DiAtomic O2xygen Info - 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
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
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
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
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
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
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
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.
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
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
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
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.
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
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.
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.
Contrast of the Q values of some normal body cells with the
Qvalues of ascites cancer cells.
QATPO2 + QATPN2
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 =
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 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).
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).
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."
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
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
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(1932)]. G. Klein (Stockholm) expanded ourknowledge about the
physiology and morphology of the ascites tumors and showed their
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- 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)].
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Stockholm, providedEhrlich strain of mouse ascites cells.
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NATURFORSCH. 9b, 371 (1954).
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- This summary of studies
was prepared by Dean Burk at ProfessorWarburg's request.
information presented above is information only. It should not be
considered as medical advice. It is supplied so that you can make an
informed decision. Please consult your Health Professional before
considering any therapy or therapeutic protocol.
Oxyrich Technical Data