Glycolytic and Respiratory Properties of Malignant and
Nonmalignant LymphoblastsCultured in Vitro
C. J. HOMBURG,* C. J. Bos,t W. M. DE BRUYN,* AND P. EMMELOTt
(Antoni van Leeuwenhoek-Huis: The Netherlands Cancer Institute, Amsterdam, The Netherlands)
SUMMARY
Two strains of lymphoblasts, the malignant MB VIA and the nonmalignant MB
HI', both derived by continuous subculture from an explant of a mouse lymphosarcoma, were cultured in large amounts. The respiration, anaerobic and aerobic
glycolysis of the cells were measured, and the effects of ~, 4-dinitrophenol and the
tumor-inhibitory compound 4(4'-dimethylaminostyryl)quinoline on the latter processes were studied. The respiration of the malignant lymphoblasts was equal to or
higher than that of the nonmalignant ones according to the medium in which the
latter cells had been cultivated. By a similar dependency the anaerobic glycolysis of
the malignant lymphoblasts was only slightly or moderately higher than the corresponding process of the nonmalignant lymphoblasts. Both the relative and the absolute
Pasteur effects were more pronounced in the malignant than in the nonmalignant
cells, whereas the inhibition of glycolysis by respiration could be completely eliminated
by 2,4-dinitrophenol or the styrylquinoline in the two types of cells.
The results stood thus in marked contradiction to the basis of Warburg's theory on
the origin of cancer cells, which holds that a respiratory impairment followed by a
gain in glycolysis leads to cancer cells.
It is concluded that Warburg's theory is not correct.
According to Warburg (24, 25) cancer cells have
a decreased oxidative and an increased fermentative metabolism, as compared with that of normal
cells. From this generalization, originally based on
the study of solid tumors and more recently of
ascites tumor cells, Warburg derived a theory on
the origin of cancer cells (~5). In this he envisaged
t h a t those body cells which, after receiving a nearfatal injur 5, in regard to their respiratory equipment, manage to survive by developing an intense
glycolysis for generating the necessary energy, automatically behave as cancer cells. This theory is
not sufficient to explain how the change in the intermediary metabolism can be made permanent
unless an extra-chromosomal autonomy of the respiratory granules is invoked--a hypothesis which,
in view of recent genetic findings, is not the only
possible one (16) (let alone the fact that it is completely obscure why the alleged change should
* Department of Experimental Cytology.
t Department of Biochemistry.
Received for publication September 8, 1960.
transform a normal cell into a cancerous one).
Moreover, it is our contention that, in contrast to
Warburg's explicit statement (25), ascites tumor
cells cannot be used to collect data for a theory on
carcinogenesis (14) and that, for reasons presented
elsewhere (9), Warburg's theory, when tested for its
consequences, cannot stand up to the experimental
results.
Nevertheless, the theory appears to have received support from certain experiments in which
the glycolytic and respiratory rates of cells cultured in vitro have been measured. Foremost
among these is an experiment, already quoted by
Warburg in his 1956 paper, in which it was found
that a low- and a high-cancer-producing strain,
derived from one and the same normal fibroblast
by continued subculture in vitro, differed in metabolic properties in that the low-cancer strain possessed a markedly higher respiration and a lower
glycolysis than did the high-cancer strain (25).
The results of other experiments, in which normal
cells cultured in vitro had been found to possess a
marked ability to glycolyze, were subsequently ex-
353
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354
Cancer Research
plained away by declaring the carcinogenicity of
tissue culture to a dogma and assuming that the
particular normal cells were actually in the process
of becoming malignant and had already acquired
what was considered as the typical pattern of tumor metabolism (26-28).
However, apart from the repeatedly reported
instances in which normal cells apparently did acquire cancerous properties which, it should be emphasized, go no further than to show that normal
cells cultured in vitro may be but are not necessarily
transformed into cancer cells, the opposite phenomenon, namely, the in vitro loss of the cancerous property, has also been observed (5-7, 20). Although it
cannot be determined whether the latter change did
result from a mutation or a selection of a pre-existing variant, the parent malignant culture not having been derived from a clone, such a nonmalignant strain may, nevertheless, form a suitable object for a metabolic comparison with the corresponding malignant strain, provided that it can
be established with reasonable certainty that the
two strains contain the same cell type. The latter
criterion seems to be satisfied in regard to the two
lymphoblast strains used in the present experiments: MB VIA, the malignant one, and MB III',
the nonmalignant one, both derived (1954/55 and
1951, respectively) by one of us from the parent
lymphosarcoma T 86157 which arose spontaneously in 1935 in a hybrid mouse (8). The natural
history of the nonmalignant lymphoblasts precludes that Warburg's interpretation of the socalled neoplastic pattern of the glycolysis and respiration of normal cells cultured in vitro (28-30)
can be applied to the present case. Some of the
present results, which are in disaccord with Warburg's theory, have already been presented briefly
in connection with other work (12), while Bailey,
Gey, and Gey, who have been studying the closely
related nonmalignant MB I I I strain of lymphoblasts also isolated by de Bruyn, recently reported
(1) that these cells, too, had retained the capacity
to produce high amounts of lactic acid, although
they had lost the ability to provoke a tumor in
mice.
M A T E R I A L S AND M E T H O D S
Lymphoblast strains.--The strains MB I I I ' and
MB VIA have been derived from the transplantable mouse lymphosarcoma T 86157 (8). In September, 1947, small pieces of a subcutaneous transplant from T 86157 were explanted by one of us
(5) in roller tubes (nos. 1-9) at the laboratory of
Dr. G. O. Gey, Baltimore. During the first weeks
of cultivation lymphoblasts and fibroblasts were
present in all the tubes. At the end of 1947 the
Vol. ~1, April 1961
fibroblasts outgrew the lymphoblasts in tubes 1-4.
The fibroblasts, designated as strain MB II, have
been maintained in continuous culture. As yet,
they never have produced tumors in susceptible,
newborn mice. In the tissue of tube 9 the lymphoblasts outgrew the fibroblasts. Subsequently a
strain was developed free from fibroblasts, bearing
the case number MB 13, later altered to MB III.
The cells can be maintained without clot as a fluid
culture in roller tubes or in slightly modifed
Erlenmeyer flasks in various nutritional fluids. In
1951 cells from strain MB I I I were transferred and
maintained as strain MB I I I ' in a medium containing 25 per cent human placental cord serum
and 75 per cent Tyrode (6, 7, 20). In 1955 a change
of medium was again made, the MB I I I ' now being
transferred to a culture medium containing 3 parts
of horse serum and 9 parts of Tyrode solution. The
latter medium is designated as medium A. The
MB I I I ' lymphoblasts when injected in large
amounts in newborn mice, which were susceptible
to tumor production by other strains of lymphoblasts, do not produce a tumor. In the tubes 5 and
6 both fibroblasts and lymphoblasts remained.
This strain, MB I, has been maintained in continuous culture. At the end of 1954, lymphoblasts of
strain MB I were transferred to a medium containing Tyrode solution, placental human cord serum,
and mouse embryo extracts, and maintained as
strain MB VIA. These lymphoblasts produce tumors in susceptible mice.
The two strains of lymphoblasts MB I I I ' and
MB VIA, consisting of free cells, differ not only in
that the first is nonmalignant whereas the second
is malignant, but also in their morphology. The
cells of strain MB I I I ' vary more in size and shape
than those of strain MB VIA. Among the former
are binucleated cells, and the relation between the
nucleus and the cytoplasm seems to be decreased.
However, no marked differences between the number of cells per mg. of dry weight of both cell types
appeared to e~xist. During investigations carried
out with the electron microscope in collaboration
with Dr. E. L Benedetti, 1 virus-like particles were
observed in the malignant lymphoblasts of strain
MB VIA. No such particles have as yet been detected in the nonmalignant MB I I I ' lymphoblasts.
For the present experiments the MB VIA cells
were cultured in a medium containing 7 parts of
Tyrode solution, 2 parts of mouse embryo extract,
4 parts of horse serum, and 1 part of human placental cord serum (medium B). The MB I I I ' cells
were cultured either in the latter medium or in
medium A as mentioned above. The cells were cull W. M. de Bruyn and E. L. Benedetti, unpublished observations.
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HOMBURG et al.--Metabolic Quotients of Lymphoblasts
tured at 37 ~ C. in modified Erlenmeyer flasks containing 50 ml. of medium with occasional shaking;
gas phase: air. Twice weekly half of the contents of
each flask was transferred to a new flask, and 25
ml. of fresh medium was added to both. This was
continued until sufficient cells were present for the
biochemical experiments. After 4 days the MB
I I I ' lymphoblasts had multiplied by an average
factor of 2 in medium A and of 3 in medium B; the
corresponding value for the MB VIA lymphoblasts
in medium B was 2. The cells used in the biochemical experiments were in good condition. Direct determinations of lactic acid produced by the two
cell types in the culture media showed no significant differences. 2
The MB I I I ' lymphoblast strain is labeled nonmalignant because of the fact that these cells fail
to give rise to tumors on inoculation in mice susceptible to the MB VIA lymphoblasts. Though the
former cells may be normal cells, they cannot be
designated as such for lack of experimental verification. However, the finding that the MB I I I ' cells
do not grow in newborn mice makes it unlikely
t h a t the failure to give rise to cancerous growth
has an immunological basis. Moreover, it may turn
out to be of considerable interest that the malignant cells, in contrast to the MB I I I ' cells, do contain virus-like bodies. Though the MB I I I ' cells
may not be the ideal type of material, on account
of the fact that the strain was not cloned, we feel
it as an advantage that these cells have been derived (or selected) from the malignant cells, because this rules out that the former cells are in a
state of becoming malignant (26-28). Since biological evidence for the cancerous nature of the
MB I I I ' cells is completely lacking, we, furthermore, feel justified to use these cells for comparative biochemical experiments.
Measurement of glycolysis and respiration.--The
cells were centrifuged and washed once with physiological saline. Aliquots of the concentrated stock
suspensions were added to manometric flasks
either containing Krebs-Ringer bicarbonate or
phosphate buffer. Gas phase: 95 per cent 02 d- 5
per cent C02, and 95 per cent N2 d- 5 per cent CO2,
or 100 per cent nitrogen and air, respectively. In
all experiments except those illustrated in Table 4
the glycolysis was assayed in bicarbonate buffer.
The respiration was always measured in phosphate
buffer. Total volume of the flasks was 1.6 ml. Incubation was carried out during 60 minutes at
37 ~ C. After this period the lactic acid was determined (2), and the data were converted to the gly2 W. M. de Bruyn and C. J. Homburg, unpublished observations.
355
o~ which are listed
colytic quotients, QoNS~and Qoo~,
in Tables 1-3. The experiments have been conducted over a period of more than 1 year; for example, 10 months elapsed between the two experiments with the MB VIA lymphoblasts listed in
Table 3. No evidence of a change in metabolism
during this period was observed. The differences in
the glycolytic rates of the MB I I I ' cells, among the
various experiments listed in Table 2, appear to be
due to the amount of cells incubated per flask. Although the results tend to show an inverse relation
between glycolysis and amount of MB I I I ' cells
per flask, this phenomenon has not been studied
further. Moreover, such an inverse relation was
not observed in the experiments with the MB VIA
and I I I ' cells cultured in medium B.
RESULTS
Table 1 illustrates three typical experiments
carried out with the parent lymphosarcoma T
86157, continuously maintained in vivo, with (i)
slices of solid transplants, (ii) cell suspensions prepared from such transplants as described previously (12), and (iii) cells of the tumor after
growth in the ascites form during several generations. Cells of the MB VIA strain of lymphoblasts,
collected from a batch of culture medium, were injected subcutaneously into newborn mice, the animals were killed 3.5 weeks later, and slices of the
solid tumors were also investigated (iv). Neither
the anaerobic nor the aerobic glycolysis, assayed
in bicarbonate buffer, of the slices from tumors
MB VIA and T 86157 and of the cell suspensions
prepared from the latter, did differ to any appreciable extent, but the corresponding values found
for the T 86157 ascites cells, including the rate of
respiration measured in the absence of glucose,
were much higher than the former. The aerobic
lactate production by the various preparations
was the same whether assayed in Krebs-Ringer
phosphate or bicarbonate buffer. Addition of glucose led to an inhibition of the endogenous respiration in most of the experiments.
The glycolytic rates of the cells of strain MB
I I I ' harvested from medium A, and of strain
MB VIA harvested from medium B, are presented
in Table 2. Included in Table 2 are the effects of
the uncoupling agent 2,4-dinitrophenol (10-4 M)
and the tumor-inhibitory compound 4(4'-dimethylaminostyryl)quinoline (1.5 X 10-4 M, only about
30 per cent being in real solution), which has earlier been found (10) to cause respiratory inhibition,
adenosine triphosphatase activation, and uncoupling of the oxidative phosphorylations in mitochondria. It is shown that the nitrophenol and the
styrylquinoline increased the aerobic glycolysis of
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356
both the malignant and the nonmalignant lymphoblasts to the anaerobic level.
Since the absolute :Pasteur effect (Q~), - Q%~)
was much more pronounced in the malignant
(compare also Table 3) than in the nonmalignant
cells, it followed that the respiratory process, v/z.
the mitochondrial oxidative phosphorylations, of
the former cells prevented the formation of a much
greater amount of lactic acid than the corresponding process of the latter cells. The anaerobic glycolysis of the I I I ' cells, cultured in medium A
Vo]. ~1, April 1961
(average, QoN:,2 = 29.6) was not very nmch different from that of the VIA cells, cultured in medium B (average QcSS,= 33.7, comprising the data
of Tables r and 3). Though such a comparison is
not strictly warranted in view of the fact that the
cell strains had been cultured in different media, it
is still of interest, since the two cell types showed
approximately the same rate of multiplication in
the two media.
A strict comparison is possible between the data
of the two cell types in Tables 3 and 4, since in
TABLE 1
GLYCOLYTIC AND RESPIRATORY RATES OF THE LYMPHOSARCOMA T 86157, UNDER VARIOUS CONDITIONS OF GROWTH AND ASSAY, AND OF THE RELATED
TUMOR M B VIA GROWN AS SOLID TRANSPLANT
Qoe
ExP.
TUMOR
((~ROWT~I/ASSAYED)
T 86157
T 86157
T 86157
M B VIA
i
ii..~
111
iv
N9
CONDITION
Solid/slices
Solid/free cells
Ascites
Solid/slices
Qc62
26.0
27.2
52.3
31.5
21.4
o~
Qcb2
9.0
13.2
30.2
1S .3
9.6
Glucose
present
Glucose
absent
3.2
4.4
5.0
S .4
5.2
8.7
5.5
5.8
TABLE 2
GLYCOLYTIC RATES OF NONMALIGNANT M B I I I ' LYMPHOBLASTS, CULTURED IN MEDIUM A, AND OF MALIGNANT M B VIA LYMPHOBLASTS,
CULTURED IN MEDIUM B
Effect of the uncoupling agent 2,4-dinitrophenol and the tumor-inhibitory compound 4(4'dimethylaminostyryl)quinoline.
D N P = 2,4-dinitrophenol;
DSQ = 4(4'-dimethylaminostyryl)quinoline.
Strain
MB I I I '
M B VIA
Dry weight of
cells/flask
(mg)
N2
O2
Qco2
N 2_QO2
co2
Addition
Qco~.
QCO2
8.0
None*
DSQ*
20.0
19.9
11.4
17.9
8.6
2.0
7.8
None*
DSQ
DNP
26.4
26.4
25.8
17.8
24.2
34.1
8.6
2.2
8.3
4.8
None
DNT
30.5
31.8
19.1
38.0
11.4
--6.2
3.8
None
DSQ
41.7
47.1
31.8
46.5
9.9
0.6
Nonef
29.6t
2o.of
9,6~
None
DNP
34.4
40.1
14.2
41.5
None
DNT
DSQ
25.6
25.6
24.8
8.7
27.6
20.1
5.2
3.2
* Qo~ mentioned in the text.
- -
- -
- -
20.2
1.4
16.9
2.0
4.7
t Averages.
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HOMBURG
et al.--Metabolic Quotients of Lymphoblasts
these cases the IIV cells had also been grown in
medium B. The anaerobic glycolysis of the latter
cells was now in general lower (average Q~,2 =
~1.7; Table 8) than that of the malignant VIA
cells, but no significant difference between the
rates of aerobic glycolysis of the two cell types
could be observed (average, Q%~ = 1~.9 for the
III', and = 14.0 for the VIA lymphoblasts [Tables
and 8]). However the comparison between the
l I I ' (cultured in medium A or B, or the latter data
taken together [Table 3, footnote]) and the VIA
cells is made, it appears that the anaerobic glycolysis of the latter is at the most about 50 per cent
357
m medmm A was similar to that of the VIA cells
cultured in medium B (Tables 3 and 4; note also
the similarities in the rate of multiplication of the
cells and their anaerobic glycolysis). However,
when the nonmalignant I I I ' cells were grown in
medium B, they multiplied faster than the VIA
cells (or the I I I ' cells in medium A) and showed a
lower respiration (Tables 3 and 4) than the latter
cells. The difference in respiration between the
two cell types, cultured in medium B, was most
pronounced in the absence of glucose, under which
condition the malignant cells took up nearly twice
as much oxygen as the nonmalignant cells. About
TABLE 3
GLYCOLYTIC AND RESPIRATORY RATES OF NONMALIGNANT M B I I I ' AND MALIGNANT i B
LYMPHOBLASTS, BOTH CULTURED IN MEDIUM g
Qo~
DRY WEIGHT
STRAIX
OF
CELLS/FLASK
N2
Qco~
Q r •_002
O9
Qcb2
CO2
~CO2
(~G.)
MB III'
MB VIA
10.3
10.2
8.7
12.8
12.6
VIA
11.1
9.3
5.8
GLucose
present
Glucose
absent
2.8
3.7
3.4
2.7
3.7
4.0
3.3_~
3.5~.
26.5
19.5
19.0
15.4
10.2
18.2
21.7*
12.9"
36.4
38.2
18.1
14.9
23.3
5.0
5.0
6.9
6.5
33.7?
14.0t
19.7t
5.0~
6.v$
8.8*
18.3
* Averages; if the data of Table '2 on tile M B I I I ' cells (medium A) are included, the averages read
26.2, 17.0, and 9.2, respectively.
t Averages, including the data of Table 2 on the M B VIA cells.
+ Averages.
higher than that of the former cells, and that the
absolute Pasteur effect is always more pronounced
in the malignant VIA than in the nonmalignant
I I I ' cells. Similar results were obtained with both
cell types when cultured in medium B and assayed
in phosphate buffer. Table 4 represents the two
series of data each representing the average values
of two closely agreeing experiments. The aerobic
glycolysis of both cell types was of the same magnitude whether assayed in phosphate or in bicarbonate buffer, but the anaerobic glycolysis was
less in the former than in the latter medium.
The respiration of the cells was always measured
in phosphate buffer. In the experiments of Table
the Qo2 was measured only in two cases with I I I '
cells suspended in the presence of glucose and
found to amount to 4.7 and 5.1. In the presence
of 4(4'-dimethylaminostyryl)quinoline the Qo~
dropped to 1.8. It is of interest that the glucosesupplemented respiration of the I I I ' cells cultured
TABLE 4
GLYCOLYSIS AND RESPIRATION OF MALIGNANT MB VIA
AND NONM&LIGNANT M B l I I ' LYMPHOBLASTS GROWN IN MEDIUM B
Both series of data represent the average values of two
closely agreeing experiments carried out in phosphate buffer.
From 10 to 12 mg. dry weight of cells were used.
~ [ O L E S LACTATE PRODUCED/
10 MG DRY WEIGHT OF CELLS/
6 0 bIIN,
STRAIN"
M B VIA
M B III'
Anaerobic
(nitrogen)
~ATOMS OXYGEN
CONSUMED/10 MG
DRY WEIGHT OF
C E L L S / 6 0 MIN.
Aerobic
(air)
Glucose
present
Glucose
present
12.6
8.0
6.9
5.6
Glucose
present
Glucose
absent
4.6
3.1
6.2
3.2
GLucose
ab____sent
I
1.5
2.8
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Cancer Research
Vol. ~1, April 1961
50 per cent more oxygen was consumed by the the Pasteur effect. It is generally agreed (4, 11, 22)
malignant than by the nonmalignant cells when that a competition between glycolytic, substratethe incubation was carried out in the presence of level phosphorylations and respiratory chainglucose. The latter was due to the fact that the linked oxidative phosphorylations for inorganic
endogenous respiration of the I I I ' cells was hardly, phosphate and adenine nucleotides exists; the
if at all, inhibited in the presence of glucose, where- former compound and adenosine diphosphate are
as that of the VIA cells was inhibited markedly being used in both types of phosphorylations,
(Tables 3 and 4). This difference may be attrib- whereas the adenosine triphosphate generated by
uted to a Crabtree effect (11), i.e., glycolysis in- the latter type of phosphorylation apparently does
hibiting the endogenous respiration, operating in not equilibrate readily with the cell sap and thus
the malignant but not, or less so, in the nonmalig- retards the hexose phosphorylation. As soon as the
nant cells. It is, however, not certain whether the mitochondrial phosphorylations are eliminated by
latter distinction is so real as it appears at first inhibiting electron transport by anaerobiosis or
sight, since the I I I ' cells showed a higher endoge- respiratory inhibition, such as brought about in
nous lactate production than the VIA cells (Table the present experiments by 4(4'-dimethylamino4), it cannot be excluded that in the former cells styryl)quinoline or by uncoupling by such agents
some measure of competition between glycolysis as dinitrophenol, the glycolytic reactions may disand respiration existed already at the endogenous pose of all inorganic phosphate and adenine nuglucose concentration. On the other hand, the cleotides available to the cell. Concomitantly, the
glycolytic potency (as shown by the anaerobic main ATP production shifts over to the cell sap,
glycolysis, Table 4) of the malignant VIA was thus guaranteeing an unimpaired glucose phosmore pronounced than that of the nonmalignant phorylation and an increased lactate production as
I I I ' cells. In all cases examined a linear uptake of compared with normal aerobic conditions. Since
oxygen during 60 minutes at 37 ~ C. was observed; these effects could be realized in the present exno evidence for an inhibition of respiration by a periments, the results corroborated the above thepH shift was found.
ory and thus unequivocally attested to the phosphorylation-linked status of the tumor mitochonDISCUSSION
drial oxidations. In this connection it is of interThe present investigation has brought out three est to consider some of the details of the latter
things very clearly. First, the anaerobic glycolysis processes. The data of Table 4 allow two kinds of
of the malignant MB VIA lymphoblasts, cultured calculations to be made. The first calculation,
in medium B, was only slightly to moderately yielding more exact values than the second, shows
higher (at the most 50 per cent) than that of the that not only the absolute 3 but also the relative
nonmalignant MB I I I ' lymphoblasts cultured in Pasteur effect (/zmoles of lactate brought to dismedium A and B, respectively. This result stands appearance per gatom oxygen consumed) is more
in marked contrast to those reported by Woods et pronounced in the case of the malignant than of
d. (31) and by Warburg (25) which showed much the nonmalignant cells: ( 1 2 . 6 - 6.9)/4.6 = 1.23
greater differences between the glycolytic rates of and (8.0 - 5.6)/3.1 = 0.8 izmoles lactate, respeclow-and high-cancer-producing fibroblast strains. tively. This difference is very probably due to a
Second, the respiration of the malignant lympho- more pronounced competition between the glyblasts was of a similar or (much) higher magnitude colytic and respiratory phosphorylations in the
than that of the nonmalignant cells, the variation malignant cells than in the nonmalignant ones,
being due to the kind of medium used for culturing given their glycolytic potencies, number and acthe nonmalignant cells (either medium A or B) and tivities of mitochondria, rates of glucose entrance,
the presence or absence of glucose in the incuba- adenosine triphosphate-consuming processes, and
tion medium used to measure the respiration of the endogenous amounts of phosphate and adenine
malignant cells. Again, this result is different from nucleotides. A comparison between some of the
that quoted by Warburg (25). Third, the absolute latter properties of the parent lymphosarcoma T
Pasteur effect was always significantly more pro- 86157 ascites and other ascites tumor cells on the
nounced in the case of the malignant than of the one hand, and the cultured lymphoblasts on the
nonmalignant cells, and in both types of cells the other, shows definite differences. Not only is the
latter effect could be completely abolished by 2,4- glycolytic potency of the former cells more prodinitrophenol, which is known to uncouple the nounced (Table 1) than of the latter, but the
oxidative phosphorylations in the respiratory anaerobic glycolysis of the ascites cells is markedly
chain of mitochondria. Normally, i.e., in the ab- enhanced by 2,4-dinitrophenol (12), whereas that
sence of uncoupling agent, the latter reactions are
3 The absolute Pasteur effect is generally found to be
for good reasons considered to be responsible for high among tumors (8, 81).
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HOMBURG et al.--Metabolic Quotients of Lymphoblasts
of the lymphoblasts is not or only slightly increased. The conclusion may therefore be drawn
(compare :13) that, whereas the glycolytic potency of the ascites cells is much higher than the
rate actually attained under anaerobic conditions
as a result of the relatively slower high-energy
phosphate turnover, such is apparently not the
case m the lymphoblasts.
Before proceeding to the second calculation, it
seems well to consider briefly the possible effect of
the virus-like particles present in the malignant
lymphoblasts. Although no evidence is as yet
available to label these particles as tumor viruses,
we may still reckon with this possibility. It has
been shown (19) that the virus capable of inducing
certain neoplasms in the chick contains an active
adenosine triphosphatase. If the present particles
were to contain such an enzyme, the mitochondrial
respiration of the malignant lymphoblasts might
function in an uncoupled state (cf. also Graffi [18],
who applied this concept in an attempt to interpret the carcinogenicity of tumor viruses along the
lines of Warburg's theory). However, this possibility may be discarded in the present case in view
of the finding that the relative Pasteur effect was
more pronounced in the malignant than in the
nonmalignant cells.
A competition of a similar kind as operating in
the Pasteur reaction but working in the opposite
direction occurs when glucose is added to the malignant VIA lymphoblasts--i.e., the glycolysis inhibits the respiration (Crabtree effect). Our second
calculation, though of an approximate and admittedly very rough nature, can be based on the
latter phenomenon and may furnish an impression
of the efficiency of the mitochondrial oxidative
phosphorylations in the intact cells (13, 31, 33).
The calculation only bears some validity if the
production and utilization of adenosine triphosphate remain constant in the absence and presence
of glucose; the latter condition appears to be satisfied in the case of ascites tumor cells (31) but.has
not been controlled in the present case. Taking
into account that the glycolytic production of 1
molecule of lactate is accompanied by the formation of 1 molecule adenosine triphosphate and the
finding that the extra production of 5.4 ~moles
lactate (6.9 - 1.5, Table 4) after addition of glucose caused a decrease in the oxygen uptake of
1.6 gatoms (6.3 - 4.6), the P : O ratio accompanying the mitochondrial oxidations may be calculated as 5.4/1.6 = 3.5.
The present results on the glycolytic and respiratory rates of two closely related cell types, the
one malignant and the other nonmalignant, and
the deductions made in the wake of the widely accepted interpretation of the connection between
359
these processes, show that the respiration of the
tumor cells is not lower than that of the nonmalignant cells and that tumor mitochondria in.
situ behave like normal mitochondria. These findings undermine the basic concept of an "irreversibly damaged," "impaired, . . . . decreased" or uncoupled respiration of tumor cells as proposed by
Warburg (35). It is exactly the latter concept,
namely, the interpretation 4 that the respiration of
tumors was deficient, that has been criticized repeatedly by several authors (9, 39, 30). Instead of
stating, as Warburg did, that the respiration of tumor tissue was too weak in comparison with the
glycolytic activity (34, 35), Weinhouse emphasized the high glycolytic potency of tumor cells,
thus: "Anaerobic glycolysis is so high in tumors
that a normal respiration and a normal Pasteur
effect is incapable of eliminating it" (30). In this
connection it should be pointed out that the possible significance of the high glycolysis of tumors,
as originally observed by Warburg, was not contested by Weinhouse (30). However, the present
results with the nonmalignant lymphoblasts, especially after their being cultured in medium A, may
cast serious doubt on the assumption that a "high"
glycolysis, either under aerobic or anaerobic conditions, is characteristic for tumors. Moreover,
other experiments in which a high lactate production by normal cells cultured in vitro has been
found, should not be explained away by the dogma
of the carcinogenicity of tissue culture for the
simple reason that in vitro culture of normal cells
does not consistently lead to cancer cells. Compare, for example, the fibroblasts, mentioned in the
experimental part of this paper, which have been
in continuous culture for 13 years without having
become cancerous. Furthermore, there are some
very good reasons why the glycolysis of tumors
should not be considered as the propelling force for
tumor growth and anabolism: HeLa carcinoma
cells grow as well in the presence of a small amount
of galactose, with only very small amounts of lactate being formed, as under conditions in which
much lactic acid is produced (~3), whereas ascites
tumor ceils do incorporate the same amount of
amino acid into their proteins irrespective of
whether glucose is present or not (13, 15, 33).
Since the latter results were obtained in vitro, they
bring out the potentiality of the tumor mitochondrial processes rather than their actuality under in vivo conditions. The possibility cannot be
ruled out that a high glycolytic potency may be of
4 That the basic assumption underlying Warburg's theory
is an interpretation without factual verification follows from
the fact that Warburg has never attempted to show that the
respiration of cancer cells was too low to support their energy
requirements.
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Research.
Cancer Research
360
Vol. ~1, April 1961
i
advan[age to {:he survival and growth of tumor
cells under in vivo conditions when the oxygen
supply m a y be scarce. However, if this were the
case, there is still no reason to assume that the socalled neoplastic pattern of metabolism is responsible for the uncontrolled growth; it rather appears
to be a symptom, favoring the growth in vivo.
However, even exceptions to the proportionality
between malignant character and extent of anaerobic glycolysis are known (17).
To sum up, the so-called neoplastic pattern of
metabolism does not appear to be confined to tumor cells, nor does the experimental verification of
the main prediction stemming from Warburg's
theory (namely, that tumor cells are dependent on
glycolytic rather than on respiratory chain-linked
phosphorylations) appear to be true. For these
reasons we conclude that Warburg's theory on the
origin of cancer cells is not correct.
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Research.
Glycolytic and Respiratory Properties of Malignant and
Nonmalignant Lymphoblasts Cultured in Vitro
C. J. Homburg, C. J. Bos, W. M. de Bruyn, et al.
Cancer Res 1961;21:353-360.
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