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On the Origin of Cancer Cells

Author(s): Otto Warburg

Source: Science, New Series, Vol. 123, No. 3191, (Feb. 24, 1956), pp. 309-314

Published by: American Association for the Advancement of Science

Stable URL: http://www.jstor.org/stable/1750066

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24 February 1956, Volume 123, Number 3191 SCIENCE

Injuring of Respiration

On the Origin of Cancer CellsOtto Warburg

Our principal experimental object forthe measurement of the metabolism ofcancer cells is today no longer the tumorbut the ascites cancer cells (1) livingfree in the abdominal cavity, which arealmost pure cultures of cancer cells withwhich one can work quantitatively as inchemical analysis. Formerly, it could besaid of tumors, with their varying cancercell content, that they ferment morestrongly the more cancer cells they con-tain, but today we can determine theabsolute fermentation values of the can-cer cells and find such high values thatwe come very close to the fermentationvalues of wildly proliferating Torulayeasts.What was formerly only qualitativehas now become quantitative. What wasformerly only probable has now becomecertain. The era in which the fermenta-tion of the cancer cells or its importancecould be disputed is over, and no onetoday can doubt that we understand theorigin of cancer cells if we know howtheir large fermentation originates, or,to express it more fully, if we know,howthe damaged respiration and the exces-sive fermentation of the cancer cellsoriginate.

Energy of Respiration and FermentationWe now understand the chemicalmechanism of respirationand fermenta-tion almost completely, but we do notneed this knowledge for what follows,since energy alone will be the center ofour considerations. We need to know nomore of respiration and fermentationhere than that they are energy-produc-

,24 FEBRUARY 1956

ing reactions and that they synthesizethe energy-rich adenosine triphosphate,through which the energy of respirationand fermentation is then made availablefor life. Since it is known how muchadenosine triphosphate can be synthe-sized by respiration and how much byfermentation, we can write immediatelythe potential, biologically utilizable en-ergy production of any cells if we havemeasured their respiration and fermen-tation. With the ascites cancer cells ofthe mouse, for example, we find an aver-age respiration of 7 cubic millimeters ofoxygen consumed per milligram, perhour, and fermentation of 60 cubic milli-meters of lactic acid produced per milli-gram, per hour. This, converted to en-ergy equivalents, means that the cancercells can obtain approximately the sameamount of energy from fermentation asfrom respiration, whereas the normalbody cells obtain much more energyfrom respiration than from fermenta-tion. For example, the liver and kidneyof an adult animal obtain about 100times as much energy from respirationas from fermentation.I shall not consider aerobic fermenta-tion, which is a result of the interactionof respiration and fermentation, becauseaerobic fermentation is too labile andtoo dependent on external conditions.Of importance for the considerationsthat follow are only the two stable inde-pendent metabolic processes, respirationand anaerobicfermentation-respiration,which is measured by the oxygen con-sumption of cells that are saturated withoxygen, and fermentation, which ismeasured by the formation of lacticacid in the absence of oxygen.

Since the respiration of all cancercells is damaged, our first question is,How can the respiration of body cellsbe injured? Of this damage to respira-tion, it can be said at the outset that itmust be irreversible, since the respira-tion of cancer cells never returns to nor-mal. Second, the injury to respirationmust not be so great that the cells arekilled, for then no cancer cells could re-sult. If respiration is damaged when itforms too little adenosine triphosphate,it may be either that the oxygen con-sumption has been decreased or that,with undiminished oxygen consumption,the coupling between respiration and theformation of adenosine triphosphate hasbeen broken, as was first pointed out byFeodor Lynen (2).One method for the destruction of therespiration of body cells is removal ofoxygen. If, for example, embryonal tis-sue is exposed to an oxygen deficiencyfor some hours and then is placed inoxygen again, 50 percent or more of therespiration is usually destroyed. Thecause of this destruction of respiration islack of energy. As a matter of fact, thecells need their respiratory energy topreserve their structure, and if respira-tion is inhibited, both structure and res-piration disappear.Another method for destroying respi-ration is to use respiratorypoisons. Fromthe standpoint of energy, this methodcomes to the same result as the firstmethod. No matter whether oxygen iswithdrawn from the cell or whether theoxygen is prevented from reacting by apoison, the result is the same in bothcases-namely, impairment of respira-tion from lack of energy.I may mention a few respiratory poi-sons. A strong, specific respiratory poi-son is arsenious acid, which, as everyclinician knows, may produce cancer.Hydrogen sulfide and many of its deriv-

Professor Warburg is director of the Max PlanckInstitute for Cell Physiology, Berlin-Dahlem, Ger-many. This article is based on a lecture deliveredat Stuttgart on 25 May 1955 before the GermanCentral Committee for Cancer Control. It wasfirst published in German [Naturwissenschaften42, 401 (1955)]. This translation was prepared byDean Burk, Jehu Hunter, and W. H. Everhardyof the U.S. Department of Health, Education, andWelfare, Public Health Service, National Institutesof Health, Bethesda, Md., with the permission ofNaturwissenschaften and with the collaboration ofProfessor Warburg, who has introduced additionalmaterial.309


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atives are also strong, specific respira-tory poisons. We know today that cer-tain hydrogen sulfide derivatives, thio-urea and thioacetamide, with whichcitrus fruit juices have been preservedin recent times, induce cancer of theliver and gall bladder in rats.Urethane is a nonspecific respiratorypoison. It inhibits respiration as a chem-ically indifferent narcotic, since it dis-places metabolites from cell structures.In recent years it has been recognizedthat subnarcotic doses of urethane causelung cancer in mice in 100 percent oftreatments. Urethane is particularly suit-able as a carcinogen, because, in con-trast to alcohol, it is not itself burnedup on the respiring surfaces and, unlikeether or chloroform, it does not cyto-lyze the cells. Any narcotic that hasthese properties may cause cancer uponchronic administration in small doses.The first notable experimental induc-tion of cancer by oxygen deficiency wasdescribed by Goldblatt and Cameron(3), who exposed heart fibroblasts intissue culture to intermittent oxygendeficiency for long periods and finallyobtained transplantable cancer cells,whereas in the control cultures that weremaintained without oxygen deficiency,no cancer cells resulted. Clinical experi-ences along these lines are innumerable:the production of cancer by intermittentirritation of the outer skin and of themucosa of internal organs, by the plug-ging of excretory ducts of glands, bycirrhoses of tissues, and so forth. In allthese cases, the intermittent irritationslead to intermittent circulatory disturb-ances. Probably chronic intermittentoxygen deficiency plays a greater role inthe formation of cancer in the body thandoes the chronic administration of respi-ratory poisons.Any respiratory injury due to lack ofenergy, however, whether it is producedby oxygen deficiency or by respiratorypoisons, must be cumulative, since it isirreversible. Frequent small doses of res-piratory poisons are therefore more dan-gerous than a single large dose, wherethere is always the chance that the cellswill be killed rather than that they willbecome carcinogenic.Grana

If an injury of respiration is to pro-duce cancer, this injury must, as alreadymentioned, be irreversible. We under-stand by this not only that the inhibi-tion of respiration remains after removalof the respiratorypoison but, even more,that the inhibition of respiration alsocontinues through all the following celldivisions, for measurements of metabo-lism in transplanted tumors have shownthat cancer cells cannot regain normal310

respiration, even in the course of manydecades, once they have lost it.This originally mysterious phenome-non has been explained by a discoverythat comes from the early years of cellphysiology (4). When liver cells werecytolyzed by infusion of water and thecytolyzate was centrifuged, it was foundthat the greater part of the respirationsank to the bottom with the cell grana.It was also shown that the respirationofthe centrifuged grana was inhibited bynarcotics at concentrations affecting cellstructures, from which it was concluded-already in 1914-that the respiringgrana are not insoluble cell particles butautonomous organisms, a result that hasbeen extended in recent years by theEnglish botanist Darlington (5) andparticularly by Mark Woods and H. G.du Buy (6) of the National Cancer In-stitute in Bethesda, Md. Woods and duBuy have experimentally expanded ourconcepts concerning the self-perpetua-ting nature of mitochondrial elements(grana) and have demonstrated the he-reditary role of extranuclear aberrantforms of these in the causation of neo-plasia. The autonomy of the respiringgrana, both biochemically and geneti-cally, can hardly be doubted today.If the principle Omne granum e granois valid for the respiring grana, we un-derstand why the respiration connectedwith the grana remains damaged whenit has once been damaged; it is for thesame reason that properties linked withgenes remain damaged when the geneshave been damaged.Furthermore, the connection of respi-ration with the grana (7) also explainsa carcinogenesis that I have not men-tioned previously, the carcinogenesis byx-rays. Rajewsky and Pauly have re-cently shown that the respiration linkedwith the grana can be destroyed withstrong doses of x-rays, while the smallpart of the respiration that takes placein the fluid protoplasm can be inhibitedvery little by irradiation. Carcinogenesisby x-rays is obviously nothing else thana destruction of respiration by elimina-tion of the respiring grana.It should also be mentioned here thatgrana, as Graffi has shown (8), fluorescebrightly if carcinogenic hydrocarbonsare brought into their surroundings, be-cause the grana accumulate the carcino-genic substances. Probably this accumu-lation is the explanation for the factthat carcinogenic hydrocarbons,althoughalmost insoluble in water, can inhibitrespirationand therefore have a carcino-genic effect.

Increase of FermentationWhen the respirationof body cells hasbeen irreversibly damaged, cancer cells

by no means immediately result. For can-cer formation there is necessary not onlyan irreversible damaging of the respira-tion but also an increase in the fermen-tation-indeed, such an increase of thefermentation that the failure of respira-tion is compensated for energetically.But how does this increase of fermenta-tion come about?

The most important fact in this fieldis that there is no physical or chemicalagent with which the fermentation ofcells in the body can be increased di-rectly; for increasing fermentation, along time and many cell divisions arealways necessary. The temporal courseof this increase of fermentation in car-cinogenesis has been measured in manyinteresting works, among which I shouldlike to make special mention of those ofDean Burk (9).Burk first cut out part of the liver ofhealthy rats and investigated the me-tabolism of the liver cells in the courseof the ensuing regeneration, in which,as is well known, the liver grows morerapidly than a rapidly growing tumor.No increase of fermentation was found.Burk then fed rats for 200 days on butteryellow, whereupon liver carcinomaswereproduced, and he found that the fer-mentation slowly increased in the courseof 200 days toward values characteristicof tumors.The mysterious latency period of theproduction of cancer is, therefore, noth-ing more than the time in which the fer-mentation increases after a damaging ofthe respiration. This time differs in vari-ous animals; it is especially long in manand here often amounts to several dec-ades, as can be determined in the casesin which the time of the respiratorydam-age is known-for example, in arseniccancer and irradiation cancer.The driving force of the increase offermentation, however, is the energy de-ficiency under which the cells operateafter destruction of their respiration,which forces the cells to replace the irre-trievably lost respiration energy in someway. They are able to do this by a selec-tive process that makes use of the fer-mentation of the normal body cells. Themore weakly fermenting body cells per-ish, but the more strongly fermentingones remain alive, and this selectiveprocess continues until the respiratoryfailure is compensated for energeticallyby the increase in fermentation. Onlythen has a cancer cell resulted from thenormal body cell.Now we understand why the increasein fermentation takes such a long timeand why it is possible only with the helpof many cell divisions. We also under-stand why the latency period is differentin rats and in man. Since the averagefermentation of normal rat cells is muchgreater than the average fermentation

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of normal human cells, the selectiveprocess begins at a higher fermentationlevel in the rat and, hence, is completedmore quickly than it is in man.It follows from this that there wouldbe no cancers if there were no fermenta-tion of normal body cells, and hence weshould like to know, naturally, fromwhere the fermentation of the normalbody cells stems and what its significanceis in the body. Since, as Burkhas shown,the fermentation remains almost zero inthe regenerating liver growth, we mustconclude that the fermentation of thebody cells has nothing to do with normalgrowth. On the other hand, we havefound that the fermentation of the bodycells is greatest in the very earliest stagesof embryonal development and that itthen decreases gradually in the courseof embryonal development. Under theseconditions, it is obvious-since ontogenyis the repetition of phylogeny-that thefermentation of body cells is the inheri-tance of undifferentiated ancestors thathave lived in the past at the expense offermentation energy.Structure and Energy

But why-and this is our last ques-tion-are the body cells dedifferentiatedwhen their respirationenergy is replacedby fermentation energy? At first, onewould think that it is immaterial to thecells whether they obtain their energyfrom respiration or from fermentation,since the energy of both reactions istransformed into the energy of adeno-sine triphosphate, and yet adenosine tri-phosphate= adenosine triphosphate.Thisequation is certainly correct chemicallyand energetically, but it is incorrectmorphologically, because, although res-piration takes place for the most partin the structure of the grana, the fermen-tation enzymes are found for a greaterpart in the fluid protoplasm. The adeno-sine triphosphate synthesized by respira-tion therefore involves more structurethan the adenosine triphosphate synthe-sized by fermentation. Thus, it is as ifone reduced the same amount of silveron a photographic plate by the sameamount of light, but in one case withdiffused light and in the other with pat-terned light. In the first case, a diffuseblackening appears on the plate, but inthe second case, a picture appears; how-ever, the same thing happens chemicallyand energetically in both cases. Just asthe one type of light energy involvesmore structure than the other type, theadenosine triphosphate energy involvesmore structure when it is formed byrespiration nhan de itoes when it is formedby fermentation.In any event, it is one of the funda-mental facts of present-day biochemis-24 FEBRUARY 1956

try that adenosine triphosphate can besynthesized in homogeneous solutionswith crystallized fermentation enzymes,whereas so far no one has succeeded insynthesizing adenosine triphosphate inhomogeneous solutions with dissolvedrespiratory enzymes, and the structurealways goes with oxidative phosphoryla-tion.

Moreover, it was known for a longtime before the advent of crystallizedfer-mentation enzymes and oxidative phos-phorylation that fermentation-the en-ergy-supplying reaction of the lower or-ganisms-is morphologically inferior torespiration. Not even yeast, which is oneof the lowest forms of life, can maintainits structurepermanentlyby fermentationalone; it degenerates to bizarre forms.However, as Pasteur showed, it is re-juvenated in a wonderful manner if itcomes in contact with oxygen for a shorttime. "I should not be surprised," Pas-teur said in 1876 (10) in the descriptionof these experiments, "if there shouldarise in the mind of an attentive hearera presentiment about the causes of thosegreat mysteries of life which we concealunder the words youth and age of cells."Today, after 80 years, the explanationis as follows: the firmer connection ofrespiration with structure and the looserconnection of fermentation with struc-ture.

This, therefore, is the physicochemi-cal explanation of the dedifferentiationof cancer cells. If the structure of yeastcannot be maintained by fermentationalone, one need not wonder that highlydifferentiated body cells lose their dif-ferentiation upon continuous replace-ment of their respiration with fermen-tation.I would like at this point to draw at-tention to a consequence of practicalimportance. When one irradiates a tis-sue that contains cancer cells as well asnormal cells, the respirationof the cancercells, already too small, will declinefurther. If the respiration falls below acertain minimum that the cells need un-conditionally, despite their increased fer-mentation, they die; whereas the normalcells, where respiration may be harmedby the same amount,will survivebecause,with a greater initial respiration, theywill still possess a higher residual respi-ration after irradiation. This explains theselective killing action of x-rays on can-cer cells. But still further: the descend-ants of the surviving normal cells may inthe course of the latent period compen-sate the respiration decrease by fermen-tation increase and, thence, become can-cer cells. Thus it happens that radiationwhich kills cancer cells can also at thesame time produce cancer or that ure-thane, which kills cancer cells, can alsoat the same time produce cancer. Bothevents take place from harming respira-

tion: the killing, by harming an alreadyharmed respiration; the carcinogenesisby the harmingof a not yet harmed respi-ration.

Maintenance EnergyWhen dedifferentiation of the bodycells has occurred and cancer cells have

thereby developed, there appears a phe-nomenon to which our attention has beencalled by the special living conditions ofthe ascites cancer cells. In extensivelyprogressed ascites cancer of the mouse,the abdominal cavity contains so manycancer cells that the latter cannot utilizetheir full capacity to respire and fermentbecause of the lack of oxygen and sugar.Nevertheless, the cancer cells remainalive in the abdominal cavity, as the re-sult of transplantation proves.Recently we have confirmed this re-sult by direct experiments in which weplaced varying amounts of energy at thedisposal of the ascites outside the body,in vitro, and then transplanted it. Thisinvestigation showed that all cancer cellswere killed when no energy at all wassupplied for 24 hours at 38?C but thatone-fifth of the growth energy was suffi-cient to preserve the transplantability ofthe ascites. This result can also be ex-pressed by saying that cancer cells re-quire much less energy to keep themalive than they do for growth. In thisthey resemble other lower cells, such asyeast cells, which remain alive for a longtime in densely packed packets-almostwithout respiration and fermentation.In any case, the ability of cancer cellsto survive with little energy, if they arenot growing, will be of great importancefor the behavior of the cancer cells in thebody.

Sleeping Cancer CellsSince the increase in fermentation inthe development of cancer cells takesplace gradually, there must be a transi-tional phase between normal body cellsand fully formed cancer cells. Thus, forexample, when fermentation has becomeso great that dedifferentiation has com-

menced, but not so great that the respira-tory defect has been fully compensatedfor energeticallyby fermentation,we mayhave cells which indeed look like cancercells but are still energetically insuffi-cient. Such cells, which are clinically notcancer cells, have lately been found, notonly in the prostate, but also in the lungs,kidney, and stomach of elderly persons.Such cells have been referred to as"sleeping cancer cells" (11, 12).The sleeping cancer cells will possiblyplay a role in chemotherapy. Fromenergy considerations, I could think that

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sleeping cancer cells could be killed morereadily than growing cancer cells in thebody and that the most suitable test ob-jects for finding effective killing agentswould be the sleeping cancer cells ofskin-that is, precancerous skin.

SummaryCancer cells originate from normalbody cells in two phases. The first phaseis the irreversible injuring of respiration.Just as there are many remote causes ofplague-heat, insects, rats-but onlyone common cause, the plague bacillus,there are a great many remote causes ofcancer-tar, rays, arsenic, pressure, ure-thane-but there is only one commoncause into which all other causes of can-cer merge, the irreversible injuring ofrespiration.The irreversible injuring of respira-tion is followed, as the second phase ofcancer formation, by a long struggle forexistence by the injured cells to maintaintheir structure, in which a part of thecells perish from lack of energy, whileanother part succeed in replacing the ir-retrievablylost respiration energy by fer-mentation energy. Because of the mor-phological inferiority of fermentationenergy, the highly differentiated bodycells are converted by this into undiffer-entiated cells that grow wildly-the can-cer cells.To the thousands of quantitative ex-periments on which these results arebased, I should like to add, as a furtherargument, the fact that there is no alter-

native today. If the explanation of a vitalprocess is its reduction to physics andchemistry, there is today no other ex-planation for the origin of cancer cells,either special or general. From this pointof view, mutation and carcinogenicagentare not alternatives, but empty words,unless metabolically specified. Even moreharmful in the struggle against cancercan be the continual discovery of miscel-laneous cancer agents and cancer viruses,which, by obscuring the underlying phe-nomena, may hinder necessarypreventivemeasures and thereby become respon-sible for cancer cases.

Technical Considerationsand Comments

Metabolism of the ascites cancer cells.The high fermentation of ascites cancercells was discovered in Dahlem in 1951(12) and since then has been confirmedin many works (13, 14). For best meas-urements, the ascites cells are not trans-ferred to Ringer's solution but are main-tained in their natural medium, ascitesserum, which is adjusted physiologicallyat the beginning of the measurement by

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addition of glucose and bicarbonate. Be-cause of the very large fermentation, it isnecessary to dilute the ascites cells thatare removed from the abdominal cavityrather considerably with ascites serum;otherwise the bicarbonate would be usedup within a few minutes after additionof the glucose, and hence the fermenta-tion would be brought to a standstill.Under physiological conditions of pHand temperature, we find the followingmetabolic quotients in ascites serum(15):

Qo2=-5 to -10QMO2 25 to 35QMN2= 50 to 70

where Qo2 is the amount of oxygen incubic millimeters that 1 milligram oftissue (dry weight) consumes per hourat 38?C with oxygen saturation, QM?2 isthe amount of lactic acid in cubic milli-meters that 1 milligram of tissue (dryweight) develops per hour at 38?C withoxygen saturation, and QMN2 is theamount of lactic acid in cubic millime-ters that 1 milligram of tissue (dryweight) develops per hour at 38?C inthe absence of oxygen.Even .higher fermentation quotientshave been found in the United Stateswith other strains of mouse ascites cancercells (13, 14).All calculations of the energy-produc-tion potential of cancer cells should nowbe based on the quotients of the ascitescancer cells, since these quotients are 2or 3 times as large anaerobically as thevalues formerlyfound for the purest solidtumors. The quotients of the normalbody cells, however, remain as they werefound in Dahlem in the years from 1924to 1929 (16-19). It is clear that the dif-ference in metabolism between normalcells and cancer cells is much greaterthan it formerly appeared to be on thebasis of measurementson solid tumors.Utilizable energy of respiration andfermentation. Since the discovery of theoxidation reaction of fermentation in1939 (20), we have known the chemicalreactions by which adenosine diphos-phate is phosphorylated to adenosinetriphosphate in fermentation; and sincethen we have found that 1 mole of fer-mentation lactic acid produces 1 mole ofadenosine triphosphate (ATP).The chemical reactionsby which ATPis synthesized in respiration are still un-known, but it can be assumed, accordingto the existing measurements (21), that7 moles of ATP can be formed when 1mole of oxygen is consumed in respira-tion.ATP quotients. If we multiply Qo2 by7 and QMN2 by 1, we obtain the numberof cubic millimeters of ATP that 1 milli-gram of tissue (dry substance) can syn-thesize per hour (22,400 cubic milli-

meters= 1 millimole of ATP). We callthese quotients QATP02 and QATpN2, ac-cording to whether the ATP is formed byrespiration or by fermentation, respec-tively.Energy production of cancer cells andof normal body cells. In Table 1, the Qvalues of some normal body cells are con-trasted with the Q values of our ascitescancer cells.The cancer cells have about as muchenergy available as the normal body cells,but the ratio of the fermentation energyto the respiration energy is much greaterin the cancer cells than it is in the nor-mal cells.

Uncoupling of respiration. If a youngrat embryo is transferredfrom the amni-otic sac to Ringer's solution, the pre-viously transparent embryo becomesopaque and soon appears coagulated(17). At the same time, the connectionbetween respiration and phosphorylationis broken;that is, although oxygen is stillconsumed and carbon dioxide is still de-veloped, the energy of this combustionprocess is lost for life. If the metabolismquotients had previously been

Qo2 = - 15, QMO2 = 0, Q1 N2= 25,QATpO2=105, QATPN2 25

in the amniotic fluid, afterward, in Ring-er's solution, they areQo2 = - 15, Q.,2 = 25, Q,N2 = 25,

QATPO2 = 0) QATPN2 = 25Because of uncoupling of respirationand phosphorylation, the energy produc-tion of the embryo has fallen from

QATPO2 + QATPN2 = 130, to 25; since theuncoupling is irreversible, the embryodies in the Ringer's solution.This example will show that the firstphase of carcinogenesis, the irreversibledamaging of respiration,need not be anactual decrease in the respiration quo-tient but merely an uncoupling of respira-tion, with undiminished over-all oxygenconsumption. Ascites cancer cells, whichowe their origin primarily to an uncou-pling of respiration, could conceivablyhave the following metabolism quotients,for example:

Qo2 = - 50, QM02 = 100, QMN2 100,QATPO2 = 0, QATPN2 = 100

which would mean that, despite greatrespiration, the usable energy productionwould be displaced completely towardthe side of fermentation. One will nowhave to search for such cancer cellsamong the ascites cancer cells. Solidtumors-and especially solid spontane-ous tumors-need no longer be subjectedto sch examinations today, of course,since the solid tumors are usually so im-pure histologically.Aerobic fermentation. Aerobic fer-mentation is a property of all growingcancer cells, but aerobic fermentation

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without growth is a propertyof damagedbody cells-for example, embryos thathave been transferred from amnioticfluid to Ringer's solution. Since it is al-ways easy to detect aerobic fermentationbut generally difficult to detect growth,or lack thereof, of body cells, aerobicfermentation should not be used as a testfor cancer cells, as I made clear in 1928(19).Nevertheless, misuse is still made ofaerobic fermentation. Thus, O'Connor(22) recently repeated our old experi-ments on the aerobic fermentationof theembryo that has been transferred intoRinger's solution, but he drew the con-clusion that the growth of normal bodycells is completed at the expense of theaerobic fermentation, even though it haslong been established that the embryodoes not ferment aerobically when itgrows in the amniotic fluid.Respiratory poisons. The specific respi-ration-inhibiting effect of arsenious acidand the irreversibility of its inhibitionswere discovered in the first quantitativeworks on cell respiration (23, 24). Thereis abundant literature on the carcino-genesis by arsenic, particularlyon arseniccancer after treatment of psoriasis andon the cancer of grape ownerswho spraytheir vineyardswith arsenic. The specificrespiration-inhibiting effect of hydrogensulfide has likewise been described byNegelein (25), and carcinogenesisby de-rivatives of hydrogen sulfide has beenrecently described by D. N. Gupta (26).The irreversible nhibition of cell respi-ration by urethane was discovered early(27) as well as the fact that the urethaneinhibition is more irreversible the higherthe temperature. In sea urchin eggs, theeffect of urethane was investigated, notonly on the metabolism, but also on celldivision in studies (28) from which thelater urethane treatment of leukemia wasdeveloped. The physicochemical mecha-nism by which urethane and other in-different narcotics inhibit cell respirationwas cleared up in 1921 (29). Only muchlater did the carcinogenic effect of ure-thane become known. Actually, multiplelung adenomas can often be producedin 100 percent of the mice treated withsmall doses of urethane (30).Oxygen deficiency. Short-period oxy-gen deficiency irreversibly destroys therespiration of embryos (16) withoutthereby inhibiting the anaerobic fermen-

tation of the embryo. If such embryosare transplanted, teratomas are formed(31). It has recently been reported that,in the development of the Alpine sala-mander, malformations occurred whenthe respiration was inhibited by hydro-cyanic acid in the early stages of em-bryonal development (32).Goldblatt and Cameron (3) reportedthat, in the in vitro culturing of fibro-blasts, tumor cells appeared when thecultures were exposed to intermittentoxygen deficiency for long periods,whereas, in the control cultures, no tumorcells appeared. In the discussion at theStuttgart convention, Lettre cited againstGoldblatt and Cameron the fact thatanother American tissue culturist, Earle,has occasionally obtained tumor cellsfrom fibroblasts for reasons unknown tohim and in an unreproduceablemanner,but this objection does not seem weighty,and the latter part is untrue (33). In anyevent, here is an area in which the meth-ods of tissue culture could prove usefulfor cancer research. But warnings mustbe given against metabolism measure-ments in tissue cultures, if and when thetissue cultures are mixtures of growingand dying cells, especially under condi-tions of malnutrition. An example of thelatter type of confusion is involved in thediscussion by Albert Fischer (34), espe-cially in the chapter "Energy exchangeof tissue cells cultivated in vitro."Rous agent. If the Rous agent is in-oculated into the chorion of chick em-bryos, tumors originate in the course ofa few days-as rapidly as in the trans-plantation of cancer cells. The tumorsformed are not chorion tumors but Roussarcomas. The Rous agent, to which aparticle weight of 150 million is ascribedat present, is therefore capable of trans-mitting the morphological properties ofthe Rous sarcoma;and whatever we callthe Rous agent-"hereditary unit," cellfragment, microcell, or spore-the trans-mission of the Rous sarcoma by the Rousagent is, in any case, nothing more thana transplantation and is to be differen-tiated strictly from the production of achicken sarcoma by methylcholanthrene,which is a neoformationof a tumor fromnormal body cells and as such takes along time.

The metabolism of the chicken sarco-mas, whether produced by the Rousagent or by methylcholanthrene, is the

Table 1. Contrast of the Q values of some normalbody cells with the Q values of ascitescancer cells.Cells Qo2 QMN QATPO2 QATPN2 QATPO2 + QATPN2

Liver -15 1 105 1 106Kidney - 15 1 105 1 106Embryo (very young) - 15 25 105 25 130Cancer - 7 60 49 60 10924 FEBRUARY 1956

same and does not differ in any way fromthe metabolism of the tumors of otheranimals (35). In the first case, however,the fermentation potential has beentransplanted with the Rous agent,whereas in the second case the fermen-tation has been intensified by selectionfrom normal body cells under the actionof the methylcholanthrene.Addendum: in vitroCarcinogenesis and Metabolism

Since this paperwas prepared, strikingconfirmation and extension of its mainconclusions have been obtained from cor-related metabolic and growth studies oftwo lines of tissue culture cancer cellsof widely differing malignancy that wereboth derived from one and the same nor-mal, tissue-culture cell (36). The singlecell was isolated some 5 years ago froma 97-day old parent culture of normalsubcutaneous adipose tissue of a strainC3H/He mouse by Sanford, Likely, andEarle (33) of the National Cancer In-stitute. Up to the time that the single-cell isolation was made, no tumors devel-oped when cells of the parent culturewere injected into strain C3H/He mice.Injections of in vitro cells of the lines1742 and 2049 (formerly labeled sub-strains VII and III, respectively) firstproduced tumors in normal C3H/Hemice after the 12th and 19th in vitrotransplantgenerations, respectively; after1Y2 years, the percentage production ofsarcomas was 63 and 0 percent, respec-tively; and after 3 years, it was 97 and 1percent, respectively, with correspond-ingly marked differences in length of in-duction period. Despite such gross dif-ferences in "malignancy" in vivo, therates of growth of the two lines of cellsmaintained continuously in vitro haveremained nearly identical and relativelyrapid. Nevertheless, the metabolism ofthe two lines of cancer cells, whose ma-lignancy was developed in vitro, has beenfound by Woods, Hunter, Hobby, andBurk to parallel strikinglythe differencesin malignancy observed in vivo, in amanner in harmony with the predica-tions and predictions of this article.The metabolic values were measuredfollowing direct transfer of the liquidcultures from the growth flasks intomanometric vessels, without notable al-teration of environmental temperature,pH, or medium composition (horseserum, chick embryo extract, glucose,bicarbonate, balanced saline). The val-ues obtained thus accurately representthe metabolism of growing, adequatelynourished, pure lines of healthy cancercells free of admixture with any othertissue cell type. The anaerobic glycolysisof the high-malignancy line 1742 wasQm2 = 60 to 80, which is virtually max-

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imum for any and all cancer cells previ-ously reported, including ascites cells(12-14). The anaerobic glycolysis of thelow-malignancy line was, however, onlyone-third as great, QMN2 20 to 30. Theaverage aerobic glycolysis values for thetwo lines were in the same order, QM2 =30 and 10, respectively, but of lowermagnitude because of the usual, pro-nounced Pasteur effect, greater in line1742 than in line 2049 (QMN - QM02 =about 40 and 15). On the other hand,the rates of oxygen consumption werein the converse order, being smaller inline 1742 (Qo2=5 to 10) than in line2049 (Qo2 = 10 to 15), corresponding toa greater degree of respiratorydefect inline 1742. The respiratorydefect in bothlines was further delineated by the find-ing of little or no increase in respirationafter the addition of succinate to eitherline of cells, in contrast to the consider-able increases obtained with virtually allnormal tissues (9); and the respiratoryincrease with paraphenylenediaminewaslikewise relatively low, compared withnormal tissue responses.A further notable difference betweenthe two cell lines was the very muchlower inhibition of glycolysis by podo-phyllin materials (anti-insulin potentia-tors) observed with line 1742 comparedwith line 2049 (for example, 10 and 70percent, respectively, at a suitably lowconcentration). This result would be ex-pected on the basis of the much greaterloss of anti-insulin hormonal restraint ofglucose metabolism, at the hexokinasephosphorylating level, as the degree ofmalignancy is increased, just as was re-ported for a spectrum of solid tumors(14).Finally, the high-malignancyline 1742cells have been found by A. L. Schadeto contain 3 times as much aldolase asthe low-malignancy line 2049 cells (11,-300 versus 3700 Warburg activity unitsper milliliter of packed cells extracted),and about 2 times as much a-glycero-phosphate dehydrogenase [2600 versus

1400 Schade activity units (13) per mil-liliter of packed cells extracted]. Thepotential significance of these indicatedenzymic differences in relation to theparallel glycolytic differences, measuredwith aliquots of the same cell cultures,is evident, and may well be connectedwith the corresponding hexokinase sys-tem differences.The new metabolic data on the tworemarkably contrasting lines of cancercells, which originated from a single, in-dividual cell and have been maintained

exclusively in vitro over a period ofyears, epitomize and prove finally themain conclusions of this article, whichare based on decades of research. Suchmetabolic analyses provide promise of apowerful tool for diagnosis of malig-nancy in the ever-increasing variety oftissue-culture lines now becoming avail-able in this rapidly expanding biolog-ical and medical field, where character-ization of malignancy by conventionalmethods (animal inoculation or other-wise) may be difficult or impracticable.This metabolic tool should be especiallyimportant in connection with the use oftissue cultures for the evaluation ofchemotherapeutic agents or other con-trol procedures.

References and Notes1. The transplantable ascites cancer was discov-ered by H. Loewenthal and G. Jahn [Z. Krebs-forsch. 37, 439 (1932)]. G. Klein (Stockholm)expanded our knowledge about the physiologyand morphology of the ascites tumors andshowed their great advantages as experimentalmaterial. [Exptl. Cell Research 2, 518 (1951)].2. F. Lynen, Naturwissenschaften 30, 398 (1942);Ann. Chem. Justus Liebig 573, 60 (1951).3. H. Goldblatt and G. Cameron, J. Exptl. Med.

97, 525 (1953).4. 0. Warburg, Pfliigers 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. 11,1105 (1951); 9, 311 (1949); 9, 325 (1949); 16,351 (1955). Science 102, 591 (1945); 111, 572(1950). AAAS Research Conf. on Cancer (Sci-ence Press, Lancaster, Pa., 1945), p. 162. J.Wash. Acad. Sci. 42, 169 (1952). Pigment CellGrowth, M. Gordon, Ed. (Academic Press,New York, 1953), p. 335. Biochem. et Biophys.Acta 12, 329 (1953). Proc. Am. Assoc. Cancer

Research 1, 7 (1954). Proc. Soc. Exptl. Biol.Med. 83, 62 (1953). Phytopathology 33, 637,766 (1943); 36, 472 (1946); 31, 978 (1941);32, 288 (1942). Am. J. Botany 33, 12a (1946);38, 419 (1951).7. A compilation of American works on the me-tabolism of the grana, in which my results of1914 (4) have been confirmed, is given by G.Hogeboom, W. Schneider, and M. Striebichin Cancer Research [13, 617 (1953)]. In a veryspecial case-nucleated red blood cells of birds,which contain no grana or only poorly visibleones-the entire respiration can be centrifugedoff with the cell nuclei [O. Warburg, Hoppe-Seyler's Z. physiol. Chem. 70, 413 (1913)].8. A. Graffi, Z. Krebsforsch. 49, 477 (1939).9. D. Burk, Symposium on Respiratory Enzymes(Univ. of Wisconsin Press, Madison, 1942), p.235; J. G. Kidd, R. J. Winzler, D. Burk,Cancer Research 4, 547 (1944).10. L. Pasteur, ttudes 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. 0. 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. I am indebted to Georg Klein of the Karo-linska Institute, Stockholm, Sweden, for hisEhrlich strain of mouse ascites cells.16. 0. Warburg, K. Posener, E. Negelein, Bio-chem. Z. 152, 309 (1924).17. E. Negelein, ibid. 165, 122 (1925).18. 0. Warburg et al., ibid 189, 114, 175, 242(1927); 193, 315 (1928); 197, 175 (1928); 204,475, 479 (1929).19. 0. Warburg, ibid. 204, 482 (1929).20. 0. 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 (1950).23. 0. Warburg, Hoppe-Seyler's Z. physiol. Chem.70, 413 (1911); M. Onaka, ibid. 70, 433 (1911);71, 193 (1911).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, Pfliiger's Arch. ges. Physiol. 147, 100(1912).28. 0. Warburg, Hoppe-Seyler's Z. physiol. Chem.66, 305 (1910); 70, 413 (1911).29. -, Biochem. 119, 134 (1921.)

30. C. D. Larsen, J. Natl. Cancer Inst. 8, 63(1947).31. 0. Warburg,Biochem. Z. 228, 257 (1930).32. H. Tiedemann and H. Tiedemann, Z. Natur-forsch. 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 (Copen-hagen, Denmark, 1946).35. 0. Warburg, Biochem. Z. 160, 307 (1925); D.Burk et al., J. Natl. Cancer Inst. 2, 201 (1941).36. This summary of studies of various collaborat-ing groups of investigators was prepared byDean Burk at Professor Warburg's request.

A university has its responsibility to the student as well as an obligation to add to knowl-edge. Let us not put our students in the position of observing or being a party to practicescontrary to the ideal for a university and for his future career. If the student sees his insti-tution engaged in fundamental research, freely supported and freely conducted, he willlearn that independence in investigation is man's right and a university's responsibility toexemplify.-N. PAULHUDSON,Ohio State University Graduate School Record, December1951.

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