The Effect of Nitrogen on the Enzymatic Pattern
of Strain L Cells*
FESTUSO. ADEBONOJO,
KLAUSG. BENSCH,ANDDONALDW. KING
(Department
of Pathology,
Vale University School of Medicine,
New Haven, Conn.)
SUMMARY
Strain L cells, incubated for 16 hours per day under an anaerobic atmosphere of 95
per cent nitrogen and 5 per cent carbon dioxide for a period of 3 months, showed a
marked increase in their aldolase and lactic dehydrogenase enzyme activities and a
marked decrease in their cytochrome oxidase activity. The changes were quickly re
versed upon reincubation in an aerobic atmosphere. Multiple inoculations of anaerobic
cells in Swiss-Webster mice throughout the period of the experiment failed to produce
malignant tumors.
It is generally believed that cancer cells have
low respiratory enzyme activities and, concomi
tant with increased glycolysis, increased fermenta
tive enzyme activities (5). It has also been sug
gested that tumors have sufficient respiratory en
zymes for their particular needs, but not the excess
found in other tissues (17). Recent work on the
cytochrome concentration of Ehrlich ascites cells
has challenged even this relative deficiency (1).
One hypothesis concerning the origin of cancer
is that anaerobiosis eventually results in the for
mation of malignant cells (21). Experimental evi
dence for this theory was provided by experiments
concerning malignant transformation of myoblasts
in tissue culture maintained under a nitrogen
atmosphere for a short period daily over an inter
val of several months (3). The present investiga
tion was initiated to study the effects of anaero
biosis on strain L cells in regard to changes in the
enzymatic pattern of the cells and their ability
to form tumors.
MATERIALS AND METHODS
Cultures.—Strain L cells were grown in suspen
sion tissue culture in Eagle's basal medium sup
plemented with 10 per cent horse serum as pre
viously described (10). Ehrlich ascites cells weregrown in a similar fashion as strain L cells, also
previously described (8). Both strain L and Ehrlich
cells were subcultured every 3-4 days and usually
* This work was supported by a grant from the USPHS
O2938 (C3).
Received for publication August 53, 1960.
showed a generation time of 24-26 hours for the
3-month period of the experiment. No significant
number of necrotic cells were ever seen in the strain
L or Ehrlich cell cultures. The control cultures
were maintained in a mixed atmosphere of 92 per
cent balanced air and 8 per cent carbon dioxide.
The experimental cultures were grown in an at
mosphere of 95 per cent nitrogen and 5 per cent
carbon dioxide for 16 hours per day. The cells
were then incubated in an atmosphere of 8 per
cent carbon dioxide and 92 per cent balanced
air for the remaining 8 hours per day.
Assays.—Total cell counts were done daily with
a standard hemocytometer. Protein was deter
mined by the method of Lowry as modified by
Oyama and Eagle (15). Enzyme activities were
determined on one million washed cells suspended
in 1 cc. distilled water according to the following
methods: lactic dehydrogenase (12), aldolase (19),
cytochrome oxidase (20), and malic dehydrogenase
(13).
RESULTS
By transferring aliquots of the cells into fresh
medium every 3-4 days, it was possible to keep
both the strain L cells and the Ehrlich ascites
cells in almost continuous logarithmic growth.
Morphologically, the nitrogen-incubated strain
L cells showed many of the characteristics of
Ehrlich ascites carcinoma ; the cells became larger,
the presence of lipide granules stained with Sudan
II became more pronounced, and the plasma mem
brane became more irregular. It has previously
been reported that lipide granules increase in
252
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ADEBONOJOet al.—Enzymatic Pattern of Strain L Cells
Ehrlich ascites cells concurrent with increased
time after inoculation and that the lipide concen
tration is highest in the old, presumably anaerobic,
tumors (14). It is also well known that lipide
accumulates in myocardial cells under anaerobic
conditions (9). Conversely, in strict log growth,
the Ehrlich cells became more regular in appear
ance, lost some of their lipide granules, and were
hard to distinguish from the strain L cells. An
aerobic strain L cultures and Ehrlich cultures
always maintained a low pH (approximately 6.9)
as seen with phenol red dye, and this relative
acidity appeared necessary to maintain the re
quired growth rate.
As shown in Charts 1 and 2, incubation with
a mixed atmosphere of 95 per cent nitrogen and
203
5 per cent carbon dioxide resulted in a marked
increase in the amount of aldolase and lactic
dehydrogenase activity in the strain L cells, both
on a unit per cell basis and a unit per mg. of
cell protein. This occurred maximally within 72
hours for all the enzymes assayed. Upon reincubation of these cells with 92 per cent balanced air
and 8 per cent carbon dioxide after a period of
3 months of relative anaerobiosis, the activities
of these enzymes returned to their previous levels
within 24 hours. Conversely, the relative anaerobic
environment resulted in a marked depression of
STRAIN
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CHART1.—The effect of anaerobiosis on aldolase activi
ty. White bars = controls; black bars = nitrogen incubated;
hatched bars = oxygen reintroduced.
The first bar represents aldolase activity in strain L and
Ehrlich cell control cultures maintained for a period of 3
months. The second bar represents aldolase activity in control
cultures before an anaerobic atmosphere was introduced. The
third bar represents aldolase activity in the cultures given
a mixture of 95 per cent nitrogen and 5 per cent carbon
dioxide for 16 hours per day for a period of 3 months. The
fourth bar represents aldolase activity in 3-month-old anaerobic
cultures 24 hours after the nitrogen atmosphere was replaced
by balanced air. Each bar represents 15-25 determinations.
The change in aldolase activity in the strain L cells is highly
significant with a P value of less than 0.001. The variations
in aldolase activity values over the 3-month period, in units/106
cells were as follows: strain L control, 90 + 20; strain L nitro
gen, 232 ±50; Ehrlich control, 186 + 39; Ehrlich nitrogen,
193 ±40.
CHART2.—Theeffect of anaerobiosis on lactic dehydrogen
ase activity. See Chart 1 for legend.
The first bar represents lactic dehydrogenase activity in
strain L and Ehrlich cell control cultures maintained for a
period of 3 months. The second bar representa lactic dehydro
genase activity in control cultures before an anaerobic atmos
phere was introduced. The third bar represents lactic dehy
drogenase activity in the cultures given a mixture of 95 per
cent nitrogen ami 5 per cent carbon dioxide for 16 hours per
day for a period of 3 months. The fourth bar represents lactic
dehydrogenase activity in 3-month-old anaerobic cultures 24
hours after the nitrogen atmosphere was replaced by balanced
air. Each bar represents 15-25 determinations. The change
in lactic dehydrogenase activity in the strain L cells is highly
significant with a P value of less than 0.001. The variations
in lactic dehydrogenase activity values over the 3-month peri
od, in units/106 cells were as follows: strain L control, 562 ±
65; strain L nitrogen, 917 + 100; Ehrlich control, 889 ±121;
Ehrlich nitrogen, 1000 ±149.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1961 American Association for Cancer Research.
254
Vol. 21, February 1961
Cancer Research
cytochrome odixase activity, which also returned
to its previous level when oxygen was reintroduced
into the culture (Chart 3). These changes have
been found to be highly significant statistically
by the Fisher "t" test with a P value of less than
0.001. As shown in the first three charts, the effect
of anaerobiosis on Ehrlich cells' enzyme activities
was not significant, but the values were never
theless important as control values, being repre
sentative of well accepted malignant cells. The
variation in the malic dehydrogenase enzyme acSTRAIN
L CELLS
EHRLICH
CELLS
tivities was found to be statistically insignificant
in both the strain L and Ehrlich carcinoma cells
(Chart 4). This was interesting in that it showed
the lack of effect of anaerobiosis on an enzyme
presumed to be part of the Krebs cycle.
The enzymatic analyses were done bi-weekly
over the entire period, and each bar in the graphs
represented the average of fifteen to 25 determina
tions. Although some variation was found from
week to week, the constancy of the culture is
indicated by the return of enzyme activity levels
to the control values after prolonged incubation
with nitrogen.
Adult male Swiss-Webster mice were given in-
300
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CHART3.—Theeffect of anaerobiosis on cytochrome oxidase
activity. See Chart 1 for legend.
The first bar represents cytochrome oxidase activity in
strain L and Ehrlich cell cultures maintained for a period of
3 months. The second bar represents cytochrome oxidase
activity in control cultures before an anaerobic atmosphere
was introduced. The third bar represents cytochrome oxidase
activity in the cultures given a mixture of 95 per cent nitrogen
and 5 per cent carbon dioxide for 16 hours per day for a period
of 3 months. The fourth bar represents cytochrome oxidase
activity in 3-month-old anaerobic cultures 24 hours after the
nitrogen atmosphere was replaced by balanced air. Each bar
represents 15-25 determinations. The change in cytochrome
oxidase activity in the strain L cells is highly significant with
a P value of less than 0.001. The variations in cytochrome
oxidase activity values over the 3-month period, in units/10*
cells were as follows: strain L control, 116 ±20; strain L
nitrogen, 68 ±16; Ehrlich control, 54 + 10; Ehrlich nitrogen,
63 ±12.
C
=1
I
CHART4.—Theeffect of anaerobiosis on malic dehydrogen
ase activity. See Chart 1 for legend.
The first bar represents malic dehydrogenase activity in
strain L and Ehrlich cell control cultures maintained for a
period of 3 months. The second bar represents malic dehydro
genase activity in a control culture before an anaerobic at
mosphere was introduced. The third bar represents malic
dehydrogenase activity in the cultures given a mixture of
95 per cent nitrogen and 5 per cent carbon dioxide for 16 hours
per day for a period of 3 months. The fourth bar presents
malic dehydrogenase activity in 3-month-old anaerobic cul
tures 24 hours after nitrogen atmosphere was replaced by
balanced air. Each bar represents 15-25 determinations. The
change in malic dehydrogenase activity is not statistically
significant in either the strain L or Ehrlich cultures. The
variations in malic dehydrogenase activity values over the
3-month period, in units/106 cells were as follows: strain L
control, 79 ±25; strain L nitrogen, 103 ±32; Ehrlich control,
110 ±32; Khrlich nitrogen, 114 + 50.
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ADEBOXOJO
et al.—Enzymatic Pattern of Strain L Cells
jections weekly, subcutaneously or intraperitoneally, of 0.3 ml. of fluid containing approximately
10 million Ehrlich ascites or nitrogenated strain
L cells. In all instances 100 per cent of the five
mice given weekly injections of Ehrlich cells de
veloped carcinoma in 7-15 days, whereas none of
the mice injected with strain L cells ever formed
tumors after a period of 5 months. The number
of cells selected for inoculation of mice is manyfold
the number usually considered necessary to pro
duce tumors in transplantation experiments.
DISCUSSION
The results on the growth of tissue culture
cells under anaerobic conditions are similar to
those previously reported (6). Since there is con
siderable controversy whether malignant cells real
ly have a deficiency of respiratory enzymes, we
were fortunate in selecting the strain L cell, which
has a markedly higher level of cytochrome oxidase
activity than the Ehrlich ascites cell under normal
conditions. The fact that cytochrome oxidase ac
tivity could be lowered and concomitantly two
of the glycolytic enzyme activities increased to
approximate that of the Ehrlich carcinoma is in
agreement with those who believe enzymes are
constantly changing their activities in response
to their environment (11). The results also agree
nicely with those of Ephrussi et al. (2), who
found that enzymatic changes in nitrogen-incu
bated yeast were completely reversible and that
permanent mutations were only produced with
acriflavin. We have speculated in a previous paper
on the effect of the acriflavin dyes on nucleic
acid metabolism resulting in a permanent change
in the template (7).
The strain L cell was originally isolated from
an expiant of subcutaneous connective tissue taken
from a strain C3H mouse. The incidence of sar
comas when the cells were reinjected into C3H
mice dropped from 68 per cent in 1943 to 1 per
cent in 1946. A later study of this cell (now called
strain 929) done after 10 years in tissue culture
showed that sarcomas could be produced in 15
per cent of untreated C3H mice given injections
of 1.5 X IO7cells (16).
There is considerable controversy as to what
constitutes a malignant cell, morphologically, bio
chemically, and indeed by transplantation experi
ments. There are some who claim that the strain
L cell represents a maligant sarcoma cell, in spite
of the relatively low incidence of takes in C3H
mice and the complete lack of tumor growth in
all other strains. Other investigators support the
concept that all tumors capable of métastases,
255
irrespective of species of origin, possess the ca
pacity to grow on heterologous transfer (4).
It had already been shown that strain L cells
would grow in an inbred host when maintained
in tissue culture under aerobic conditions (16).
Since such drastic changes in the enzymatic ac
tivity were noted under anaerobic conditions, it
was decided to apply the strictest criteria of malig
nancy and attempt to grow the cells as tumors in
a different strain of mice. These experiments are,
therefore, not strictly analogous to those of Goldblatt and Cameron, who found that rat myoblasts
incubated daily for short periods with nitrogen
would grow in the anterior chamber of the eye
of a rat of the same strain (3). They do show
that the incubation of cells with nitrogen for
several hours per da,y over scores of generations
will not produce permanent enzymatic changes
in the enzymes assayed. The cells also fail to pro
duce tumors in strains of mice different from the
strain from which the cell was originally isolated.
The results of the effect of nitrogen on the gly
colytic enzymes of Ehrlich cells are somewhat
different from the results of Schade (18), since he
found a marked increase in several glycolytic en
zymes when incubated with nitrogen. However,
we are most willing to admit that the inclusion of
5 per cent carbon dioxide with the nitrogen mix
ture, necessary because of the bicarbonate buffer
of the tissue culture medium, does not permit
absolute anaerobiosis such as he was able to ob
tain with pure, high dry nitrogen.
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The Effect of Nitrogen on the Enzymatic Pattern of Strain L Cells
Festus O. Adebonojo, Klaus G. Bensch and Donald W. King
Cancer Res 1961;21:252-256.
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