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Dr. Warburg

Dr. Otto Warburg, "On The Origin of Cancer Cells," SCIENCE, (24FEB1956) Volume 123, Number 3191, pp. 309-314.

Professor Warburg is director of the Max Planck Institute for Cell  Physiology, Berlin-Dahlem, Germany.  This article is based on a lecture delivered at Stuttgart on 25 May 1955 before the German Central Committee for Cancer Control.  It was first published in German [Naturwissenschaften 42, 401 (1955)].  This translation was prepared by Dean Burk, Jehu Hunter, and W. H. Everhardy of the U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Md., with permission of Naturwissenschaften and with collaboration of Professor Warburg, who has introduced additional material.

Our principal experimental object for the measurement of the metabolism of cancer cells is today no longer the tumor but the ascites cancer cells (1) living free in the abdominal cavity, which are almost pure cultures of cancer cells with which one can work quantitatively as in chemical analysis.    Formerly, it could be said of tumors, with their varying cancer cell content, that they ferment more strongly the more cancer cells they contain, but today we can determine the absolute fermentation values of the cancer cells and find such high values that we come very close to the fermentation values of wildly proliferating Torula yeasts.

What was formerly only qualitative has now become quantitative. What was formerly only probably has now become certain. The era in which the fermentation of the cancer cells or its importance could be disputed is over, and no one today can doubt that we understand the origin of can cercells if we know how their large fermentation originates, or, to express it more fully, if we know how the damaged respiration and the excessive fermentation of the cancer cells originate.

Energy of Respiration and Fermentation

We now understand the chemical mechanism of respiration and fermentation almost completely, but we do not need this knowledge for what follows, since energy alone will be the center of our consideration. We need to know no more or respiration and fermentation here than that they are energy-producing reactions and that they synthesize the energy-richadenosine triphosphate through which the energy of respiration and fermentation is then made available for life. Since it is known how much adenosine triphosphate can be synthesized by respiration and how much by fermentation, we can write immediately the potential, biologically utilizable energy production of any cells if we have measured their respiration and fermentation. With the ascites cancer cells of the mouse, for example, we find an average respiration of 7 cubic millimeters of oxygen consumed per milligram, per hour, and fermentation of 60 cubic millimeters of lactic acid produced permilligram, per hour. This, converted to energy equivalents, means that the cancer cells can obtain approximately the same amount of energy from fermentation as from respiration, whereas the normal body cells obtain much more energy from respiration than from fermentation. For example, the liver and the kidney of an adult animal obtain about 100 times as much energy from respiration as from fermentation.

I shall not consider aerobic fermentation, which is a result of the interaction of respiration and fermentation, because aerobic fermentation is too labile and too dependent on external conditions. Of importance for the considerations that follow are only the two stable independent metabolic processes, respiration and anaerobic fermentation- respiration, which is measured by the oxygen consumption of cells that are saturated with oxygen, and fermentation, which is measured by the formation of lactic acid in the absence of oxygen.

Injuring of Respiration Since the respiration of all cancer cells is damaged, our firm questionis, How can the respiration of body cells be injured? Of this damage to respiration, it can be said at the outset that it must be irreversible, since the respiration of cancer cells can never returns to normal. Second, the injury to respiration must not be so great that the cells are killed, for then no cancer cells could result. If respiration is damaged when it forms too little adenosine triphosphate, it may be either that the oxygen consumption has been decreased or that, with undiminished oxygen consumption, the coupling between respiration and the formation of adenosine triphosphate has been broken, as was first pointed out by Feodor Lynen (2).

One method for the destruction of the respiration of body cells is removal of oxygen. If, for example, embryonal tissue is exposed to an oxygen deficiency for some hours and then is placed in oxygen again, 50 percent more or more of the respiration in destroyed. The cause of this destruction of respiration is lack of energy. As a matter of fact, the cells need their respiratory energy to preserve their structure, and if respiration is inhibited, both structure and respiration disappear.

Another method for destroying respiration is to use respiratory poisons.>From the standpoint of energy, this method comes to the same result asthe first method. No matter whether oxygen is withdrawn from the cell or whether the oxygen is prevented from reacting by a poison, the result is the same in both cases - namely, impairment of respiration from lackof energy.

I may mention a few respiratory poisons. A strong, specific respiratory poison is arsenious acid, which as every clinician knows, may produce cancer. Hydrogen sulfide and many of its derivatives are also strong, specific respiratory poisons. We know today that certain hydrogensulfide derivatives thiourea and thioacetamide, with which citrus fruit juices have been preserved in recent times, induce cancer of the liver and gall bladder in rats.

Urethane is a nonspecific respiratory poison. It inhibits respirationas a chemically indifferent narcotic, since it displaces metabolitesfrom cell structures. In recent years it has been recognized that subnarcotic does of urethane cause lung cancer in mice in 100 percent of treatments. Urethane is particularly suitable as a carcinogen, because in contrast to alcohol, it is not itself burned up on the respiring surfaces and, unlike ether or chloroform, it does not cytolize thecells. Any narcotic that has these properties may cause cancer upon chronic administration in small doses.

The first notable experimental induction of cancer by oxygen deficiency was described by Goldblatt and Cameron (3), who exposed heart fibroblasts in tissue culture to intermittent oxygen deficiency for long periods and finally obtained transplantable cancer cells, where as in control cultures that were maintained without oxygen deficiency, no cancer cells resulted. Clinical experiences along these lines are innumerable: the production of cancer by intermittent irritation of the outer skin and of the mucosa of internal organs, by the plugging of the excretory ducts of glands, by cirrhoses of tissues, and so forth. In all these cases, the intermittent irritations lead to intermittent circulatory disturbances. Probably chronic intermittent oxygen deficiency plays a greater role in the formation of cancer in the body than does the chronic administration of respiratory poisons.

Any respiratory injury due to lack of energy, however, whether it is produced by oxygen deficiency or by respiratory poisons, must be cumulative, since it is irreversible. Frequent small doses of respiratory poisons are therefore more dangerous than a single large dose, where there is always the chance that the cells will be killed rather than that they will become carcinogenic.

If an injury of respiration is to produce cancer, this injury must, as already mentioned, be irreversible. We understand by this not only that the inhibition of respiration remains after removal of the respiratory poison but, even more, that the inhibition of respiration also continues through all the following cell divisions, for measurements of metabolismin transplanted tumors have shown that cancer cells cannot regain normal respiration, even in the course of many decades, once they have lost it.

This originally mysterious phenomenon has been explained by a discovery that comes from the early years of cell physiology (4). When liver cells were cytolized by infusion of water and the cytolyzate was centrifuged, it was found that the greater part of the respiration sank to the bottom with the cell grana. It was also shown that the respiration of the centrifuged grana was inhibited by narcotics at concentrations affecting cell structures, from which it was concluded-already in 1914- that the respiring grana are not in soluble cell particles but autonomous organisms, a result that has been extended inrecent years by the English botanist Darlington (5) and particularly by Mark Woods and H.G. du Buy (6) of the National Cancer Institute in Bethesda, Md. Woods and du Buy have experimentally expanded our concepts concerning the self-perpetuating nature of mitochondrial elements (grana) and have demonstrated the hereditary role of extranuclear aberrant forms of these in the causation of neoplasia. The autonomy of the respiring grana, both biochemically and genetically, can hardly be doubted today.

If the principle Omne granum e grano is valid for respiring grana, we understand why the respiration connected with grana remains damaged when it has once been damaged; it is for the same reason that properties linked with genes remain damaged when the genes have been damaged.

Furthermore, the connection of respiration with the grana (7) also explains carcinogenesis that I have not mentioned previously, the carcinogenesis by x-rays. Rajewsky and Pauly have recently shown that the respiration linked with the grana can be destroyed with strong doses of x-rays, while the small part of the respiration that takes place in the fluid protoplasm can be inhibited very little by irradiation.Carcinogenesis by x-rays is obviously nothing else than destruction of respiration by elimination of the respiring grana.

It should also be mentioned here that grana, as Graffi has shown (8), fluoresce brightly if carcinogenic hydrocarbons are brought into their surroundings, because the grana accumulate the carcinogenic substances. Probably this accumulation is the explanation for the fact that carcinogenic hydrocarbons, although almost insoluble in water, can inhibit respiration and therefore have a carcinogenic effect.

Increase of Fermentation

When the respiration of body cells has been irreversibly damaged, cancer cells by no means immediately result. For cancer formation there is necessary not only an irreversible damaging of the respiration but also an increase in the fermentation -- indeed, such an increase of the fermentation that the failure of respiration in compensated for energetically. But how does this increase of fermentation come about?

The most important fact in this field is that there is no physical or chemical agent with which the fermentation of cells in the body can beincreased directly; for increasing fermentation, a long time and many cell divisions are always necessary. The temporal course of this increase of fermentation in carcinogenesis has been measured in many interesting works, among which I should like to make special mention of those of Dean Burk (9).

Burk first cut out part of the liver of healthy rats and investigated the metabolism of the liver cells in the course of ensuing regeneration, in which, as is well known, the liver grows more rapidly than a rapidly growing tumor. No increase of fermentation was found. Burk then fed rats for 200 days on butter yellow, where upon liver carcinomas were produced, and he found that the fermentation slowly increased in the course of 200 days toward values characteristic of tumors.

The mysterious latency period of the production of cancer is, therefore nothing more than the time in which the fermentation increases after adamaging of the respiration. This time differs in various animals; it is essentially long in man and here often amounts to several decades, ascan be determined by the cases in which the time of the respiratorydamage is known -- for example, in arsenic cancer and irradiation cancer.

The driving force of the increase of fermentation, however, is theenergy deficiency under which the cells operate after destruction oftheir respiration which forces the cells to replace the irretrievably lost respiration energy in some way. They are able to do this by a selective process that makes use of the fermentation of the normal body cells. The more weakly fermenting body cells perish, but the more strongly fermenting ones remain alive, and this selective process continues until respiratory failure is compensated for energetically by the increase in fermentation. Only then has a cancer cell resulted from the normal body cell.

Now we understand why the increase in fermentation takes such a long time and why it is possible only with the help of many cell divisions. We also understand why the latency period is different in rats and in man. Since the average fermentation of normal rat cells is much greater that the average fermentation of normal human cells, the selective process begins at a higher fermentation level in the rat and, hence is completed more quickly than it is in man.

It follows from this that there would be no cancers if there were no fermentation of normal body cells, and hence we should like to know, naturally, from where the fermentation of the normal body cells stemsand what its significance is in the body. Since, as Burk has shown, the fermentation remains almost zero in the regenerating liver growth, we must conclude that the fermentation of the body cells has nothing to do with normal growth. On the other hand, we have found tat the fermentation of the body cells is greatest in the very earliest stages of embryonal development and that it then decreases gradually in the course of embryonal development. Under these conditions, it is obvious--since ontogeny is the repetition of phylogeny-- that the fermentation of body cells is the inheritance of undifferentiated ancestors that have lived in the past at the expense of fermentation energy.

Structure and Energy

But why -- and this is our last question -- are the body cells differentiated when their respiration energy is replaced by fermentation energy? At first, one would think that it is immaterial to the cellswhether they obtain their energy from respiration or from fermentation, since the energy of both reactions is transformed into the energy of adenosine triphosphate, and yet adenosine triphosphate=adenosinetriphosphate. This equation is certainly correct chemically andenergetically, but it is incorrect morphologically, because, although respiration takes place for the most part in the structure of the grana, the fermentation enzymes are found for a greater part in the fluid protoplasm. The adenosine triphosphate synthesized by respiration therefore involves more structure than the adenosine triphosphate synthesized by fermentation. Thus, it is as if one reduced the same amount of silver on a photographic plate by the same amount of light, but in one case with diffused light and in the other with patterned light. In the first case, a diffuse blackening appears on the plate, but in the second case, a picture appears; however, the same thing happens chemically and energetically in both cases. Just as one type of light energy involves more structure than the other type, the adenosinetriphosphate energy involves more structure when it is formed by respiration than it does when it is formed by fermentation.

In any event, it is one of the fundamental facts of present-day biochemistry that adenosine triphosphate can be synthesized in homogeneous solutions with crystallized fermentation enzymes, whereas sofar no one has succeeded in synthesizing adenosine triphosphate in homogeneous solutions with dissolved respiratory enzymes, and the structure always goes with oxidative phosphorylation.

Moreover, it was known for a long time before the advent of crystallized fermentation enzymes and oxidative phosphorylation that fermentation--the energy supplying reaction of the lower organisms-- is morphologically inferior to respiration. Not even yeast, which is oneof the lowest forms of life, can maintain its structure permanently by fermentation alone; it degenerates to bizarre forms. However, as Pasteur showed, it is rejuvenated in a wonderful manner, if it comes incontact with oxygen for a short time. "I should not be surprised,"Pasteur said in 1876 (10) in the description of these experiments, if there should arise in the mind of an attentive hearer a presentiment about the causes of those great mysteries of life which we conceal under the words youth and age of cells." Today, after 80 years, the explanation is as follows: the firmer connection of respiration with structure and the looser connection of fermentation with structure.

This, therefore, is the physiochemical explanation of the dedifferentiation of cancer cells. If the structure of yeast cannot bemaintained by fermentation alone, one need not that highly differentiated body cells lose their differentiation upon continuous replacement of their respiration with fermentation.

I would like at this point to draw attention to a consequence of practical importance. When one irradiates a tissue that contains cancer cells as well as normal cells, the respiration of the cancer cells, already too small, will decline further. If the respiration falls below a certain minimum that the cells need unconditionally, despite their increased fermentation, they die; whereas the normal cells, where respiration may be harmed by the same amount, will survive because, with a greater initial respiration, they will still possess a higher residual respiration after irradiation. This explains the selective killing action of of x-rays on cancer cells. But still further: the descendants of the surviving normal cells may in the course of the latent period compensate the respiration decrease by the fermentation increase and, thence, become cancer cells. Thus it happens that radiation which kills cancer cells can also at the same time produce cancer or that urethane, which kills cancer cells, can also at the sametime produce cancer. Both events take place from harming respiration: the killing, by harming an already harmed respiration; the carcinogenesis by the harming of a not yet harmed respiration.

Maintenance Energy

When differentiation of the body cells has occurred and cancer cells have thereby developed, there appears a phenomenon to which our attention has been called by the special living conditions of ascites cancer cells. In extensively progressed ascites cancer cells of the mouse, the abdominal cavity contains so many cancer cells that the latter cannot utilize their full capacity to respire and ferment because of the lack of oxygen and sugar. Nevertheless, the cancer cells remain alive in the abdominal cavity, as the result of transplantation proves.

Recently, we have confirmed this result by direct experiments in which we placed varying amounts of energy at the disposal of the ascites outside the body, in vitro, and then transplanted it. This investigation showed that all cancer cells were killed when no energy at all was supplied for 24 hours at 38 degrees C but that one-fifth of the growth energy was sufficient to preserve the transplantability of the ascites. This result can also be expressed by saying that cancer cells require much less energy to keep them alive than they do for growth. In this they resemble other lower cells, such as yeast cells, which remain alive for a long time in densely packed packets -- almost without respiration and fermentation.

In any case, the ability of cancer cells to survive with little energy, if they are not growing, will be of great importance for the behaviourof the cancer cells in the body.

Sleeping Cancer Cells

Since the increase in fermentation in the development of cancer cells takes place gradually, there must be a transitional phase between normal body cells and fully formed cancer cells. Thus, for example, when fermentation has become so great that dedifferentiation has commenced, but not so great that the respiration defect has been fully compensated for energetically by fermentation, we may have cells which indeed look like cancer cells but are still energetically insufficient. Such cells,which are clinically not cancer cells, have lately been found, not only in the prostate, but also in the lungs, kidney, and stomach of elderlypersons. Such cells have been referred to as "sleeping cancer cells."(11,12)

The sleeping cancer cells will possibly play a role in chemotherapy.>From energy considerations, I could think that sleeping cancer cellscould be killed more easily than growing cancer cells in the body and that the most suitable test objects for finding effective agents would be the sleeping cells of the skin -- that is, precancerous skin.

Summary

Cancer cells originate from normal body cells in two phases. The first phases is the irreversible injuring of respiration. Just as there are many remote causes of plague --heat, insects, rats-- but only one common cause, the plague bacillus, there are a great many remote causes of cancer --tar, rays, arsenic, pressure, urethane-- but there is only one common cause into which all other causes of cancer merge, their reversible injuring of respiration.

The irreversible injuring of respiration is followed, as the second phase of cancer formation, by a long struggle for existence by the injured cells to maintain their structure, in which a part of the cells perish from lack of energy, while another part succeed in replacing thei rretrievably lost respiration by fermentation energy. Because of the morphological inferiority of fermentation energy, the highly differentiated body cells are converted by this into undifferentiated cells that grow wildly -- the cancer cells.

To the thousands of quantitative experiments on which these results are based, I should like to add, as a further argument, the fact that there is no alternative today. If the explanation of a vital process is its reduction to physics and chemistry, there is today no other explanation for the origin of cancer cells, either special or general. From this point of view, mutation and carcinogenic agent are not alternatives, but empty words, unless metabolically specified. Even more harmful in the struggle against cancer an be the continual discovery of miscellaneous cancer agents and cancer viruses, which, by obscuring the under lyingphenomena, may hinder necessary preventive measures and thereby become responsible for cancer cases.

Technical Ethical Considerations And Comments

Metabolism of the Ascites Cancer Cells

The high fermentation of ascites cancer cells was discovered in Dahlemin 1951 (12) and since then has been confirmed in many works (13,14) Forbest measurements, the ascites cells are not transferred to Ringer's solution but are maintained in their natural medium, ascites serum, which is adjusted physiologically at the beginning of the measurement by addition of glucose and bicarbonate. Because of the very large fermentation, it is necessary to dilute the ascites cells that are removed from the abdominal cavity rather considerably with ascitesserum; otherwise the bicarbonate would be used up within a few minutes after addition to the glucose, and hence the fermentation would be brought to a standstill.

Under physiological conditions of pH and temperature, we find the following metabolic quotients in ascites serum (15):

QO2 = -5 to -10
QMO2 = 25 to 35   
QMN2 = 50 to 70

where QO2 is the amount of oxygen in cubic millimeters that 1 milligram of tissue (dry weight) consumes per hour at 38* C with oxygen saturation, QMO2 is the amount of lactic acid in cubic millimeters that 1 milligram of tissue (dry weight) develops per hour at 38* C in the absence of oxygen.

Even higher fermentation quotients have been found in the United Stateswith other strains of mouse ascites cancer cells (13,14).

All calculations of the energy-production potential of cancer cells should now be based on quotients of the ascites cancer cells, since these quotients are 2 or 3 times as large anaerobically as the values formerly found for the purest solid tumors. The quotients of the normal body cells, however, remain as they were found in Dahlem in the years from 1924 to 1929 (16-19). It is clear that the difference inmetabolism between normal cells and cancer cells is much greater than it formerly appeared to be on the basis of measurements of solid tumors.

Utilizable Energy of Respiration and Fermentation

Since the discovery of the oxidation reaction of fermentation in 1939(20), we have known the chemical reactions by which adenosinediphosphate is phosphorylated to adenosine triphosphate in fermentation;and since then we have found that 1 mole of fermentation lactic acidproduces 1 mole of adenosine triphosphate (ATP).

The chemical reactions by which ATP is synthesized in respiration are still unknown, but it can be assumed, according to the existing measurements (21), that 7 moles of ATP can be formed when 1 mole of oxygen is consumed in respiration.

ATP Quotients

If we multiply QO2 by 7 and QMN2 by 1, we obtain the number of cubicmillimeters of ATP that 1 milligram of tissue (dry substance) cansynthe size per hour (22,400 cubic millimeters=1 millimole of ATP). Wecall these quotients QATPO2 and QATPN2, according to whether the ATP is formed by respiration or by fermentation, respectively.

Energy Production of Cancer Cells and Normal Body Cells

In Table 1, the Q values of some normal body cells are contrasted withthe Q values of our ascites cancer cells.

The cancer cells have about as much energy available as the normal body cells, but the ratio of the fermentation energy to the respirationenergy is much greater in the cancer cells than it is in the normal cells.

 

Table 1. Contrast of the Q values of some normal body cells with the Qvalues of ascites cancer cells.
Cells QO2 QMN2 QATPO2 QATPN2 QATPO2 + QATPN2
Liver -15 1 105 1 106
Kidney -15 1 105 1 106
Embryo
(Very Yong)		 -15 1 105 1 106
Cancer -7 60 49 60 109

Uncoupling of Respiration

If a young rat embryo is transferred from the amniotic sac to Ringer's solution, the previously transparent embryo becomes opaque and soon appears coagulated (17). At the same time, the connection between respiration and phosphorylation is broken; that is, although oxygen is still consumed and carbon dioxide is still developed, the energy of this combustion process is lost for life. If the metabolism quotients had previously been.

QO2 = 15, QMO2 = O, QMN2 = 25
QATPO2 = 105, QATPN2 = 25

in the amniotic fluid, afterward in Ringer's solution they are

QO2 = -15, QMO2 = 25, QMN2 = 25
QATPO2 = O, QATPN2 = 25

Because of uncoupling of respiration and phosphorylation, the energy production of the embryo has fallen from QATPO2 + QATPN2 = 130, to 25; since the uncoupling is irreversible, the embryo dies in the Ringer's solution.

This example will show that the first phase of carcinogenesis, their reversible damaging of respiration, need not be an actual decrease in the respiration quotient but merely an uncoupling of respiration, with undiminished over-all oxygen consumption. Ascites cancer cells, which owe their origin primarily to an uncoupling of respiration, could conceivably have the following metabolism quotients, for example:

QO2 = -50, QMO2 = 100, QMN2 = 100
QATPO2 = O, QATPN2 = 100

which would mean that, despite great respiration, the usable energy production would be displaced completely toward the side of fermentation. One will now have to search for such cancer cells among the ascites cancer cells. Solid tumors --and especially solid spontaneous tumors-- need no longer be subjected to such examinations today, of course, since the solid tumors are usually so impure histologically.

Aerobic Fermentation

Aerobic fermentation is a property of all growing cancer cells, butaerobic fermentation [p. 313 -->] without growth is a property ofdamaged body cells -- for example, embryos that have been transferred from amniotic fluid to Ringer's solution. Since it is always easy to detect aerobic fermentation but generally difficult to detect growth, or lack thereof, of body cells, aerobic fermentation should not be used as a test for cancer cells, as I made clear in 1928 (19).

Respiratory Poisons

The specific respiration-inhibiting effect of arsenious acid and the irreversibility of its inhibitions were discovered in the firstquantitative works on cell respiration (23,24). There is abundant literature on the carcinogenesis by arsenic, particularly on arsenic cancer after treatment of psoriasis and on the cancer of grape owners who spray their vineyards with arsenic. The specific respiration-inhibiting effect of hydrogen sulfide has likewise been described by Negelein (25), and carcinogenesis by derivatives of hydrogen sulfide has been recently described by D. N. Gupta (26).

The irreversible inhibition of cell respiration by urethane was discovered early (27) as well as the fact that the urethane inhibitionis more irreversible, the higher the temperature. In sea urchin eggs,the effect of urethane was investigated, not only on the metabolism, but also on cell division in studies (28) from which the later urethane treatment of leukemia was developed. The physiochemical mechanism by which urethane and other indifferent narcotics inhibit cell respiration was cleared up in 1921 (29). Only much later did the carcinogenic effect of urethane become known. Actually, multiple lung adenomas can often be produced in 100 percent of the mice treated with small doses of urethane (30).

Oxygen Deficiency

Short-period oxygen deficiency irreversibly destroys the respiration ofembryos (16) without thereby inhibiting the anaerobic fermentation of the embryo. If such embryos are transplanted, teratomas are formed(31). It has recently been reported that, in the development of the Alpine salamander, malformations occurred when the respiration was inhibited by hydrocyanic acid in the early stages of embryonal development (32).

Goldblatt and Cameron (3) reported that, in the in vitro culturing of fibroblasts, tumor cells appeared when the cultures were exposed tointermittent oxygen deficiency for long periods, whereas, in the control cultures, no tumor cells appeared. In the discussion at the Stuttgart convention, Lettre cited against Goldblatt and Cameron the fact that another American tissue culturist, Earle, had occasionally obtained tumor cells from fibroblasts for reasons unknown to him and in anun reproduceable manner, but this objection does not seem weighty, and the latter part is untrue (33). In any event, here is an area in which the methods of tissue culture could prove useful for cancer research. But warnings must be given against metabolism measurements in tissue cultures, if and when the tissue cultures are mixtures of growing and dying cells, especially under conditions of malnutrition. An example ofthe latter type of confusion is involved in the discussion by Albert Fischer (34), especially in the chapter "Energy exchange of tissue cells cultivated in vitro."

Rous Agent

If the Rous agent is inoculated into the chorion of chick embryos, tumors originate in the course of a few days -- as rapidly as the transplantation of cancer cells. The tumors formed are not chorion tumors but Rous sarcomas. The Rous agent, to which a particle weight of 150 million is ascribed at present, is therefore capable of transmitting the morphological properties of the Rous sarcoma; and whatever we call the Rous agent -- "hereditary unit," cell fragment, micro cell, or spore-- the transmission of the Rous sarcoma by the Rous agent is, in any case, nothing more than a transplantation and is to be differentiated strictly from the production of a chicken sarcoma by methylcholanthrene,which is a neoformation of a tumor from normal body cells and as such takes a long time.

The metabolism of the chicken sarcomas, whether produced by the Rousagent or by methylcholanthrene, is the same and does not differ in anyway from the metabolism of the tumors of other animals (35). In the first case, however, the fermentation potential has been transplantedwith the Rous agent, whereas in the second case, the fermentation has been intensified by selection from normal body cells under the action ofmethylcholanthrene.

Addendum: in vitro Carcinogenesis and Metabolism

Since this paper was prepared, striking confirmation and extension of its main conclusions have been obtained from correlated metabolic and growth studies of two lines of tissue culture cancer cells of widely differing malignancy that were both derived from one and the samenormal, tissue-culture cell (36). The single cell as isolated some 5 years ago from a 97-day old parent culture of a strain C3H/He mouse bySanford, Likely and Earle (33) of the National Cancer Institute. Up to the time that the single-cell isolation was made, no tumors developed when cells of the parent culture were injected into strain C3H/He mice. Injections of in vitro cells of the lines 1742 and 2049 (formerlylabelled substrains VII and III, respectively) first produced tumors innormal C3H/He mice after the 12th and 19th in vitro transplant generations, respectively; after 1.5 years, the percentage production of sarcomas was 63 and 0 percent, respectively; and after 3 years, itwas 97 and 1 percent, respectively, with correspondingly marked differences in length of induction period. Despite such gross differences in "malignancy" in vivo, the rates of growth of the two lines of cells maintained continuously in vitro have remained nearly identical and relatively rapid. Nevertheless, the metabolism of the two lines of cancer cells, whose malignancy was developed in vitro, has been found by Woods, Hunter, Hobby, and Burk to parallel strikingly the differences in malignancy observed in vivo, in a manner in harmony with the predications and predictions of this article.

The metabolic values were measured following direct transfer of the liquid cultures from the growth flasks into manometric vessels, without notable alteration of environmental temperature, pH, or mediumcomposition (horse serum, chick embryo extract, glucose, bicarbonate, balanced saline). The values obtained this accurately represent the metabolism of growing, adequately nourished, pure lines of healthy cancer cells free of admixture with any other tissue cell type. The anaerobic glycolysis of the high-malignancy line 1742 was QMN2 = 60 to 80, which is virtually maximum for any and all cancer cells previouslyreported, including ascites cells (12-14). The anaerobic glycolysis of the low-malignancy line was, however, only one-third as great, QMN2 = 20to 30. The average aerobic glycolysis values for the two lines were in the same order, QMO2 = 30 and 10, respectively, but of lower magnitude because of the usual, pronounced Pasteur effect, greater in line 1742 than in line 2049 [QMN2 - QMO2 = about 40 and 15]. On the other hand, the rates of oxygen consumption were in converse order, being smaller in line 1742 [QO2 = 5 to 10] than in line 2049 [QO2 = 10 to 15], corresponding to a greater degree of respiratory defect in line 1742. The respiratory defect in both lines was further delineated by the finding of little or no increase in respiration after the addition of succinate to either line of cells, in contrast to the considerable increases obtained with virtually all normal tissues (9); and the respiratory increase with paraphenylenediamine was likewise relatively low, compared with normal tissue responses.

A further notable difference between the two cell lines was the very much lower inhibition of glycolysis by podophyllin materials (anti-insulin potentiators) observed with line 1742 compared with line 2049 (for example, 10 and 70 percent, respectively, at a suitably low concentration). This result would be expected on the basis of the much greater loss of anti-insulin hormonal restraint of glucose metabolism, at the hexokinase phosphorylating level, as the degree of malignancy is increased, just as was reported for a spectrum of solid tumors (14).

Finally, the high-malignancy line 1742 cells have been found by A. L.Schade to contain 3 times as much aldolase as the low-malignancy line 2049 cells (11,300 versus 3700 Warburg activity units per millimeter of packed cells extracted), and about 2 times as much a-glycerophosphatedehydrogenase [2600 versus 1400 Schade activity units (13) permillimeter of packed cells extracted]. The potential significance of these indicated enzymic differences in relation to the parallel glycolytic differences, measured with aliquots of the same cellcultures, is evident, and may well be connected with the corresponding hexokinase system differences.

The new metabolic data on the two remarkably contrasting lines of cancer cells, which originated from a single, individual cell and have been maintained exclusively in vitro over a period of years, epitomize and prove finally the main conclusions of this article, which are based on decades of research. Such metabolic analyses provide promise of a powerful tool for diagnosis of malignancy in the ever-increasing variety of tissue culture lines now becoming available in this rapidly expanding biological and medical field, where characterization of malignancy by conventional methods (animal inoculation or otherwise) may be difficultor impracticable. This metabolic tool should be especially important inconnection with the use of tissue cultures for the evaluation of chemotherapeutic agents or other control procedures.

REFERENCES AND NOTES

  1. The transplantable ascites cancer was discovered by H. Loewenthal andG. Jahn [Z. KREBSFORSCH, 37, 439 (1932)]. G. Klein (Stockholm) expanded ourknowledge about the physiology and morphology of the ascites tumors and showed their great advantages as experimental material. [EXPTL. CELL RESEARCH, 2, 518 (1951)]
  2. F.Lynen, NATURWISSENSCHAFTEN 30 (1942); ANN. CHEM. JUSTUSLIEBIG (1951).
  3. H. Goldblatt and G. Cameron, J. EXPTL. MED. 97, 525 (1953).
  4. O. Warburg, PFLUGER'S. ARCH. GES. PHYSIOL. 154, 599 (1913); 158, 19(1914).
  5. C. D. Darlington, BRIT. J. CANCER 2, 118 (1948).
  6. M. W. Woods et al., J. NATL. CANCER INST, (1951); SCIENCE 102,591 (1945); AAAS RESEARCH CONF. ON CANCER (1945),WASH. ACAD. SCI. (1952).PIGMENT CELL GROWTH, M. Gordon, Ed. (1953), BIOCHEM. ET BIOPHYS. ACTA 12,(1953). PROC. AM. ASS. CANCER RESEARCH 1, 7 (1954). PROC. SOC. EXPTL.BIOL. MED. (1953). PHYTOPATHOLOGY (1941, 1943, 1946); AM. J. BOTANY 33, 12a(1946); 38, 419 (1951).
  7. A compilation of American works on the grana, in which my results of1914 (4) have been confirmed, is given by G. Hogeboom, W. Schneider, and M. Striebich in CANCERRESEARCH [13, 617 (1953)]. In a very special case -- nucleated red blood cells of birds, which contain no grana or only poorly visible ones --the entire respiration can be centrifuged off with the cell nuclei [O.Warburg, HOPPE-SEYLER'S Z. PHYSIOL. CHEM. (1913)].
  8. A. Graffi, Z. KREBSFORSCH, 49, 477 (1939).
  9. D. Burk, SYMPOSIUM ON RESPIRATORY ENZYMES (University of WisconsinPress, Madison, 942), p. 235. J. G. Kidd, R. J. Winzler, D. Burk, CANCERRESEARCH (1944).
  10. L. Pasteur, ETUDES SUR LA BIERE (Masson, Paris, 1876), p. 240.
  11. J. Craigie, J. PATHOL. BACTERIOL. 63, 177 (1951); 64, 251 (1952). H.Hamperl, VERHANDL. DEUT. GES. PATHOL. 35, 29 (1951).
  12. O. Warburg and E. Hiepler, Z. NATURFORSCH 7b, 193 (1952).
  13. A. L. Schade, BIOCHIM. ET. BIOPHYS. ACTA 12, 163 (1953).
  14. M. Woods, J. Hunter, D. Burk, J. NATL. CANCER. INST. 16, 351 (1955).
  15. Georg Klein, Karolinska Institute, Stockholm, providedEhrlich strain of mouse ascites cells.
  16. O. Warburg, K. Posener, E. Negelein, BIOCHEM. Z. 152, 309 (1924).
  17. E. Negelein, IBID, 165, 122 (1925).
  18. O. Warburg et al., IBID. 189, 114, 175, 242 (1924); 193, 315 (1928);197, 175 (1928); (1929).
  19. O. Warburg, IBID. 204, 482 (1929).
  20. O. Warburg et al., IBID. 303, 40, 132 (1939).
  21. H. A. Krebs et al., BIOCHEM. J. LONDON 54, 107 (1953).
  22. R. J. O'Connor, BRIT. J. EXPTL. PATHOL. 31, 390 (1951).
  23. O. Warburg, HOPPE-SEYLER'S Z. PHYSIOL. CHEM. (1911); M.Onaka, IBID.
  24. K. Dresel, BIOCHEM. Z. 178, 70 (1926).
  25. E. Negelein, IBID. 165, 203 (1925).
  26. D. N. Gupta, NATURE 175, 257 (1955).
  27. R. Usui, PFLUGER'S ARCH. GES. PHYSIOL. 147, 100 (1912).
  28. O. Warburg, HOPPE-SEYLER'S Z. PHYSIOL. CHEM. 66, 305 (1910); 70, 413(1911).
  29. ----------, BIOCHEM. 119, 143 (1921).
  30. C. D. Larsen, J. NATL. CANCER INST. 8, 63 (1947).
  31. O. Warburg, BIOCHEM. Z. 228, 257 (1930).
  32. H. Tiedemann and H. Tiedemann, Z. NATURFORSCH. 9b, 371 (1954).
  33. K. K. Sanford, G. D. Likely, W. R. Earle, J. NATL. CANCER INST. 15,215 (1954).
  34. A. Fischer, BIOLOGY OF TISSUE CULTURE (Copenhagen, Denmark, 1946).
  35. O. Warburg, BIOCHEM. Z. (1925); D. Burk et al., J. NATL. CANCERINST. (1941).
  36. This summary of studies was prepared by Dean Burk at ProfessorWarburg's request.

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