mouse.

Seoplastic Tissues (Mouse) Hepatoma 98/15 Rhabdom yosarcoma Sarcoma 37 Mammary carcinoma Ascites sarcoma 37 .4scites carcinoma (Ehrliclr) 0

c

Human Xormal Tissues 1.28 2.35 23.5 5.51 0.11 0.04 8i7 2.86 1.4

1.80 8.10 31.2 10.3 0.52 0.18 632

TABLE 9 B Vitaniins in Human, Rat, and Mouse Seoplasms (51) (B vitamin levels pg./g. moist tissue.)

Vitamin Thiamine Riboflavin Nicotinic acid Pantothenic acid Pyridoxine Biotin Inositol Folk acid

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

291

of transplanted tumors ranged from 60 to 202 pg./g. The tumors displayed values in the range of such normal tissues as lung, spleen, pancreas, and epidermis. Similar results have been reported in abstract form by Strength and Seibert (50). That tumors may have a relatively low content of factors concerned in electron transport is suggested also by an investigation of the B vitamin content of human and rat tumors, carried out by Pollock, Taylor, and Williams (51). A summary of their extensive data is given in Table X. All of the vitamins except inositol and folic acid were lower in human and rat tumors than in normal tissue of the same species. Especially noteworthy for this discussion is the lowered riboflavin and nicotinic acid content. 1. Cytochrome c and Cytochrome Oxidase

Evidence for a relative deficiency of cytochrome c in neoplastic cells is available from a number of studies. DuBois and Potter (52), using a TABLE XI Cytochrome c Content of Normal and Neoplastic Tissues of Rat Data of Rosenthal and Drabkin (53)

Data of DuBois and Potter (52)

Tumors

Cyt. c (alp. fresh tissue)

Flexner-Jobling 12 carcinoma Walker 256 carcinoma 9 Jensen sarcoma 12 Yale #1 mouse tumor 16 Ultraviolet ear tumor 11 Rous chicken sarcoma 12 Rat liver tumor 20 61 Tumorous rat liver

Normal tissues, rat

Cyt. c (alg. fresh tissue)

Heart

371

Kidney Skeletal muscle Liver Brain Spleen Lung

247 97 90 50 43 21

Tissue

Cyt. c (l.4g.k. fresh tissue)

Kidney cortex 1430 Liver 607 Brain cortex 375 Submaxillary 378 Colon mucosa 136 Mammary gland 32 Lung 24 Spontaneous mam51 mary adenoma Transplanted adeno26 carcinoma Walker 256 carcinoma 71

spectrophotometric assay procedure, found that the cytochrome c content (based on an estimated molecular weight of 16,500) ranged from 9 to 61 pg./g. of fresh tissue for a series of transplanted tumors as com-

292

SIDXEP WEINHOUSE

pared with values ranging from 21 t o 371 pg./g. for a variety of normal rat tissues (Table XI). These authors conclude th a t their results lend weight t o It-arburg’s idea th at tumor tissue may have a n anaerobic pattern of metabolism. In a similar study, Rosenthal and Drabkin (53) found t ha t liver, kidney, and brain cortex (in descending order) had highest contents of cytochrome c, and a series of tumors had only about 2% of the cytochrome c content of kidney cortex (Table XI). These differences are not due to ‘‘ cellularity ’’ differences, since the relative cytochrome c content does not change materially when calculated on the basis of protein phosphorus content. Though a low cytochrome c content was displayed by tumor tissues, this is not a distinguishing feature, since similar lorn cytochrome c levels were found in such normal tissues as colon mucosa, mammary gland, and lung. Shack (54) measured relative cytochrome oxidase activity of various tumors and normal tissues, using oxygen uptake, measured manometrically in the presence of excess phenylenediamine and cytochrome c, as a measure of cytochrome oxidase activity. Cytochrome oxidase activity was four t o five times higher in liver than in hepatoma or in other neoplastic cells. h tenfold higher D-amino acid oxidase content was also found in liver than in hepatoma or other tumors. Extensive investigations of the cytochrome oxidase activity of normal and neoplastic tissues have been carried out by Elliott and Greig (55) and Schneider and Potter (56), and the pertinent information is collected in Table XII. Though the values given by Schneider and Potter are far higher than those obtained by Elliott and Greig, both studies are in agreement in indicating a rather wide range in variation of cytochrome oxidase activity in normal tissues, and a much narrower range of activities in neoplastic tissues, of a magnitude similar to th a t of the less active normal tissues. It can be seen, from Schneider and Potter’s data, th a t the cytochrome oxidase activity ranged from 92 t o 974, whereas that of a variety of tumors of the rat, mouse, and chicken ranged from 44 t o 136. Schneider and Potter concluded that oxidative metabolism is deficient in tumors, but that succinic dehydrogenase was not the weakest link in the electron transport chain. Despite its low activity in tumors it would appear th a t the cytochrome oxidase activity is not a limiting factor in electron transport. Greenstein et al. (57) have shown th at a t least one factor which is lower in activity in a variety of rat and human tumors is cytochrome c. These investigators noted, in agreement with others, th a t in the presence of p-phenylenediamine the addition of cytochrome c t o a fixed amount of cytochrome oxidase preparation produced a n increased velocity of oxygen consumption up t o a point beyond which further additions are

293

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

The Succinic Dehydrogenases

TABLE XI1 and Cytochrome Oxidase (QJ Content of Normal and Cancer Tissues (56) (Q8)

Average Q.

Average

QOx &OX

Tissue

Etiology

S & P a E & G b S & PE & G & .

Rat heart Normal 219 62 974 Rat kidney 195 Normal 112 549 Rat liver 87.7 Normal 66 392 Rat brain Normal 48.7 18 420 Rat muscle 35.5 Normal 6 . 6 180 Rat spleen 23.3 Normal 0 . 5 195 Rat lung Normal 17.9 7 . 5 92.3 26.3 - 134 Rat liver tumor Orally ingested BYd 25 .O Rat liver tumorC 67.2 Orally ingested BYd Hepatoma 31 rat Originally oral BYd 21.7 136 Transplantable hepatoma Originally oral BYd 18.1 I24 Walker 256 rat 9.4 61.5 carcinosarcoma Originally spontaneous Walker 256c rat 0 , 6 77,8 carcinosarcoma Originally spontaneous 1 2 , 3 Flexner-Jobling rat carcinoma Originally spontaneous 15.5 - 91.3 Flexner-Jobling rat carcinomac Originally spontaneous 14.8 12.8 75.9 Yale No. 1 mouse tumor Estrin 20.2 - 106 Yale No. 1 mouse 19.0 87.5 tumorc Estrin Mouse ear tumorc Ultraviolet irradiation 19.1 64.0 Rous chicken sarcomaC Virus 11.1 44.4 Mouse mammary 27.7 0 . 5 87.8 tumorsc Spontaneous Jensen rat sarcoma Originally spontaneous 17.8 13 129 a

506 288 167 134 38 32 31 -

4.4 2.8 4.5 8.6 5.1 8.4 5.2

5.1

-

2.7 6.3

-

6.9

-

6.6

15

6.3

-

5.9

28

5.2

-

5.2

-

4.8 3.3 4.0

43

3.2 7.2

-

Schneider and Potter.

a Elliott and Greig.

4 These tissues were homogenized in 0.033 If phosphate buffer at pH 7.4. -411 other tissues were homogenized in glass-redistilled water. d BY = p-dimethylaminoazobenzene.

without effect. This maximum velocity represents a measure of the cytochrome oxidase activity under conditions in which the enzyme is operating a t highest efficiency. Since calculation of the Michaelis constant for cytochrome oxidase in tissue suspensions gave the same value obtained by others for preparations of this enzyme, this author concluded that the respiratory activity of the tumor suspension truly represents cytochrome

294

SIDNEY WEINHOUSE

osidase activity. From the Pllichaelis-Menten expression

’ where T’,

VmaxS

=

K F S

velocity of the reaction without added cytochrome c, = maximal velocity with escess cytochrome c, S = cytochrome c concentration of the tissues, it should be possible to calculate values for u from the cytochrome c content of tissues and from the difference between T’, and 1’. It was found th at the observed values corresponded quite closely with those calculated by the Jlichaelis-Menten equation. It was also found, using the observed values for t i , th at the calculated values for the cytochrome c content corresponded reasonably well with observed values. The authors reasoned, therefore, th at the difference between the observed oxygen consumption rates, with and without added cytochrome c, represents the disparity between the cytochrome oxidase activity and 2:

=

the cytochrome c content. The value

(”””’~- ’) 100, called the per cent

response t o cytochrome c addition, was calculated for the tissues studied, and it was found to fall into four categories. C’ategory 1, with per cent responses ranging from 100 t o 400, included only normal tissues, viz., heart, muscle, liver, kidney, and brain. Categories 2 and 3, with per cent responses ranging from 600 t o 1200, included some normal and some neoplastic tissues. (’ategory 4, with per cent responses ranging from 1500 to 6,000 included only neoplastic tissues. It mas concluded th at normal tissues are characterized by generally high values for cytochrome oxidase and cytochrome c, whereas neoplastic tissues have a generally low activity of cytochrome oxidase with a great disparity between cytochrome osidase activity and cytochrome c content. The earlier studies of the cytochrome system in tumors failed t o take into account the question of intracellular distribution of the oxidative enzymes and cofactors. The more recent results of Schneider and Hogeboom (58) on the intracellular distribution of cytochrome oxidase activities are of particular interest. These authors have compared these activities in four well-defined fractions of the transplantable mouse hepatoma 98/15 and the liver of its host; their data are in Table XIII. Despite generally much low\.er activities in the tumor fractions the distribution was remarkably similar. The very high specific activity of the mitochondria indicates th at essentially all of the cytochrome oxidase activity is in this fraction. Such comparisons between normal and neoplastic cells with respect to intracellular distribution of enzyme activities

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

295

are as yet few in number but promise t o yield a rich harvest in our understanding of relationships between over-all metabolic patterns and individual enzyme activities. Since all of these published data are in agreement with respect to a relative deficiency of cytochrome c and cytochrome oxidase in a wide variety of tumor cells, it might be thought that this would definitely represent a distinct characteristic of tumor cells, lending weight t o the idea that respiratory activity may be impaired in tumors. A recent paper by Chance and Castor (59), however, indicates that the whole question of cytochrome deficiency will probably need reinvestigation. Using sensitive TABLE XI11 Cytochrome Oxidase Activities of Mouse Liver and Hepatoma Fractions (58) (The total values reported are for 100 mg. of fresh tissue or an equivalent amount of each fraction, and each figure represents the average of 3 experiments.)

C3H Mouse Liver Cytochrome oxidase activity

Tissue Fraction Homogenate Nuclei Mitochondria Microsomes Supernatant

Qo, Per cent of (mm.3 0 2 per hr. Total total (mm.3 0 2 homogenate per mg. nitrogen) per hr.) activity 6860 1360 5390 292 0

(100) 19.8 78.6 4.1 0

2060 2440 6460 351 0

Hepatoma 98/15 Cytochrome oxidase activity

Total (mm.3 0 2 per hr.) 1520 195 964 247 0

Per cent of total homogenate activity

Qo~ (mm.3 0 2 per hr. per mg. nitrogen)

(100) 12.8 63.4 16.3 0

633 379 3300 624 0

spectrophotometric methods which allow the measurement of light absorption through whole cells, these investigators measured the changes in optical density a t various wave lengths corresponding to reduction of the cytochromes a, b, c, and a3,in several ascites tumor cells, when respiration is stopped by exhaustion of oxygen in solution. A comparison of the behavior of three ascites tumors with that of other cell-types is given in Table XIV. The quantity, K , measures the rate of removal of oxygen from solution and therefore gives an indication of the intensity a t which cytochrome oxidase is operating. The values for the ascites tumor cells are within the range of values for the normal cells, not as high as yeast, but higher than the sarcosomes of rabbit heart or fly muscle. The patterns of cytochrome distribution are rather similar except that cytochrome b is apparently lower in the tumors than in the normal cells (it was undetectable in the Ehrlich and Krebs 2 tumors). The most significant result with

296

SIDXEP WEIWHOUSE

respect t o the present discussion is the high relative cytochrome c content of the ascites tumors, actually exceeding th a t of the highly respiring yeast cells. Another point of interest is that there is a wider disparity between cytochromes c and a 3 (cytochrome oxidase) in the normal cells than in the tumor cells. In commenting on these discrepancies with previous reports, Chance states the belief th at the direct spectrophotometric determination in freely suspended, living cells is probably more decisive than the various indirect assay methods th a t have led t o the earlier conclusions. TABLE XIV Comparison of the Pattern of Respiratory Pigments of Tumor and Other Cells (59)

k‘= pJI 02/sec.

Material Keilin and Hartrw heart muscle preparation Rabbit heart sarcosomes Fly muscle sarcosomes Bakers’ yeast cells Ehrlich ascites Krehs 2 ascites dba thymoma

Relative optical density change

a

Cytochrornes b C

a3

DPNH

160

1 1 1 1

0.6 0.8 0.6 1.7

0.8 0.9 1.5 2.7

9 11 9 11

60

37 40 32

1 1 1

0.5 0.5 0.4

4 4

12 6 6

-

D115-180 (25°C.)

49 14 12

2

13 11

Relatively little attention has been given to studies of other factors involved in electron transport. Lenta and Riehl (GO) have recently made a study of the enzyme systems involved in the transfer of electrons from reduced diphosphopyridine nucleotide t o oxygen. The source of the activities measured was a phosphate buffer extract of homogenized tissues, precipitated by acetate at p H 4.6, and taken u p in phosphate buffer a t pH 8.5. Over-all DPS-oxidase activity was determined by spectrophotometric measurement of the oxidation of reduced DPN by 2,6-dichlorophenol indophenol; diaphorase activity was measured by oxidation of reduced D P N in the presence of methylene blue, and cyanide to prevent functioning of the cytochrome system; and cytochrome c reductase activity was determined by spectrometric measurement of the reduction of cytochrome c by reduced DPN in the presence of cyanide. The relative activities are designated in Table XV. Over-all coenzyme I oxidase activity was present in the three tumors studied but was on the low side of the normal tissues. This behavior could not be specifically localized

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

297

in one particular step, since low activities were observed generally in the tumor cells, except for cytochrome c reductase, which appeared in one tumor at least to be as high as in the highly active normal tissues. The high activity of DPN-cytochrome c reductase in the hepatoma 98/15 is in accord with a study of the intracellular distribution of this enzyme by Hogeboom and Schneider (61)) who previously found a somewhat higher content of this enzyme in the hepatoma than in the C3H mouse TABLE XV General Summary of the Coenzyme I Oxidase System and Its Components in Normal and Tumor Tissue Extracts (60)

Liver Kidney Heart Brain Muscle 5-37 Adenocarcinoma Hepatoma 98/15

Coenzyme I Oxidase Diaphorase

Cytochrome c Reductase

CytoCytochrome chrome c Oxidase

+++ ++++ +++++ ++ + + + +

+++++ ++++ ++++ +++ + ++ ++ +++++

++ +++ ++++ + +? + +? + + +

++++ ++++ ++++ ++ + + + +++

+++ ++++ ++++ +++ +++ + + ++

liver. I n contrast with cytochrome oxidase, cytochrome c reductase was present in all cell fractions, but highest specific activity was observed in the microsomes. In a previous study Rhian and Potter (62) found a low activity of cytochrome c reductase in tumors, but the manometric procedure used leaves open the possibility that some factor other than the enzyme per se may have been limiting the rate of reaction. 2. Dehydrogenases

Many efforts have been made t o relate the high glycolytic activity of tumor cells t o some peculiarity of lactic dehydrogenase, and though widely divergent results have been reported with respect to the occurrence of this enzyme in neoplastic tissues, a recent study by Meister (63) of a very wide variety of tumor tissues failed t o reveal any consistent differences among tumors, tissues of origin, or various normal tissues in their content of this enzyme. A highly condensed summary of his results is given in Table XVI. Similar results have been reported for a number of mouse and rat tissues by Wenner et al. (64) (Table XVII). Both of these studies employed spectrophotometric measurement of the oxidation of reduced DPN in the presence of pyruvate as substrate, and hence the

298

SIDKET WEINHOUSE

TABLE XVI Data of JIeister on Lactic Dehydrogenase A4ctivityof Various Normal and Seoplastic Tissues (63) Tissue

Kurnber of Samples

Activityo Range

12 13 13 13 13 13 54 19 57

36 1-490 390-523 284-426 296-405 900- 1040 940-1100 78-770 172-642 133-565 367-47 1 599-679 476-516 390-444

Mouse liver Liver of tumor-bearing miw llouse kidney Kidney of tumor-bearing mice Skeletal muscle, mouse Skeletal muscle of tumor-bearing mice Other tissues of mouse Primary mousc tumors Transplanted mouse tumors Adult rat liver Fetal rat liver Primary hepatoma Transplanted fibrosarcoma

5 4

3 3

Activity is expressed as moles X 10-8 pyrurate reduced per nig. total nitrogen per minute.

TABLE SVII Ilehydrogenase Assays i n .4cetone Powder Extracts (64) Deh ydrogenase (units/mg. acetone powder) a-Ketoglutaric Tissue Normal : Heart (rat) l i v e r (mouse) Kidney (rat) Nusclc (mouse) Neoplastic: Rhabdomyosarcoma (mouse) Mammary tumor (mouse) Hepatoma (rat) Hepatoma (mouse) Xscites (mouse) n

Lactic

Nalic

320 200 104 ,520

383 256 Ii 3 330

160,220 148, 208 380 168 238 540 108 288 165 200

Isocitric 56 10 ti6 15

0 8 0 6

6 7 16 0 12 5 1 2 8" 14 8 4 8

Qo2

7.7 4.0

3 3 2 2

3 2 4 1

Range of three determinations

enzyme assays were independent of the presence of other electron transport factors. Because of the question, t o be discussed later, of the participation of the citric acid cycle in oxidation processes in tumors, assays of various citric acid cycle enzymes mere made by Wenner et al. (64). Among these

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

299

were malic, isocitric, and a-ketoglutaric dehydrogenases (Table XVII) . All three were found to be present in a variety of transplanted mouse tumors, though the last was present in rather low activity when compared with several normal tissues. Malic and isocitric dehydrogenases were assayed by spectrophotometric observation of the oxidation or reduction of the pyridine nucleotide coenzyme, and the results were therefore independent of other electron transport factors. It is of interest that when such specific assay methods were employed, as was the case with lactic dehydrogenase (63) and DPN-cytochrome c reductase (61), high values were obtained for the enzyme activities in tumors, whereas, when manometric methods were employed, as for example for DPNcytochrome c reductase (62) or for malic dehydrogenase (65), invariably low results were obtained. These findings suggest that some factor involved in electron transport from the substrates in question to oxygen is either diminished in tumor cells or is destroyed more rapidly in such tissues during preparation of the enzyme. As we shall see later, this substance is probably DPNf itself. To summarize the present status of electron transport, it seems certain that the same factors, namely, the pyridine and flavin nucleotides and the cytochromes, are involved in oxidative reactions in both normal and neoplastic tissue types. No evidence is available to indicate that other means of electron transport are employed in the cancer cell. Although quantitative differences in some of these enzymes and cofactors have been observed, and some of these seem so marked as to suggest that they may account for some of the peculiarities of metabolism exhibited by tumors, no decisive information is available to justify any definite statements. Although the low cytochrome activity of tumor cells can be cited in support of the idea that respiration may be deficient in tumor cells, such a deficiency if it exists is not manifested in any marked quantitative diminution in oxygen consumption of such cells in vitro. The situation is further clouded by the above-cited spectroscopic data of Chance and Castor (59), which suggest that perhaps the earlier assays may not have measured the total cytochrome content of neoplastic tissues. 3. Respiration in the Intact Tumor Cell

Although, as we have already seen, Warburg, before 1930, abandoned his earlier idea that respiration was quantitatively impaired in tumor slices, this view has tenaciously persisted and is still often expressed or implied. A careful exploration of the available literature convincingly refutes such a concept. Studies of oxygen consumption of tumor slices by Crabtree (66), Murphy and Hawkins (67), and Warburg et al. (68), which

300

SIDNEY WEINHOUSE

are included in the data of Table I, clearly demonstrated that respiration of tumor slices is about as high as that of various representative normal tissues; and a large body of data to be found in the monographs of Greenstein ((3) and Stern and Willheim (10) have, without exception, confirmed and extended these findings. Elliott, whose work will be discussed presently, has stated (69) : " . . . Most cancer tissues show only a moderately high respiration rate, a moderately low respiratory quotient, and a definitely high, sustained aerobic and anaerobic glycolysis . . . ? 9 ., and Dean Burk, i n comparisons of various liver tumors with homologous normal, embryonic, aged, cirrhotic, and regenerating liver (70), has left no doubt that the neoplastic transformation of the liver cell is not associated with any regular, significant., qunntitativc diminution of oxygen consumption. There thus appears to be no basis in fact for the proposition that, when shaken in a saline medium, slices of tumor tissue consume appreciably less oxygen than do normal tissues similarly handled. Unfortunately this represents the only manner which a t present we have to measure respiratory activity of tumor cells, and it is, of course, possible that these methods do not convey an accurate picture of the behavior of tumor tissues in sitzi. By the same token, however, there is no evidence that the studies in vitro portray a false picture of the respiratory capability of living cells.

4. Nature of Substrates for Respiration

of Tumor Cells

Prior to the use of isotopically labeled substrates, relatively few criteria could be applied to determine whether or not a substance underwent oxidation by tissues. Perhaps the most direct means a t the disposal of earlier investigators was to test whether addition of a substance to respiring tissue slices in vitro brought about an increase in the rate of oxygen consumption. Virtually all animal tissue slices display a rather high endogenous oxygen Consumption. When the addition of a metabolite increased the oxygen consumption, the evidence was clear and unequivocal; when, as often happened, oxygen consumption was not increased, it could not be stated definitely that the added substance was inert, since it was conceivable that its oxidation replaced that of some metabolite endogenous to the tissue slice. Even when oxygen consumption is increased on the addition of a subst.rate, it cannot easily be ascertained from oxygen consumption data alone to what extent the added substance may be replacing the endogenous tissue metabolites. It is sometimes possible, by means other than oxygen consumption measurements, t o obtain the desired information indirectly. For example, Quastel and Wheatley (71) and Leloir and hIunoz (72) demonstrated the ability of liver slices t o oxidize fatty acids by measuring fatty acid disap-

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

30 1

pearance and ketone body formation, even though increases in oxygen consumption were minimal. These studies paved the way for later demonstration of fatty acid oxidation in homogenates and mitochondria. Other indirect criteria of the type of substrate undergoing oxidation may also be applied; for example, the respiratory quotient may indicate whether the oxidation is predominantly that of fatty acid or carbohydrate; or the appearance of ammonia or urea may disclose the oxidation of protein or amino acids. I n general, however, this type of approach has provided no definite answers to the fundamental question of what types of substrates are oxidized by tumor tissues. Another approach to this question is the study of known or presumed intermediates. The Thunberg technique (73) has shown that tissues contain dehydrogenases for many substances; on the assumption that substances are not entirely foreign to cells which can rapidly carry out their oxidation, this technique has been instrumental in tracing reaction sequences in carbohydrate metabolism. This procedure can be extended further to imply that tissues which oxidize such intermediates also utilize carbohydrate for oxidation. The usefulness of this type of procedure is well demonstrated by the fact that such reasoning applied t o oxidation of various intermediates in pigeon breast muscle supplied some of the basic information from which Krebs (74) formulated the citric acid cycle. So far, however, this type of approach has not yielded any decisive data which would allow definite conclusions concerning the types of foodstuffs oxidized by tumor cells. Such evidence as is available is rather indirect. We have already referred to Dickens’ studies of the respiratory quotient which suggested that glucose did not undergo oxidation readily in tumor cells. In line with this conclusion is the finding that oxygen consumption of tumor slices is not increased in the presence of glucose (66, 75). Somewhat later, Elliott, Benoy, and Baker (76) observed that whereas the addition of lactate, pyruvate, succinate, fumarate, or malate increased the oxygen consumption of rat kidney cortex slices, they had no effect on the oxygen uptake of slices of Philadelphia number 1 sarcoma or Walker 256 carcinoma (Table XVIII). Though these investigators carefully refrained from drawing the obvious conclusion that carbohydrate oxidation is impaired in these tumors, this paper has often been cited in support of such a thesis. A more detailed investigation of the problem of respiration in tumor slices was undertaken by Salter and his colleagues, some of which has already been discussed in connection with electron transport. Craig, Bassett, and Salter (77) made an interesting observation which formed the basis of a detailed investigation. It was found (Table XIX) that

302

SIDSEY WEINHOUSE

whereas various normal tissues such as liver, muscle, and “ benign” granulation tissue exhibited large increases in oxygen consumption on addition of sucrinate t o the medium, the oxidative response of the corresponding malignant counterpart was far lower. This phenomenon appeared to be rather general, being noted also in a comparison of a TABLE XVIII &o, Values in Presence of Various Substrates (76)

Suhstl-ate Xone m-Lact a t e Pyruvat e None Succinatc Fumarate hlfalate

Rat Kidney Cortex 21 32 33 19 32 24 21

5 1 6 9 8 5 0

Phila. Sarcoma 13 13 12 14 13 12 13

Walker 256 Carcinoma

6 2 8

11 11 13 11 11 12 10

1 8 2 3

5 5 5 7 0 0 8

TABLE X I X Effect of Succinatp on Oxygen Consumption of Tissue Slices (77) (Succinate when present was 0.02 M . Each value is the mean of approximately 10 observations.) &or Succinate: Per Cent Absent Present Change

Sormal liver Hepatoma Muscle Rhabdom yosarcoma Granulation tissue (bcnign) Sarcoma 180 “Benign” sarcoma 180 (presumably slow growing)

10.6 10.5 7 0 8 7 1 52 8 2

25 2 12 2 209 122 4 14 102

137 17 242 41 192 27

6 4

8 6

37

variety of normal and neoplastic human tissues (78). The oxidative response of the normal tissues ranged from 100% t o 158%, whereas 22 different tumors displayed responses ranging from -25% t o +76%. A similar effect was found in the “precancerous” liver of the rat fed butter yellow. With increasing periods of feeding of the dye, there was a steady diminution in the Oxidative response, ultimately reaching values characteristic of neoplastic tissues. Since p-phenylenediamine exhibited a similar behavior when used as a substrate, and since both p-phenylenediamine and succinic acid transfer electrons t o cytochrome c, these results were interpreted, in the light of their lowered cytochrome c, already discussed,

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

303

to indicate that the tumor cell has a lower “oxidative reserve”; that is, its oxidative capacity is sufficiently high to maintain a high respiratory rate, but it cannot respond by increased oxygen consumption to the stress of higher substrate concentrations. These findings provide further support for the idea that a possible bottleneck in electron transport in tumors is the cytochrome level. 5. Isotope Tracer Studies o n Tumor Respiration-Glucose Oxidation

The value of the tracer technique is perhaps nowhere better exemplified than in its application to the question of the metabolic fuel of the cancer cell. The availability of carbon-14-labeled glucose has finally made it possible to establish, without uncertainty or equivocation, that the neoplastic cell can convert glucose to carbon dioxide. Using this method, the appearance of radioactivity in the respiratory carbon dioxide affords a simple, direct, and reliable criterion for the oxidation of a substrate. By carrying out such experiments with tissue slices in Warburg vessels it is possible to correlate oxygen consumption and other respiratory data with the extent of oxidation determined quantitatively by measurement of the incorporation of isotopic carbon in the COz. I n 1949 Olson and Stare (79) reported that glucose, labeled uniformly with C14 in all of its carbon atoms, was oxidized to CO2 in slices of primary tumor more rapidly than in the adjacent normal liver, and that both the carboxyl and the CY carbons of pyruvic acid likewise were oxidized by the hepatoma slices. A more detailed report of these studies was given at a symposium on carbohydrate metabolism in tumors, held under the auspices of the American Association for Cancer Research (80). Since most of the data were presented graphically, their reproduction is somewhat inconvenient. Briefly summarized, the data showed that glucose, D( -) and L( +) lactate, acetate, succinate, and pyruvate are oxidized both by hepatoma and liver cells; and though quantitative differences were observed between the two tissue types, no uniformity in response was exhibited. Thus, although glucose was oxidized far more rapidly by hepatoma slices, rates of oxidation of methyl- and carboxyl-labeled acetates, uniformly labeled L ( + ) and D( -) lactate, and 1- and 2-C14pyruvate were about the same in liver and hepatoma slices, and succinate (carboxyl-labeled) was oxidized more readily by liver than by hepatoma. The results of Olson will be discussed later in other connections. In a parallel study of a variety of transplanted rat and mouse tumors, Weinhouse (81) and Weinhouse et al. (82) made similar findings. As shown in Table XX the oxidation of glucose in slices of such representative normal tissues as kidney, brain, liver, and muscle was compared with that displayed by slices of four transplanted tumors: a mouse and rat hepatoma,

304

SIDNEY WEIXHOUSE

a rhabdomyosarcoma, and a mammary adenocarcinoma. The relative specific activity of the respiratory carbon dioxide revealed that an appreciable percentage of the respiration of all of the tissues studied arose from the oxidation of C'4-labeled glucose added to the medium. This ranged from 34% for brain down to 8.5% for liver. Though somewhat lower than those of brain and heart, the relative specific activities of the respiratory C 0 2 produced by the tumor slices were in the range of those of kidney and heart and decidedly higher than the values displayed by skeletal TABLE X X CL4-GluroseOxidation by S o r m a l and Kcoplastic Tissue Slices (82)

(per cent.)

0.C.b (microatoms carbon)

19.9 34.2 26.8 8.5 10.0

111 93 87 23 7.0

16.5 17.0 23.7 17.0

43.8 38.3 35 25.5

Respiratory CO,

R.S.4.a

Tissue Sormal, rat:

Kidney Brain (homogenate) Heart

Liver Skeletal niusrle (homogenate) Neoplastic: Hepatoma (mouse) Hepatoma (rat) Rhahdornyosarcoma (mouse) Maminary adrnocarciuorns (~iiouse)

0 The relative sperific artivity is the ratio of activity of COz to substrate ( X 100) and represents the relative nmount of substrate carbon in the cnrhon of C o t . b 0.C. (Oxidative Capacity) represents the microatoms of substrate carbon oxidized to CO, per grain dry tissue per hour at 38'C. at a substrate concentration of 0.005 M.

muscle and liver. Thus, there is no suggestion from these data of any quaiititative impairment of glucose oxidation in the tumors studied. -4similar picture emerges from a comparison of the oxidative capacities given i n the last column (O.C.). This value is defined as the microatoms of labeled substrate carbon which have been converted to COZ per gram of tissue per hour. It is calculated from the relative specific activity and quantity of respiratory COT.This value applies only to the conditions of these experiments. I t may \*ary greatly, particularly with concentration of the substrate, but is of value for comparative purposes when conditions of experiments are similar. In this table me see that the oxidative capacity of the neoplastic tissues is within the range of values exhibited by the normal slices. Essentially the same conclusions were reached with respect to the

305

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

oxidation of lactic acid (Table XXI). Normal tissues showed a very wide range of oxidative capacity toward lactate, and the tumors studied gave values which fell within the normal range-lower than the most active, but higher than the less active, normal tissues. Here again, the data provide no confirmation of a respiratory disturbance in tumor cells which leads to quantitative impairment in the oxidation of lactic acid. These data are of limited value, since they were carried out with lactate labeled TABLE XXI Lactic Acid Oxidation by Normal and Tumor Tissues (82) (Substrate concentration, 0.005 M.)

0.c.a

Normal Tissue Rat liver Rat kidney Rat heart Rat muscle Rat brain Rat spleen Mouse liver Mouse kidney Mouse heart Mouse muscle a O.C. = oxidative capacity per hour.

44

152 148 3 112 130 32 440 108 5 =

Neoplastic Tissue Rat hepatoma Mouse hepatoma Mouse Andervont hepatoma Mouse Sarcoma 37 Mouse rhabdomyosarcoma Mouse mammary adenocarcinoma Ehrlich ascites

.

0.c.a 61 87 48 10.1 72 65 116

microatoms substrate carbon converted t o CO, per gram dry tissue

only in the carboxyl carbon and, therefore, give no indication of the rate of Oxidation of the a! and P carbons (the acetyl moiety) ; however, they supplement and extend the studies of Olson (80), which were carried out with other types of labeled lactate and pyruvate but which were limited to a comparison of liver with primary hepatoma. 6. Oxidation of Fatty Acids in Neoplastic Tissues

For want of any specific information to the contrary, it has been generally assumed that the major fuel for the tumor cell is glucose. We have already referred to the studies of Dickens and Simer (14) in which the low respiratory quotient of tumor slices was interpreted as indicative that fat may supply, a t least partially, the energy needs of the cancer cell. Direct studies of the capacity of neoplastic cells to oxidize fatty acids are relatively few. Kirsch (83) found that none of the fatty acids ranging from formate to octanoate increased the respiration of the Jensen sarcoma. Ciaranfi (84) attempted a manometric study of the effect of fatty acids on oxygen consumption by slices of a variety of human and animal tumors. None of the acids tested, ranging in chain length from 4

306

SIDNEY WEINHOUSE

to 18 carbons, gave any significant increase in oxygen upt,ake or the slightest indication of the presence of ketone bodies. The only effect observed was a slight increase displayed by the Ehrlich adenocarcinoma in the presence of /3-hydroxybutyric acid, in which case acetoacetic acid was detected as the oxidation product. I n a brief note (85) amplified in a more detailed study (86) the same investigator, however, reported th a t both normal and neoplastic tissues slices readily oxidized methyl esters of fatty acids ranging from one t o eight carbons. H e found also that various normal tissues oxidized the methyl esters a t considerably greater speed than they oxidized the sodium salts. Dickens and Weil-Malherbe (87) in a comparison of the metabolism of hepatomas with their tissue of origin found that the liver tumor arising in rats by butter yellow feeding largely lost the capacity for acetoacetate formation from caproic acid. A decrease was also noted for spontaneous mouse hepatoma, but in these experiments the tumors retained to a considerable degree their ketogenic ability. A study by Baker and Meister (88) revealed that homogenates of various mouse and rat hepatomas largely lost the capacity of the tissue of origin for oxidation of shortchain fatty acids. Oxygen consumption values in the presence of octanoic and hexanoic acids ranged from 0% t o 14% of the values obtained with normal liver. It appeared in this study that the neoplastic tissue contained some inhibitor of fatty acid oxidation, since the addition of the hepatoma homogenate decreased fatty acid oxidation in the liver homogenate. I n similar experiments with leukemic infiltrated liver homogenates, Vestling et al. (89) observed a marked diminution in octanoic acid oxidation, which was greater proportionately than the extent of leukemic infiltration. Though these data suggest th at fatty acid oxidation may be impaired in the neoplastic cell, they provide no basis for a categorical answer. ;\lore recently the application of the isotope tracer method has left no doubt that tumor cells can oxidize fatty acids, but the possibility still remains that there may be a quantitative diminution in fatty acid oxidation in neoplastic as compared with normal cells. Olson (80) found a somewhat lower capacity for oxidation of carboxyland methyl-labeled acetate by slices of primary rat hepatoma in comparison with normal tissue of origin; similar findings have been reported by Pardee, Heidelberger, and Potter (90). These investigators found th a t COOH-labeled acetate was oxidized much less rapidly by slices of Flexner-Jobling and Walker 236 tumors than by kidney, liver, or lung slices. A more extensive study of the oxidation of C'J-labeled fatty acids by tumor slices was carried out in the author's laboratory (82). As shown i n Table S X I I , C'3-carboxyl-labeled palmitic acid is oxidized about as readily by slices of four tumor types as by slices of liver and kidney. A

307

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

TABLE XXII Oxidation of 1 4 - 1 4 Palmitate by Slices of Normal and Neoplastic Rat Tissues (91) (Substrate Concn. = 0.0005 M.)

o.c.5 Normal Rat Tissue Kidney Liver (fasted) Liver (fed) Liver of tumor-bearing animal Neoplastic Tissue Hepatoma (mouse) Hepatoma (rat) Mammary adenocarcinoma (mouse) Rhabdomyosarcoma (mouse)

39.8 18.6 10.0 8.7 20.3 13.6 11.0 16.5

O.C. represent8 the number of microatoma of substrate carbon oxidized to carbon dioxide per gram dry tiesue weight per hour.

further investigation of fatty acid oxidation in neoplastic tissue (91) was devoted t o the following questions: (1) Does the hepatoma, the neoplastic counterpart of liver, display the same specialized features of fatty acid metabolism characteristic of normal liver? (2) Does the hepatoma resemble other neoplastic tissue types in its fatty acid metabolism? (3) Does the liver of the tumor-bearing host show any deviations from the normal liver? TABLE XXIII Oxidation of Fatty Acids by Liver and Tumor Slices of the Mouse (91) Oxidative Capacity Acetic

Butyric

Octanoic

Palmitic

Normal liver

58

59

43

11

Host liver Hepatoma 98/15

49 19

83 42

104 45

17 11

Host liver Mammary adenocarcinoma

82 6

54 12

90 0.6

16 5

Host liver Sarcoma 37

40 6

54 10

63 0.2

13 9

Host liver Rhabdomyosarcoma

67 7

30 16

72 0.6

15 11

308

SIDNEY WEINHOUSE

To summarize the data shown in Table XXIII, oxidation of all four fatty acids is more rapid in the liver slices than in the tumor slices, the differences being more marked with the short-chain acids than with palmitate. I t appears that the apparently lower oxidative capacity of the TABLE XXIV Conversion of 1-C-14 Butyrate to Acetoacetate by Liver and Hepatoma Slices (91) .4cetoacetate Kesp. COZ C.C.b pstoms

Total C.C. patoms

n’ornial liver

59.5

122

Host liver Hepat oma

82.8 42.0

87.5

Host liver 1Iammary adenorarcirioma

53.5 12 0

125 3.1

Host liver Sarcoma 37

53.8 9.8

Tumor lix-er Rhabdomyovnrcoma

4.4

B C.C. patorns

COOH

56

66

36 -0.4

C.C.

patoms

52 3.9

55 -1

70 -2

1.2

-0

-1

30.2 15.8

90.3 6.1

46 -1

44 -5

Host liver, rat Hepatoma, rata

159 19.3

152 4.5

58 -1

94 N4

Host liver. rat Hepatoma, rat0

190 180

47 -2

117

164 -5

-3

Pool of unlabeled acetoacetate added. = conversion capacity, represents the number of microatoms of substrate carbon converted t o the product in question per gram dry tissue per hour. 0

* C.C.

tumor tissues for the short-chain acids is due less t o ail inability t o oxidize these substances than t o powerful inhibitory effects exerted by the fatty acids on the tumor cells. When octanoate was used as a substrate in lower concentration than the levels used in the experiments shown in Table XXIII, its oxidation by various tumor slices was of the same magnitude as was displayed by liver slices. Thus, although a case could be made for a lowered fatty acid oxidation in tumor slices as compared with liver, 011 the whole it seems fair t o conclude that by and large fatty acid oxidation is not markedly impaired in neoplastic cells. It also seems

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

309

fair to state, on the basis of rather scant evidence, it is true, that the liver of the tumor-bearing animal has no impairment in its pattern of fatty acid metabolism. Some interesting differences were disclosed, however, in ketogenesis and in acetoacetate oxidation. Inasmuch as the liver has a much greater ketogenic capacity than nonhepatic tissues, it was of interest t o test whether the hepatoma would resemble liver or nonhepatic tissue in this respect. I n contrast with liver slices ketogenesis was very low in neoplastic tissue slices. Even with butyrate, the most ketogenic of the fatty acids, as the substrate (Table XXIV), none of the neoplastic slices, including the hepatoma, showed a degree of ketogenesis approaching that of liver slices. The possibility was considered that acetoacetate was being produced by tumor cells but was being rapidly metabolized. To check on this possibility a “trapping” procedure was employed in which the isotopic butyrate was oxidized in the presence of a pool of unlabeled acetoacetate. Under these conditions i t was expected that any labeled metabolic acetoacetate would mix with the unlabeled material and would be detected by radioactivity of the recovered acetoacetate. As shown in the last two experiments of Table XXIV, no appreciable activity was trapped by this procedure, and, therefore, the conclusion seems warranted that ketogenesis is of low or questionable occurrence in these tumor cells. Lack of ketogenesis in the hepatoma is of particular interest in that it represents a marked deviation from the metabolism of the normal liver cell.

Y . Oxidation of Acetoacetate in Liver and Tumor Slices Since tumors, including the hepatoma, appeared to display a nonhepatic metabolic pattern with respect to ketogenesis, it was of further interest to compare the rates a t which these tissues oxidize acetoacetate. The results, as shown in Table XXV, leave no doubt that tumor slices can oxidize acetoacetate. A considerable range of variation in the ability t o oxidize acetoacetate was displayed by liver slices, the C.C. values ranging from 6.7 to 26.8. I n every experiment except one, however, higher values than these were observed for the tumor slices. This is particularly significant, since in every instance the over-all respiratory rate, as shown in columns 2 and 3, was considerably lower in the tumor than in the liver slices. This may be more clearly seen in a comparison of the respective R.S.A.’s (column 4). The respiratory COa samples from the tumor slices contained much more acetoacetate carbon than did the COZ derived from the liver slices. Here again, tumor cells, including the hepatoma, displayed a metabolic pattern which resembles that of nonhepatic rather than liver cells.

310

SIDKEY WEINHOUSE

The results of this investigation leave no doubt that neoplastic cells can carry out the oxidation of fatty acids t o COZ, sharing this property with hepatic and nonhepatic normal cells. Though the results with shortchain acids provide some justification for the belief that oxidation of fatty acids may not proceed as readily in tumor cells, such a conclusion is probably unwarranted in view of various uncertainties of the in vitro TABLE XXV Oxidation of 2.4-C-11 Acetoacetate by Liver and Yeoplastic Tissues (01 ) (Substrate concentration = 0.002 M.)

O2 uptake Amt.

Respiratory COz

(pmoles)

Amt. (crmoles)

R.S.A. (per cent)

C.C. (patoms)

Host liver Hepatonla

455 36-1

371 310

7.2 16.2

26.8 55.1

Host liver Mnmmary adenocarcinoma

482 136

319 155

5.7 13.0

18.1 20.2

Host liver Sarcoma 37

392 217

312 216

5.6 1.5.2

17.5 32.8

Host liver Rhahdom yosarcoma

382 219

268 I70

3.3 12.0

8.8 20.2

Host liver Hepatoma, rat

3-19 132

287 161

6.3 7.8

18.2 12.6

Host liver. rat Hepatoma, rat

272 142

310 165

2.2 8.1

6.7 13.3

Normal mousr lirrr

356

26 1

7.8

20.4

experiments. It is noteworthy th at with respect t o acetoacetate formation and utilization the neoplastic transformation of the liver cell is associated with a metabolic transformation to a pattern characteristic of nonhepatic tissue. T he loss in the ability of the neoplastic liver cell t o produce acetoacetate from fatty acids is in line with many examples of the loss of specific metabolic function when normal cells become neoplastic (9). It is of particular interest, in this connection that the neoplastic transformation of the liver cell is associated also with an enhancement in a metabolic function, namely, acetoacetate oxidation-an observation which merits further exploration.

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

311

8. The Citric Acid Cycle

When the studies of Krebs and others led to the formulation of the citric acid cycle as the mechanism by which pyruvic acid is completely oxidized t o COZ, it was natural to assume that the alleged defect in respiration of tumor cells might have its origin in their inability to carry out certain reactions of the citric acid cycle. An observation which pointed in this direction was made by Potter et al. (92). These investigators found that whole homogenates of tumor tissues failed to carry out the oxidation of oxalacetate under conditions which resulted in its rapid oxidation by homogenate‘s of normal tissues. Since oxidation processes in tissue homogenates require the maintenance of a high level of organic phosphate, and since tumors have been found to contain very active dephosphorylating enzymes, the possibility was considered that the inactivity of oxalacetate in tumor homogenates may have been due t o inadequacy of oxidative reactions t o maintain the organic phosphate level. I n a direct test of this assumption Potter and LePage (93) found that there was no significant disappearance of oxalacetate in a Flexner-Jobling tumor homogenate, in which the ATP level was being maintained by glycolysis. This observation led them to conclude that a “bottleneck” existed somewhere on the pat,h of oxalacetate oxidation, presumably the introductory step of the citric acid cycle. The interesting hypothesis was advanced that oxalacetate, blocked in its oxidative path via the citric acid cycIe, may foIlow alternate pathways leading t o synthesis of building blocks for cell components, such as pyrimidines. Another observation was made by Potter and Busch (94)) also pointing to a defect of the citric acid cycle in tumors. Buffa and Peters found that when fluoracetate was given to rats in toxic doses, citric acid accumulated in heart, kidney, and brain. It has already been amply demonstrated that fluoracetate inhibits certain of the reactions of the citric acid cycle (now known t o be a t the aconitase stage (95)). The accumulation of citrate thus results whenever its rate of formation exceeds its rate of disappearance. Utilizing this observation as a means of testing for citric acid formation in tissues, Potter and Busch (94) injected fluoracetate into tumor-bearing rats and determined the citric acid content of the tissues one hour later. From the results shown in Table XXVI most tissues responded with a large increase in citrate content, the notable exceptions being liver, testis, and tumor. Since liver is known to display high citric acid cycle activity, its failure to accumulate this acid was explained on the basis of an alternate metabolic pathway, namely, acetoacetate formation. The lack of citrate accumulation in the tumors was

312

SIDNEY WEINHOUSE

regarded as “strong evidence th at the ability of tumor tissues to form citrate is very low in comparison with a variety of normal tissues.” Recognizing the possibility that the fluoracetate technique may be subject to uncertainty in interpretation, and taking into consideration other evidence t o be presented later for the occurrence of the citric acid cycle in tumors, Busch and Potter (96) developed another technique for TABLE XXVI Meet of Fluoracetate on the Citrate Content of Korinal Tissues in Normal and Tumor-Bearing Rats (94) (Values are presentcd in micrograms per gram wet weight of tissues.) Lninjected .4nimals

Tissue Brain Heart Lung Thymus Liver Kidney Spleen Testis Blood 31llscle

Panweas Walker 256 Tumor Flexrier-Jobling Jenseri Hepatoma, primary

so. of Samples Average 5 5 5 5 5 5 5 5 5 4 3 4 4 4 2

57 49 i5 55 4i 56 59

73 54 31 53 49 121 85 95

Range 3tk 65 24- 73 40-114 24- 79 20- 86 16-123 28- 87 58-1 13 35-114 25- 38 29- 78 38- 6 1 92-144 53-141 89-110

Tumor-Bearing Animals 1 Hour after Injection s o . of Samples Average

5 5 5 5 5 5 5 4 5 2

1GO 448 206 275 39 714 514 79 79 42

4 4 3 2

42

90 6G 60

Range

234

134289133230-

638 295 337

6-

77

445-1039 306- 754 49- 119 48- 102 41- 42 31- 52 61- 119 40- 116 52- 67

testing the occurrcnce of the citric acid cycle in viuo. This method consisted in injecting malonate into tumor-bearing rats. Since malonate inhibits succinic dehydrogenase, its presence in tissues should lead to the accumulation of succinate in those tissues in which succinat,e is a metabolite (i.e., those which carry on the reactions of the citric acid cycle). With the use of column chromatography for isolation of citric acid cycle components, the succinate contents of the tissues of malonate-poisoned rats were determined. Despite a wide range of levels, all tissues except brain, muscle, and blood accumulated succinate, including five tumors. The low content in the blood discounts the possibility of a migration from one tissue t o another and thus provides support for the idea that suc-

313

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

cinate represents an intermediary metabolite in those tissues in which it accumulates. The authors caution against the obvious conclusion that these data can be interpreted only in favor of the occurrence of the citric acid cycle. In discussing the uncertainties of in vivo techniques they point out that succinate may arise from sources other than citrate, for example, from glutamate and related compounds. 9. Isotopic Studies When isotopic studies conducted by the author and his colleagues (82) demonstrated the complete oxidation of glucose and fatty acids by tumor slices, it was of interest to ascertain whether citric acid was an intermediate. This was successfully shown by a “trapping” procedure. Slices of tissue were incubated with labeled substrates, under conditions which result in their oxidation to COO, together with a “pool” of unlabeled citrate. It was expected that any metabolic citrate would mix TABLE XXVII Radioactive Citrate from Tumor Oxidations (82) ~~

Tissue

Substrate

Normal, mouse: Heart Liver Kidney

Glucose Glucose Glucose

Neoplastic, mouse: Hepatoma Mammary tumor Rhabdomyosarcoma Ehrlich ascites Mammary tumor Mammary tumor Hepatoma Rhabdomyosarcoma

Glucose Glucose Glucose Glucose Acetate Palmitate Palmitate Palmitate

Specific Activity (counts/min.) 5.5 5.5 5.5

x x x

Quinidine Citrate Specific Activity (counts/min.)

105 105 10‘

150 126 2480

1.37 X 106 1 . 3 7 X 108 5 . 5 x 106 5 . 5 x 106 1 . 4 X 10’ 66,200 66,200 66,200

1225 1750 1000 910 293 74 138 83

with the large quantity of added citrate and its further oxidation be prevented thereby. By isolating and assaying the citric acid remaining at the close of the experiment, it was found that appreciable activity had been converted to citric acid. From Table XXVII it is seen that glucose, acetate, and palmitate all yielded citkate. Though the data are

314

SIDNEY WEINHOUSE

regarded as only qualitative, giving no indication whether citrate is a major or minor metabolite, there is also, by the same token, no indication from these radioactivity data th at citrate is less important quantitatively as an intermediate in the tumor cells than in the normal ones. Further evidence for the intermediary formation of citric acid was obtained by a study of the effect of trans-aconitate on the respiration of tumor slices (82). Saffran and Prado (97) showed that trans-aconitate is an inhibitor of aconitase; when this acid is added to tissue slices, respiration is impaired and citrate accumulates. Similar treatment of tumor slices also resulted in lowering of respiration and accumulation of citrate. hgain, these results must be regarded as primarily qualitative, demonstrating only th at citric acid is an intermediary metabolite in tumor cells, without indicating the magnitude of its formation. 10. The “Condensing” Enzyme

One of the most decisive points i n favor of the operation of the citric acid cycle is the presence in tumors of the “condensing” enzyme which catalyzes the introductory step of the cycle by bringing about the condensation of acetyl coenzyme A with oxalacetate to yield citrate. Wenner, Spirtes, and Weinhouse (64) found activity of this enzyme in several transplanted tumors t o be of the same order as in normal mouse liver. Further evidence for the operation of the citric acid cycle in neoplastic cells has been advanced by Kit and Greenberg (98). I n a study of the uptake of labeled amino acids by the Gardner lymphosarcoma, these investigators showed that inhibitors of the cycle, i.e., fluoracetate, arsenite, and malonate inhibited the respiration of the tumor and also the uptake of glycine into the cell proteins. They also showed that suspensions of the malignant cells can produce citrate from oxalacetate and acetate. I n a subsequent paper these authors (99) demonstrated a rapid incorporation of radioactivity from lactate-2-C14 into aspartic and glutamic acids-a result which is most easily interpreted on the basis of its conversion t o the corresponding keto acids via the cycle. I t is possible that further careful studies of individual enzyme activities may reveal differences between normal and neoplastic tissues; a t present, however, there is no reason t o assume on direct experimental grounds t ha t there is an impaired or decreased occurrence of the citric acid cycle in tumors; nor is there any evidence which might be interpreted as indicating the presence of a respiratory pathway other than that of the citric acid cycle. The results of Potter and Busch (94) still remain unexplained; however, they can no longer be regarded as indicative of impairment in the citric acid cycle in the face of the considerable body of information in vitro in favor of its occurrence in tumor cells.

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

VII.

OXIDATION IN

315

TUMOR HOMOGENATES

1 . Citric Acid Cycle Intermediates

We have already referred to the failure of tumor homogenates to carry out the oxidation of citric acid cycle components under conditions which lead to their oxidation by normal. tissues (92). When the isotope tracer studies revealed the ability of intact tumor cells to oxidize fatty acids and glucose and demonstrated the participation of the citric acid cycle therein, it was obvious that some factor essential to oxidation was being destroyed in homogenizing the tumor tissue. In an investigation of this matter it was found (100, 101) that good oxygen consumption TABLE XXVIII Oxygen Uptake in Tumor Homogenates Stimulated by DPN+ (100) (The medium contained the following in a total volume of 1.6 ml.: tumor homogenate, equivalent to 50 mg. of dry tissue; phosphate, pH 7.4, 0.016 M ; ATP, 0.002 M ; cytochrome c, 0.1 mg.; MgSOa, 0.003 M ; yellow enzyme, 0.1 ml.; and where specified, either DPN, 0.0015 M , or oxalacetate (OAA), 0.005 M , or both. Experiments run 1 hour at 38°C.) Oxygen Consumption (micro liters) Additions None DPN OAA DPN OAA 45 152 28 252 40 133 44 118 33 142 48 136 63 168 66 167

+

Hepatoma (mouse) Hepatoma (rat) Mammary tumor Rhabdomyosarcoma

(of the same order exhibited by slices) could be obtained if whole homogenates were fortified by addition of DPN+ in rather high concentration. As seen in Table XXVIII, DPN+ greatly stimulated the oxygen consumption of whole homogenates of four tumors. In fact, so great was the stimulation of endogenous respiration that further addition of oxalacetate did not always lead to increased oxygen consumption. Though demonstrating the ability of DPN+ to stimulate respiration in tumor homogenates, these results left undecided the question whether added substrates underwent oxidation. However, the use of washed tissue residues or mitochondria derived therefrom by differential centrifugation soon demonstrated unequivocally the ability of DPN+-fortified tumor tissues to oxidize pyruvic acid and all members of the citric acid cycle. A fractionation study, shown in Table XXIX, showed that of the four cellular fractions of hepatoma 98/15, obtained by differential centrifugation according to the procedure of Schneider (102), the mitochondria had a specific activity so much higher than the other fractions that there seemed to be

316

SIDNEY WEINHOUSE

no doubt that the enzyme systems responsible for the oxidation of pyruvic acid are principally or exclusively localized in this fraction. The low activities observed in the other fractions are probably due to incomplete separation of mitochondria. As seen in this table, the mitochondria contained 13% of the tissue nitrogen, but were responsible for 55% of the total oxygen consumption. The relative effectiveness of the four fractions is best seen in the last column, in which oxygen consumption is given per milligram nitrogen; the mitochondria consume 7.5 times more oxygen per milligram nitrogen than the next higher (the nuclear) fraction. In localization of oxidative enzymes, tumors thus display the same behavior as normal tissues (103-107). TABLE XXIX Pyruvate Oxidation in Cellular Constituents of Mouse Hepatoma 98/15 (101) Oxygen Consumption

Tissue Fractionsa

02,p1./100 mg. tissue 0 2 , pl./mg. N Per Cent Additions Additions of Whole DPN DPN Total Sb IIomog. S o n e D P N Pyruvate None D P N Pyruvate

+

+

~

Homogenate Nuclei Mitochondria hlicrosomes Supernatant a b

2 0 0 0 1

08 508 275 222 150

100 24 4 13 2 10 7 55 3

46 2 6 2 3 1 6 5 8

195 14

14 4 5 41

164 14 58 2 1 31

19 8 5 1 8 4 7 2 5 0

84 28 52 20 36

72 28 211 9 5 27

Notation IS that of Schneider (61) Mg. mtrogen per 100 mg. fresh tissue.

Since the reactions of the citric acid cycle are localized in the mitochondria, it appears probable that many of the quantitative differences in oxidative activity between different tissues can be referred to differences in their content of mitochondria. Schneider and Hogeboom (108) have discussed evidence, based on nitrogen content, which indicates that the mitochondria1 content of rat and mouse hepatomas may be lower than that of normal liver. In an attempt to establish relations between mitochondrial content and oxidative activity, a study was made of pyruvate oxidation by isolated mitochondria of various normal and neoplastic cells. The oxygen consumed per hour by mitochondria representing 1 g. of fresh tissue was calculated and compared with the amount of mitochondrial nitrogen recovered per gram of tissue (101). Though the correlation was not perfect, on the whole there was a fair agreement between the oxygen consumed and the amount of nitrogen present in the mitochondria. Because of difficulties in obtaining pure fractions of cell com-

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

317

ponents and in view of our lack of knowledge of the nitrogen content of mitochondria, these data can be regarded as only extremely rough approximations of the mitochondria1 activity of cells. However, the data seem t o justify the belief that mitochondria from both normal and neoplastic cells have about the same capacity to oxidize pyruvic acid. They also suggest that previous reports of low succinic dehydrogenase and cytochrome oxidase activity as well as low oxidative activity of certain tumors may be due in part at least to a low content of mitochondria. A broad survey of the relative abilities of normal and neoplastic mitochondria to carry out oxidations of citric acid cycle components is displayed in Table XXX. The oxidation of all members of the cycle was observed in the mitochondria of the four tumors studied, and succinate was the only substrate whose oxidation did not display stimulation by added DPN. Of the normal tissues, brain was the only one which exhibited a pronounced requirement for DPN addition; however, in other normal mitochondria DPN usually increased oxygen consumption somewhat, this being due to better maintenance of oxidation rates rather than to initial stimulation.

2. The DPN+ Efect Though other factors doubtless play a part in the oxidative reactions of mitochondria, the studies of the present author suggest that the principal cause of the failure of citric acid cycle oxidations in. tumor homogenates is a lack of DPN+. In our hands DPN+ was the only factor whose absence yielded negligible oxygen uptake values for homogenates or mitochondria. The effect was consistent and reproducible; and in the presence of optimal concentrations of DPN+ and substrate, oxidation in mitochondria proceeded for a t least 2 hours with undiminished rate (100). Further support for the necessity of DPN in oxidation processes by tumor homogenates has been advanced from phosphorylation studies of R. Kielley (log), who showed that phosphorylation associated with oxidation of succinate, a-ketoglutarate, and glutamate proceeded about as readily in mitochondria of the mouse hepatoma 98/15 as in liver mitochondria, but the former tissue displayed a pronounced DPN requirement for phosphorylation associated with oxidation of those substrates, namely, a-ketoglutarate and glutamate, whose oxidation requires this factor as a coenzyme. Williams-Ashman, Kennedy, and Lehninger (110-112) likewise have shown that DPN+ exerts a pronounced effect on glutamate oxidation by washed tumor particles. The observation of the DPN+ dependence of tumor mitochondria has emerged as an interesting by-product of these studies of oxidation processes, the significance of which has yet to be evaluated; however, it is

TABLE X X S DPN Requirement for Oxidation of Krcbs Cycle Components (101) (Values are in microliters oxygen consumed per milligram mitochondria nitrogen per 30 minutes.) Normal Tissue Mitochondria Mouse Liver xa % 0 Q % I

W

& Substrate

I

None 37 Pyruvate 150 Citrate 176 a-Ketoglutaratc 142 Succinate 145 Fumarate 130 Malate

+

Rat Liver Z

a

Mouse Kidney

E

E

a

Tumor Mitochondria

n

z 8

E

+

Rat Brain

I

+

49 145 215

43 139 146

45 154 153

53 279 299

91 249 296

40 38 22

180 165 156

118 147 106 160

154 128 154 164

212 230 95

335 284 220

43 114 35 69

Q

Q

I

I

za

a

+ 50

146 116 116 104 101

127

Hepatoma 7A77

z a

Z

Hepatoma 98/15

F

a

8

+

E

15 30 14

44 250 153

19 17

100 125 15 33

214 116 143 176

I

I

82 48

Z a

a

+

36 196 153 119 79 98 82

Sarcoma 37

% a

a I

28 51

170

Z

S

+

45 150 68

Rhabdomyosarcoma

z8

E0 +

I

16 21

36 105 137

4

99 204

170

85

172 118

136

56

99

OXIDATIVE METABOLISM OF NEOPLASTIC TISSUES

319

quite evident that it represents a quantitative rather than qualitative point of departure from the normzl cell. Though freshly prepared mitochondria from liver and kidney can carry out the oxidation of fatty acids and citric acid cycle components without the necessity of additional DPNf, activating effects of added DPN+ on oxidations by normal tissue preparations have been reported. Potter et al. (113) and Kennedy and Lehninger (114) have reported that DPN+ can partially replace the adenine nucleotide requirement for mitochondria1 oxidations. Moore and Nelson (115) have found that washed residue of lactating mammary gland of the guinea pig requires DPN+ for oxidation of malate and TPN+ for oxidation of citrate. Plaut and Plaut (116) noted that DPN+ was required for activation of citrate oxidation by guinea pig heart mitochondria. Even under circumstances which ordinarily do not require DPN+ addition, a requirement for this nucleotide may be induced by relatively mild physical or chemical treatment. Lehninger (117) has shown that sedimentable particles of rat liver lose their activity on “aging” for a short period a t low temperature but can be reactivated by addition of DPN+; similar findings were made by Pardee and Potter (118), who also observed that loss in oxidative activity of kidney homogenates was paralleled by a loss in particle-bound DPN+. Freezing or thawing of the particles or treatment with distilled water sufficed to cause a DPN+ requirement for oxidation by the “ cyclophorase system’’ of rabbit liver and kidney (119, 120). These investigators concluded that the pyridinoproteins occur in the mitochondria tightly conjugated to the apoenzymes, and the various treatments effectively dissociated the nucleotide. On this basis it appears that in the tumor mitochondria the homogenization process itself is sufficient to lower the DPN concentration below the effective level. Four reasons may be cited to explain the more pronounced DPN+ requirements of tumor mitochondria: (1) The DPN+ content of the intact tumor cell may be so low that the loss and dilution which occurs on homogenization may lower its level below that required for oxidative activity. We have already discussed evidence in this direction, and some preliminary data of Dr. Jedeikin in our own laboratory have confirmed these findings. (2) The nucleotide may be bound less tightly by the apoenzymes of tumor mitochondria, so that it leaches away and is highly diluted by the suspending medium. Though this seems improbable, it is conceivable that tumor mitochondria may have different proportions of oxidized and reduced DPN from those of normal tissues. Since the two forms of the nucleotide should have different affinities for the apoenzymes, it is reasonable to suppose that such differences might lead to lowering of the DPN content of the mitochondria. (3) The DPNase activity of tumor tissues may be higheithan that of other cell

320

SIDNEY WEINHOUSE

types. Though this point requires further study, the only direct information does not support this postulate. Quastel and Zatman (121) have shown the presence of a nicotinamide-sensitive DPNase in various human and animal tumors. However, no clear-cut differences in activity were observed between neoplastic and nonneoplastic tissues. In contrast with the usual pattern of enzyme activities displayed by tumor tissue, a much wider range of variation in DPXase activity was found in neoplastic than in nonneoplastic tissue. (4) The membrane of tumor mitochondria may be excessively permeable to DPN+, or the tumor mitochondria may be so fragile that slight changes in osmolarity may produce sufficient injury to cause the loss of DPN+ without otherwise impairing their oxidative activity. None of these possibilities is yet on a firm basis, and a choice as to the reasons for the DPK requirement must await further investigation. 3. E$ects of Phosphorylation and Dephosphorylation on Oxidation in

Tumor Homogenates Potter and his colleagues have advanced evidence in favor of the reasonable hypothesis that oxidations in homogenates or mitochondria may be controlled by the balance between rates of phosphorylation and dephosphorylation. Observing a high ATP breakdown in homogenates of several rat tumors, Potter and Lyle (122) found that such homogenates, which were oxidatively inert, could be made by the addition of fluoride to consume oxygen for 20 t o 40 minutes at rates comparable with those for slices. The action of this inhibitor was attributed t o its well-known inhibitory action on dephosphorylation processes. Siekevitz, Simonson, and Potter (123) extended this concept by showing that homogenates of different tumors may display considerable variation in rates of organic phosphate breakdown and synthesis. Good oxidative activities toward pyruvate and fumarate were observed when these opposing effects were balanced by the addition either of 2,4-dinitrophenol, which inhibits phosphate esterification, or fluoride, which inhibits phosphate ester breakdown. However, Siekevitz and Potter (124) in a more detailed test of this hypothesis were unable to obtain clear-cut opposing effects of dinitrophenol and fluoride on oxidations in a variety of tumor homogenates or mitochondria. The Flexner-Jobling carcinoma homogenate was the only one whose oxygen consumption was markedly stimulated by fluoride and inhibited by DNP. The others exhibited a variety of behaviors; in some, both stimulated or both depressed; in others, no particular effects were observed. Wenner et al. (125) were likewise unable t o obtain any clear-cut differences in glucose oxidation by whole homogenates of a variety of normal and neoplastic tissues. In slices of both tissue

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

32 1

types D N P stimulated glucose oxidation a t low concentrations and inhibited a t higher concentrations. In whole homogenates of all tissues except brain D N P inhibited glucose oxidation a t all concentrations tested. Fluoride was found t o be a powerful inhibitor of glucose oxidation in all tissues tested. Despite the plausibility of the concept, there is no clear indication from these studies of any major difference in phosphorus metabolism between normal and neoplastic cells; nor is there anything in the metabolism of tumor cells which could conceivably be attributable t o such differences. The occurrence of oxidative phosphorylation (109112, 124) by mechanisms which are indistinguishable, by the techniques employed, from those in normal tissues, clearly demonstrates that tumor tissues can obtain energy through phosphorylation associated with oxidations in the citric acid cycle. This again emphasizes the fundamental similarity of oxidative mechanisms in both tissue types.

4. Possible Causes of High Glycolysis in Tumors It is evident from the foregoing discussions that there is little in favor of the Warburg hypothesis. The high glycolysis of tumor tissue, whatever its cause, does not appear to be due t o a radically altered res'piratory metabolism-whether of electrons or carbon. It is possible, of course, that certain tumors, low in certain enzymes or cofactors of respiration, may have a " bottleneck" in electron transport which might conceivably tend t o raise the level of lactic acid accumulation. We have already seen that many are low in pyridine nucleotides and cytochromes. On the other hand, we have no idea as yet what constitutes an optimal content of enzymes or coenzymes for the proper functioning of an intact cell. There is no good basis for the assumption that a tissue containing a small amount of a coenzyme or exhibiting a low assay value for a particular enzyme has a necessarily impaired metabolism. At any rate, it is difficult t o see how any such effects can play an important part in the high glycolysis of the large bulk of tumors of the most varied origins, which have a moderate to high rate of oxygen consumption and hence have no apparent difficulty in transferring electrons. The opposite suggestion has been offered on occasion that the high glycolysis might be due t o excessive quantities of a rate-controlling enzyme in tumor tissues. This theory has no experimental support and is rendered unlikely in the light of LePage's studies showing that glycolysis was about as rapid in homogenates of normal tissues as in the FlexnerJobling carcinoma, when these were adequately fortified with cofactors and substrates. It seems t o the author that a proper understanding of the high glycolysis of neoplastic tissue will require a knowledge of the factors

322

SIDNEY WEINHOUSE

which regulate and control cellular metabolism. Unfortunately, we have little knowledge of any sort as yet concerning how metabolic processes are regulated in cells. It is assumed th at many aspects of carbohydrate and lipid metabolism are controlled or directed by various hormonesparticularly those of the pancreas, pituitary, thyroid, and adrenal glands. Th a t these substances may not exert the same effects on metabolism in tumors as they do in the nonneoplastic tissues of the host may be considered an intriguing possibility. Unfortunately, we are faced with the fact tha t we still have no idea how any single hormone affects a particular enzymatic reaction. Until we learn more concerning the metabolic sites of action of hormones, it is inipossible t o do more than speculate concerning their possible regulatory role in cells. A plausible means of metabolic regulation in cells involves the availability of cofactors. It is now well recognized th a t certain metabolic processes are localized within the cell. However, though geographically isolated, they are interconnected by common cofactors. The process of glycolysis, for example, occurring in the soluble portion of the cell cytoplasm, is dependent on A T P and adenosine diphosphate (ADP), which are in turn formed and utilized during oxidative reactions in the mitochondria. The high energy of the pyrophosphate bonds of A T P is also utilized in synthetic processes occurring in nuclei and microsomes. Hence, there must be a constant circulatioii of A T P and its breakdown products t o and from all of the various components of the cell. Similarly, D P S + , which is required in the oxidative and reductive steps of glycolysis occurring in the soluble part of the cytoplasm, is produced in the nucleus, and is required in a multitude of oxidation-reduction reactions taking place in the mitochondria. Hence, there must also be a constant flow of this nucleotide among the cell components. The same situation must exist for many other organic cofactors as well as for a variety of inorganic ions. If i t is recognized th at each of these has a profound effect on more than one and perhaps many different enzymes, it is not difficult to see how their distribution may exert sensitive control over the metabolic behavior of the cell. The fact th at no less than five such substances, viz., diphosphothiamine, a-lipoic acid, coenzyme A, DPN+, and magnesium, are concerned in the oxidative decarboxylation of pyruvate t o acetyl C o h , a process which is simultaneous with or possibly even competitive with the reduction of pyruvic t o lactic acid, emphasizes the intimate relationships between glycolysis and the availability of cofactors. It seems safe t o assume that when we have a proper understanding of the complex interplay between catabolic and synthetic reaction mechanisms, and their integration, we shall have gone far in disclosing the secrets of the neoplastic process.

OXIDATIVE METABOLISM O F NEOPLASTIC TISSUES

323

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