Download STUDIES ON THE GROWTH OF TISSUES IN VITRO

232
STUDIES ON THE GROWTH OF TISSUES IN VITRO
VII. CARBOHYDRATE METABOLISM AND MITOSIS
BY C. M. POMERAT AND E. N. WILLMER
Physiological Laboratory, Cambridge
(Received 2* December 1938)
(With Sixteen Text-figures)
IN a previous paper of this series (Trowell & Willmer, 1939) an account was given
of the effects on the growth of chick periosteal fibroblasts of the addition of saline
extracts of various organs from the growing and fully grown bird. Attention was
called to the fact that those organs and tissues which yielded growth-promoting
extracts were just those which have been shown by Warburg and his school
(Warburg, 1930) to possess considerable powers of glycolysis. The parallelism is
illustrated in Fig. 1. In the upper series are plotted the mean mitotic indices for the
fibroblast cultures during the period from the tenth to the twentieth hour after the
addition of the tissue extracts, at which time the effects produced by such extracts
have been shown to be most comparable. In the lower series, the anaerobic glycolytic activities, as measured by lactic acid production, of the corresponding tissues
are plotted. Comparison of these two sets of figures at once suggests that the organ
extracts may exert their effects by influencing the carbohydrate metabolism of the
tissues to which they are applied. The only apparent exception is the thyroid, and
the effects of extracts of that organ on the growth of cultures were very variable
and probably other factors were involved.
The work of Ashford & Holmes (1929), Needham & Nowinski (1937), Needham
& Lehmann (1937 a, b) and Baker (1938) now emphasizes the correlation between
highly glycolysing tissues and the direct breakdown of glucose to lactic acid by
some mechanism which does not involve the intervention of the phosphorylating
systems characteristic of the muscle cycle. Moreover, it is significant that tissues
which show this property of direct glucose breakdown are again precisely those
whose extracts are growth-stimulating (see Fig. 1). It is, of course, not yet known
why they should be growth-stimulating, but it seems conceivable that the extracts
may contain substances which affect the direct utilization of glucose by the tissues,
and that this altered or increased carbohydrate metabolism may then affect the
growth. Or possibly the extracts may contain substances which link up direct nonphosphorylating glucolysis with protein synthesis. In either case the carbohydrate
metabolism would affect growth and the following working hypothesis would seem
to be justifiable. It may be most easily appreciated by reference to the highly
simplified diagram in Fig. 2.
Studies on the Growth of Tissues in vitro
233
Three possible major sources of energy from carbohydrate are illustrated. (1)
Glucose being converted directly to lactic acid, a process particularly characteristic
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Fig. 1. Comparison between the effects of organ extracts on growth of chick periosteal fibroblasts, with the glycolytic powers of the organs from which the extracts were made. The effect of
glyceraldehyde on the anaerobic glycolysis is indicated by + and — signs indicating acceleration
or inhibition respectively. Values for Q&J, from Warburg. Glyceraldehyde effects from Baker.
Process 1
Glucose
Lactic acid
Process 3
Glycogi
Fig. a.
of embryo and brain. (2) Glucose being first phosphorylated, or passing to glycogen
and then being phosphorylated before being converted into lactic acid as in anaerobic
muscle metabolism. (3) Glucose or lactic acid being directly oxidized to CO, and
jEB-XVlii
16
234
C. M. POMERAT and E. N. WILLMER
H,0 as in most tissues under aerobic conditions. Process (3) necessitates a supply
of oxygen, while the other two are essentially anaerobic.
All or any of these processes might be involved in the phenomenon of growth
by cell division. Experiments to be described in this paper, together with other
accumulated data, make it probable that growing tissues make use primarily of
process (1), glucolysis. The evidence is as follows:
If, as has been suggested, the important process in growth is connected with the
direct breakdown of glucose (glucolysis) it should be possible to inhibit it by any
means which inhibits glucolysis. Now it is known (Mendel, 1929; Needham &
Lehmann, 193 7 a and b; Baker, 1938; Lehmann & Needham, 1938) that this process
can be specifically inhibited in tissues and tissue breis by the addition of glyceraldehyde, and that this inhibition becomes almost complete at a concentration of about
O-OO2 M glyceraldehyde. Growth also, if the assumption be correct, should be
inhibited by the addition of glyceraldehyde in this concentration, assuming that the
reagent can penetrate the cell walls of living healthy cells, or at least come into contact
with the reacting system, and that it is not toxic in any other way.
Cultures of chick periosteal fibroblasts were therefore grown in Carrel flasks,
by the usual technique required for photographic recording, with a fluid phase
containing embryo juice (10% in Tyrode solution). The periphery of the culture
was photographed every 6 min. from the twenty-fourth hour after planting. This
was carried on for from 4 to 6 hr., so that the normal growth rates of the cultures
could be established. Individual variation between different cultures was found to
be such as to make this procedure advisable. Then the embryonic juice was removed
and replaced by a fresh quantity (1 c.c.) containing the substance to be investigated,
in this case glyceraldehyde. Different concentrations of the glyceraldehyde were
tried. The results of a typical experiment are shown in Fig. 3. The curve at the
bottom of the diagram shows the effect of adding 0-002 M glyceraldehyde which had
been previously boiled in alkaline solution and then neutralized. This process
caramelized the glyceraldehyde, and the absence of any effect upon the growth
rate shows that the solution used was not toxic or in any way injurious because of
impurities. The other curves show clearly that glyceraldehyde very strongly inhibits
growth, and the inhibition becomes maximal in the same concentration as that
necessary to produce complete inhibition of glucolysis. Smaller concentrations
were proportionately less effective. The result might have been due to the conversion of the glyceraldehyde into lactic acid, but the effect of 0002 M lactic acid on the
growth of cultures was found to be negligible. In other words the cessation of growth
was genuinely due to the glyceraldehyde itself or to some other process in which
glyceraldehyde is involved. The inhibition by fresh solutions was always complete
and generally irreversible, but by older solutions it was less complete and was then
found to be reversible as illustrated in Fig. 4. This is almost certainly connected
with the fact that glyceraldehyde slowly, in the course of a few weeks, polymerizes
into an inactive substance. Under the influence of an effective dose all visible
activity of the cells was brought to a standstill; the cells ceased to migrate and became
rounded off. Many of the cells in the culture whose behaviour is illustrated in Fig. 4
1
4
V
0002 Af glyceraldehyde
(16 experiments)
•
0001 Af glyceraldehyde
(5 experiments)
I
1
2
1
1
3
0
4
3
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o
I
00005 Af glyceraldehyde
(2 experiments)
f\
0002 Af caramelised glyceraldehyde
(3 experiments)
i
i
0
2
4
6
10
12
Time in hours
Fig. 3. The effect of glyceraldehyde on the growth of chick periosteal fibroblasts. Glyceraldehyde
added at zero hour. There is 10 % embryo juice in the medium throughout the experiment.
O002 Af glyceraldehyde
10 % embryo j uice
•8 3
i2 2
5
15% embryo juice added after
wmhing with 1*5 <ui tyiode
solution
_ 10% embryo
juice
i
6
Y 4
6
8 10 12 14
Time in hours
16
18
20
22 24
26 28
Fig. 4. Recovery of a culture after inhibition by glyceraldehyde. This does not regularly occur,
but the older the solution of glyceraldehyde the more easily does it take place.
16-2
C. M. POMERAT and E. N. WILLMER
were rounded off but recovered completely when the glyceraldehyde was removed
and replaced by fresh embryo juice.
These results were definitely encouraging and lent support to the hypothesis
concerning the relation between growth and glucolysis, but further investigations
produced results not so satisfactory to the hypothesis, although interesting in
Propyl aldehyde washed away and
replaced by 10% embryo juice
(4 experiments)
0002 Af propyl aldehyde
10 % embryo juice
(8 experiments)
8
-a
i
2
is
8
12
T~
1
1
14
16
18
~20~
22
24
Time in hours
Propyl aldehyde
4r
0-002 M methyl glyoxal
10 % embryo juice
(7 experiments)
0001 M methyl glyoxal
10 % embryo juice
(2 experiments)
Time in hours
Time in hours
Methyl glyoxal
0-002 M benialdehyda
10 % embryo juice
(2 experiments)
4
2
O
l
Time in hours
Benzaldehyde
Fig- 5-
themselves. They are not as yet fully explained. By boiling glyceraldehyde in acid
solution it is converted into methyl glyoxal and, as such, has no effect on glucolysis
(Needham & Lehmann, 1937 b), nor indeed do any other aldehydes (Needham &
Lehmann, 1937 b; Baker, 1938) which have so far been tried. On the other hand,
the addition of 0-002 M concentrations of methyl glyoxal, propyl aldehyde, butyl
aldehyde and benzaldehyde to tissue cultures produced complete inhibition of
Studies on the Growth of Tissues in vitro
237
growth, migration and cellular activity (Fig. 5). The corresponding acids, pyruvic,
propionic and benzoic and also glyceric and lactic, when neutralized with NaOH
and applied in the same molar concentrations, produced no similar deleterious effects
(Figs. 6, 7). The inhibition seems therefore to be a property of aldehydes as such.
The reversibility of the effect has only been tested in the case of propyl aldehyde,
81-
7
6
s
o
Is
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2 A
_
0'002 M glyceric acid
10 % embryo joice
(2 experiments)
10 % embryo
juice
2
I
2
9
4
6
8
Time in hoars
10
12
14
16
14
16
8
0002M benzoic acid
10 % embryo juice
(2 experiments)
7
6
3
1 5 .growth rate in
o
S 4
10% embryo
juice
§
3
4
6
2
T i m e in hours
10
12
Fig. 6. Glyceric and benzoic acids. The solutions were brought to pH 7-5 by means of NaOH.
but then the inhibition was found to be completely reversible even after it was
produced by a fresh 0-002 M solution, so that it differs slightly in this way from the
effect of glyceraldehyde.
This property of aldehydes to inhibit growth and cell activity, though in itself
extremely interesting, is disturbing to the hypothesis when compared with the
C. M. POMERAT and E. N. WILLMER
238
specificity of glyceraldehyde for the glucolytic process. So far it is unexplained, but
a study of other aldehydes and their effects should prove illuminating. Perhaps two
mechanisms are involved, as is suggested by the fact that recovery after propyl
0-0O2 M 6odium pyruvate
10 % embryo juice
3 3
•o
c
i
2
10
12
14
16
Time in hours
18
20
22
24
26
16
18
20
0002 M sodium pyruvate
0-002 Af glyceraldehyde
10 % embryo juice
6
4
2
0
2
4
6
8
10
12
14
Time in hours
5
0-002 M glyceraldehyde
10 % embryo juice
4
A / \
1o 3 - /A\ //
o 2
11
0-001 Af glyceraldehyde
0001 M sodium pyruvate
10% embryo juice
\\ A
-/
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6
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T
10% embryo
juice
4
2
C
2
4
6
8
lb
12
14
16
18
20
T i m e in hours
Fig. 7.
aldehyde is certain, but it is not so from glyceraldehyde at the same molar concentration. It is interesting to find that Boyland & Mawson (1938) have recently
shown that certain aldehydes, notably citral, phloroglucinaldehyde and 3-4-dimethoxybenzaldehyde have been able to reduce the growth rate of Crocker Sarcoma
180 in the living animal.
Studies on the Growth of Tissues in vitro
239
It may be significant that the concentration of 0-002 M for the glyceraldehyde
is the same for growth inhibition as it is for glucolysis, but on this evidence alone
it is obviously impossible to link growth with glucolysis. Moreover, the blocking
of embryonic glucolysis by glyceraldehyde is reversed by the addition of pyruvate.
This does not appear to be the case with growth, for pyruvic acid has been found
to have no influence on growth of cultures either when applied alone, in conjunction
with glyceraldehyde or after glyceraldehyde (Fig. 7). Finally, Sullmann (1938)
concludes that the formation of hexose phosphate from glucose by lens tissue is
inhibited by o-oi M glyceraldehyde, propyl aldehyde, benzaldehyde and formaldehyde. This concentration is, of course, considerably higher than those used in this
investigation, but in growth experiments the reagents are allowed to act for a
considerably longer time and so might eventually become effective. On the other
hand, the evidence adduced by Lehmann & Needham (1938) seems to rule out the
possibility of phosphorylation of glucose in the embryo, at any rate under anaerobic
conditions; and further evidence now to be described points also to the same
conclusion.
The glucolytic process is affected, like the phosphorylating glycolysis, by the
addition of sodium fluoride and sodium iodoacetate, but the concentrations required to produce inhibition are not the same for the two processes. Both substances
can be added to cultures, and an investigation of the concentrations necessary to
inhibit growth has produced very suggestive results. At the same time it should
be remembered that these substances may also cause other effects in the cell, as,
for example, the poisoning by iodoacetate of enzyme systems not related to the
metabolism of glucose.
Sodium fluoride is found to inhibit phosphorylation in the muscle cycle at a
concentration of 0-005 M, but at this concentration a considerable amount of direct
glucose breakdown still occurs, namely about 45 % (Needham & Lehmann, 1937 b).
This latter process is not stopped completely until a concentration of 0-02 M is
reached. Fig. 8 shows the results of experiments in which sodium fluoride was
added to cultures of periosteal fibrpblasts growing in vitro. It may be concluded
from these results that mitoses although considerably reduced in number and after
a time suppressed by concentrations between 0-003 and o-oi M are not immediately
knocked out until a concentration of 0-02 M is reached. Direct observation of the
cultures confirms this. The cells are manifestly disorganized by a concentration of
0-02 M almost at once, while at 0-007 M they remain healthy in appearance for a
considerable time. There is thus a very striking parallel between the concentrations
of fluoride necessary to produce inhibition of growth and inhibition of direct
glucose breakdown. No such parallel exists between growth and phosphorylating
glycolysis, for mitoses still occur when this is presumably completely suppressed
(0-007 M)- This result becomes even more significant when taken in conjunction
with similar results obtained in experiments in which sodium iodoacetate took the
place of fluoride (Fig. 9). The results, though similar, show one striking and important difference. Growth is checked when the concentration reaches 0-00005 M,
the concentration at which non-phosphorylating glucolysis is checked. Ten times
0-0033 M
(2 experiments)
0
2 4
6 8 10 12
Time in hours
Fig. 8. The effect of different concentrations of sodium fluoride in io% embryo juice
on the growth rate of chick periostea! fibroblasts.
•B
o
0-00005 M
0-00002 M
3
10% embryo
juice
2
B
4
.3
3
4 6
8
10
12
8
10
12
0-0002 M
u
1
- 10 7 ? embryo
juice
4
6
8
10
12
Time in hours
Time in hours
Fig. 9. The effect of different concentrations of sodium iodoacetate in 10% embryo juice on the
growth rate of chick penosteal fibroblasts. Two experiments at each concentration.
Studies on the Growth of Tissues in vitro
241
this concentration is necessary to stop the phosphorylating mechanism. It should
be noted that these results are in marked contrast to those obtained by Ellis (1933)
on segmenting eggs of Urechis caupo and of Strongylocentrotus purpuratus, in which
he found that concentrations of o-oi M sodium iodoacetate and fluoride had no
effect on cleavage. The mechanism in these eggs must be very different.
There is another substance which characteristically stops the process of phosphorylation, namely, phlorizin. It can be effectively used for this purpose in concentrations of 0-005 M. On applying it to cultures in this and even stronger
concentrations it produced no obvious decrease in the growth rate. The results are
shown in Fig. 10. It is just possible that phlorizin does not reach the part of the cell
where it can exert its effect, but, on the other hand, it acts on the kidney in vivo.
•o
'V
10 % embryo juice
0
2
4
Time in hours
10
12
Fig. 10. Effect of adding phlorizin in concentrations between o-oo8 M and 00033 M
in 10% embryo juice (7 experiments).
Lastly, it has been shown by Mendel (1937) that the addition of sodium ferricyanide to tissues, especially sarcoma, reduces their power of aerobic glycolysis.
Anaerobic glycolysis is unaffected. The exact significance of this has not yet been
worked out in terms of direct glucose breakdown, but the two may be connected.
The effects of ferricyanide on growth are precisely similar to its effects on aerobic
glycolysis. The latter is inhibited to the extent of about 25 % by solutions of o-ooi M
and completely suppressed at o-oi M. Growth is quite definitely checked at
0-005 M and is irreversibly stopped at o-oi M (Fig. 11).
Thus by the use of these inhibitors there is considerable evidence that growth
of fibroblasts in cultures depends upon energy obtained from direct glucolysis
rather than from phosphorylating glycolysis. Growth proceeds more or less independently of the latter, if indeed the latter process is occurring at all in these cultures;
but, should the former be checked, then the growth suffers with it.
Referring back once again to the simplified diagram on page 233, it may be concluded that growth depends on process (1) rather than on process (2). The former
is probably the more fundamental method, as witnessed by its apparent dominance
in the embryo. The second process which is clearly more elaborate may depend
C. M. POMERAT and E. N. WILLMER
242
upon greater complexity in the tissues than exists in embryonic cells or in cells
growing in culture.
It is, of course, possible that growth might depend on the more direct oxidative
mechanisms which occur in process (3). It is certainly true that tissues fare much
better when well supplied with oxygen, but, although the results have been disputed
•a
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Air
90% CO, 10% 0,
1
1
1
1
8
10
12
14
8
10
12
14
8
10
12
14
0-002 M sodium ferricyanide
9 4
XI
c
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3
I 2
_
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0-01 M sodium ferricyanide
-
10% embryo
juice
0
2
4
Time in hours
Fig. 11.
8
10
0
2
4
6
Time in hours
Fig. 12.
Fig. 11. The effect of sodium ferricyanide in 10% embryo juice on the growth of periosteal
fibroblasts.
Fig. 12. The effect of introducing gas mixtures containing carbon monoxide into the air space above
hanging drop cultures of chick periosteal fibroblasts.
by Havard & Kendal (1934), it should be remembered that Laser (1933) has obtained
the growth of fibroblasts in the complete absence of oxygen. The cells apparently
needed training for it, by being cultured in vitro for some time first, but they did it,
and we are thus compelled to believe that growth, once initiated, can continue by
means of a completely anaerobic mechanism. Burrows (1921) and Wind (1926)
Studies on the Growth of Tissues in vitro
243
have also shown that very embryonic cells can display activity under anaerobiosis,
but it is not clear from much of this work whether mitoses were occurring or not.
In this connexion it is significant to note that Ephrussi et al. (1929), who examined
cultures after treatment with atmospheres containing only minimal quantities of
oxygen, found that mitoses were arrested in prophase, although the later stages
could, like the migration of the cells, go on almost unchecked. It is, however, not
clear for how long the cells had been treated and the acidity produced in the medium
in the absence of oxidations might be sufficient to account for the results. On the
other hand, sarcoma cells, which produce far more lactic acid, can survive anaerobiosis more easily than normal cells. The results from experiments on the effects
of anaerobiosis are therefore still somewhat equivocal.
It need hardly be pointed out that both processes (1) and (2) are anaerobic, and the
evidence so far adduced seems to stress the importance of process (1). This becomes
still more significant when attempts, other than by altering the oxygen pressure,
are made to vary the rate at which process (3) is occurring. There are several ways in
which the respiratory exchanges of a tissue may be affected both favourably and
adversely. Unfortunately few of the reagents are so specific that they affect respiration alone, but the addition of such substances to tissues growing in vitro brings
further evidence in favour of the independence of growth and respiration, for they
have but little immediate effect upon the rate of growth. Before these experiments
are described it should be made quite clear that there is no question of denying the
importance of respiratory exchanges in the metabolism of tissues growing in vitro,
which would be absurd, but the evidence shows that they are not the primary
changes concerned with the growth process. They rather, as in muscle, allow catabolites to be removed and may supply energy necessary for the continuous working
of the first process. That is to say that growth can go on at least for a time on glucolysis alone, and at a time when the respiration has been reduced to a minimum.
In the first place the action of carbon monoxide on the growth of cultures is
interesting. The actual part played by carbon monoxide in the chemistry of the cell
is still somewhat obscure. It appears (Laser, 1937), in some tissues at least, to affect
the Pasteur reaction and increase the production of lactic acid rather than to decrease
the respiratory exchanges, although the cytochrome system is most probably put
out of action. It is likely therefore that the increase in lactic acid is produced rather
by accumulation of this substance through faulty removal than by an increase in
sugar breakdown. It is interesting to note therefore that the introduction of a
mixture of 95 % carbon monoxide and 5 % oxygen into culture chambers of the
type described by Gill (1938) had no immediately deleterious effect upon the growth
of the cells, which were growing on cover-slips in the chamber. The results with
carbon monoxide are illustrated in Fig. 12.
Similarly, the addition of cyanide to cultures produced striking results. Again,
its action on cellular metabolism as a whole is complex and it may even act as a
stimulant to certain processes. Experiments, however, clearly show that, in those
cultures to which it was added, growth continued in concentrations of cyanide at
which the respiration of the cells must have been profoundly altered (Fig. 13).
C. M. POMERAT and E. N. WILLMER
244
0-0005 M HCN still allowed considerable growth and even in a concentration of
0-002 M mitoses were observed to be occurring as long as 10 hr. after the addition
of the cyanide. That is to say the cells were definitely entering and completing the
process of mitosis under these conditions.
0-002 M
(2 experiments)
Time in hours
Fig. 13. The effect of different concentrations of HCN in 10% embryo juice
on the growth of chick periosteal fibroblastg.
The results with sodium azide were similar. This substance has actions very
like those of cyanide in inhibiting cellular respiration and oxidation of cytochrome
(Keilin, 1936). Cytochrome oxidase is 80-95 % inhibited by o-ooi M NaN3. The
effects of azide on growth are seen in Fig. 14 and it is clear that this concentration
still allows of considerable growth. With both azide and cyanide the growth rate
shows a progressive diminution, but undiminished activity would be no more
likely under these conditions than that a muscle should go on contracting anaerobically for an indefinite period.
Studies on the Growth of Tissues in vitro
245
00001 a
2
4 £
10 12 6
4
Time in hours
2
0 2 4 6 8
Time in hours
10 12
Fig. 14. The effect of different concentrations of sodium azide in 10% embryo juice
on the growth of chick periosteal fibroblasts.
5f-
I'
u
•3
O '
I2
10 % embryo juice
6
I
I
4
2
I
0
2
4
Time in hours
6
8
10
12
Fig. 15. The effect of o-oi M sodium malonate on the growth of chick periosteal fibroblasts.
Sodium malonate has a specific action in inhibiting succinodehydrogenase and
reducing respiration (Quastel, 1926; Gozsy & Szent-Gyorgi, 1934). Its effects on
growth are negligible (Fig. 15).
In contrast with all the former substances, sodium fumarate stimulates respiration (Gozsy & Szent-Gyorgi, 1934; Stare & Baumann, 1936). But it too has no
246
C. M. POMERAT and E. N. WILLMER
effect that is demonstrable on the growth of fibroblasts. This was tested in a slightly
different way, namely by adding it to cultures growing at a minimal rate in plasma
alone, and also by adding it to such cultures in addition to 10% embryo juice. In
neither case did the cultures show any increase above normal in their growth rates
(Fig. 16).
The addition of fresh raw material in the form of glucose, sodium pyruvate, or
sodium lactate did not cause any acceleration of growth. Probably there is always
a sufficiency of glucose in the plasma and embryo juice and no effects would there4
r
s 3
-o
c
.52 2
10 % embryo juke + 0006 M sodium fumarate
(2 experiment*)
S
.s ->
10% embryo juice
(8 experiments)
u
2 2
0
2
4
6
8
10 12 14
Time in hours
Fig. 16. The effect of C006 M sodium fumarate on the growth of chick periosteal fibroblasts.
Cultures were previously grown in plasma alone and embryo juice and sodium fumarate or embryo
juice alone were added at zero hour.
fore be observable until that supply had been exhausted. It may however be stated
that osteoblasts in hanging drop cultures in Tyrode solution when entirely deprived
of glucose derive benefit from the addition of sodium lactate, at any rate under
aerobic conditions. This has been shown by substituting sodium lactate for the
glucose present in Tyrode solution and using this as a medium for fluid hanging
drop cultures of fresh embryonic bone fragments. Such cultures showed cellular
activity comparable to that shown in ordinary Tyrode solution and far in excess of
that shown by cultures in Tyrode without either glucose or lactate. Similar results
were obtained when fresh heart and skin fragments were explanted. It would be
interesting to see if lactate is of any use to the tissues under anaerobic conditions.
It would be surprising if it were. Experiments so far carried out on these lines have
not yet proved of any value owing to technical difficulties.
It may be significant that brain, testis and tumour tissue, three tissues which
provide growth-promoting extracts, are incapable of synthesizing sugar from lactic
Studies on the Growth of Tissues in vitro
247
and pyruvic acids whereas other tissues like liver and kidney can do so (Benoy &
Elliott, 1937). This might suggest that one of the actions of the extracts in
promoting growth may be in stimulating carbohydrate breakdown rather than
synthesis.
All these observations therefore provide evidence for the correctness of the
hypothesis that the fundamental metabolic process concerned with growth is direct
glucose breakdown, rather than respiration, or phosphorylation as it occurs in
anaerobic muscular metabolism. No single piece of evidence is in itself sufficient,
but taken all together the strength of the case is apparent.
DISCUSSION
The significance of these findings may now be shortly discussed. In the first
place Needham & Nowinski (1937) have shown fairly clearly that the embryo chick
does not metabolize glycogen but relies on glucolysis or direct sugar breakdown.
Secondly, growth can occur at extremely low oxygen pressures and, Laser (1933)
believes, even under conditions of complete anaerobiosis. Moreover many workers
on tissue culture have shown that glucose is necessary for tissue growth, and that
glycogen is of comparatively rare occurrence in the cells and, certainly, it is not a
necessity that it should be there in detectable amounts before growth can occur
freely. Glucose has been shown to be used by tissues in vitro (Krontowski,
1929; Krontowski & Bronstein, 1926), and it may have a pronounced protein
sparing action (Watchorn & Holmes, 1927; Litter et al., 1937). This effect is greater
in tissues in which mitoses are abundant than it is in tissues whose growth rates are
less. The experiments described in this paper make it clear that respiratory poisons
do not immediately put a stop to mitotic activity, nor does fumarate which increases
respiratory activity increase the mitotic rate. Conditions which interfere with the
process of phosphorylation do not at the same time affect growth. There would
therefore seem to be two remaining possibilities; either the requirements for growth
energy are so small or so little concerned with carbohydrates that gross disturbance
of the metabolism of the latter may occur without depriving the cells of their energy
supplies, or else the essential process in the carbohydrate breakdown is neither
respiration nor phosphorylation but glucolysis. Direct evidence for this is certainly
scanty. Besides the above-mentioned utilization of sugar by the embryo, and by
tissue cultures, there is the evidence from the sources of active growth-promoting
extracts, which always seem to be from glucolysing tissues, at least three of which
are unable to synthesize sugar from lactic or pyruvic acids. There is also the fact that
growth is inhibited by glyceraldehyde, though this is somewhat discounted by the
inhibition caused by other aldehydes. And finally, there is the inhibition caused
by appropriate doses of sodium fluoride and of sodium iodoacetate, which gains
considerable strength as evidence from the fact that the amount of inhibitor
necessary to stop growth is in the one case larger and in the other smaller than the
amount necessary to stop phosphorylation, in this way following the dosage required
for the inhibition of glucolysis.
248
C. M. POMERAT and E. N. WILLMER
It may be concluded therefore that the hypothesis that growth by cell division
is intimately connected with direct glucose breakdown is not without foundation.
One final point should be emphasized. There is nothing in the evidence presented
in this paper to suggest that the actual process of mitosis is the thing which is
affected by glucolysis, but rather is it clear that cells when glucolysing are capable
of dividing and that the characteristic of actively growing tissues is their power of
glucolysis, upon which they rely for growth.
SUMMARY
1. Growth of chick periosteal fibroblasts is inhibited by 0-002 M solutions of
glyceraldehyde, benzaldehyde, butyl aldehyde, propyl aldehyde and methyl glyoxal.
2. The growth is not inhibited by sodium pyruvate, lactate, propionate,
glycerate or benzoate.
3. Sodium pyruvate does not affect the growth under the conditions of the
experiments, nor does it alter the inhibition brought about by glyceraldehyde.
4. Growth is inhibited by sodium fluoride and sodium iodoacetate in the
concentrations in which these inhibit glucolysis. Growth inhibition does not
correspond to inhibition of phosphorylation.
5. Growth is not inhibited by 0-008 M phlorizin.
6. Growth is not immediately inhibited by an atmosphere containing 95 % CO
and5%02.
7. Growth is not immediately inhibited by 0-002 M sodium azide, 0-002 M
HCN or by o-oi M sodium malonate. Azide and cyanide reduce the growth rate
after some hours.
8. Growth is not accelerated by the addition of 0-006 M sodium fumarate.
9. Growth is inhibited by o-oi M sodium ferricyanide.
10. The relationship between carbohydrate metabolism and growth by cell
division is discussed in the light of these results.
The expenses of this research have been in part defrayed by a grant from the
British Empire Cancer Campaign, to whom the authors wish to express their
gratitude. The work was performed while C M . Pomerat was holding a Rockefeller
Travelling Fellowship.
The authors also wish to express their sincere thanks to Dr H. Lehmann for
constant advice and for providing many of the reagents in suitable form, and also to
Mrs Simon-Reuss for invaluable technical assistance.
Studies on the Growth of Tissues in vitro
249
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