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The Competitive Effect of Canavanine on Utilization of
Arginine in Growth of Walker Carcinosarcoma 256
Cells in Vitro*
PAULF. KRUSE,JR., ANDTHOMASA. McCov
(Biomédical
Division, The Samuel Roberts Noble Foundation, Inc., Ardmore, Okla.)
aseptically; solutions were sterilized by passage
through Selas filters. Each treatment was made in
triplicate, for a duration of 72 hours. In the canav
anine experiments it was necessary to replenish
amino acids differ structurally
only in the replace
the media at the end of 48 hours, but this was not
necessary in the ornithine or citrulline studies.
ment of a méthylène group by oxygen:
After 72 hours' incubation, growth response was
NH
NH2
determined by cell counts.
I
Arginine<->H2N—C—NH—CH2—CH2—CHr-CH—COOH L-Canavanine •
H2S042 was added to produce
canavanine/arginine molar ratios between 0.5 and
NH
NH2
10.0, and these ratios were studied at three differ
II
I
Canavanine<-*H2N—C—NH—O—CH2—CH2—CH—COOH
ent arginine concentrations (0.2, 0.6, and 1.0 HIM).
In the ornithine-citrulline studies, arginine was
Accordingly, it was of interest to study arginine- replaced by 0.02-10.0 mM concentrations of either
canavanine relationships in actively multiplying
L-ornithine-HCl or L-citrulline. Later, 0.02 mM
tumor cells. Arginine has been shown to be essen
L-arginine-HCl (one-tenth the usual concentra
tial for the growth of the Walker carcinosarcoma
tion in Medium 3) was included to insure the
256 in vitro (8) ; and, with the recent developments
maintenance of approximately two-thirds of the
in tissue culture (9, 11) in this laboratory, it was initial inoculum during the 72-hour incubation
possible to study the effects of canavanine on argi
period. Thus, sufficient cells were present to yield
nine metabolism during tumor cell growth. The
a response, if any, to the supplements. Further
present report describes this investigation as well
tests with citrulline included the addition of vary
as related growth experiments with ornithine and
ing amounts of aspartic acid or ammonium chlo
citrulline.
ride, with and without adenosine triphosphate
(ATP).
MATERIALS AND METHODS
C14 experiments.—Twelve replicate cultures
Tissue cultures.—The experimental technics of
were
made. After incubation for 48 hours, two of
tissue culture of the Walker tumor in T-15 tissue
the
cultures
were counted; the medium in the
culture flasks have been described (9). Except for
other
ten
was
replaced with fresh medium con
the changes in concentration of arginine and the taining 1.0 HIML-arginine-U-C14-HCl,
ca. 350,000
additions of canavanine, the amino acid composi
counts/min/mg.3 The medium in five of the flasks
tion of the medium was composed of only the four
also contained 1.0 mM L-canavanine'HjSO.!. At
teen essential amino acids of Medium 3 (8), 1.0
the end of 24 hours of incubation, the cultures were
mM DL-serine, and 0.5 mM glycine. The concentra
tion of vitamins, antibiotics, salts, glucose, and chilled in a refrigerator for several hours to free the
dialyzed human serum was the same as that re cells from the glass. Cell counts showed that the
ported for Medium 2 (11), except that inositol was cultures had progressed uniformly within each
raised to 9 fig/ml.1 All operations were performed group, and their contents were combined. The
cells in each group were then centrifuged, washed
* Presented in part at the 132d National Meeting of the
Some striking examples of antagonism
between
amino acids in microbiological
systems have been
those dealing with the interference
of arginine by
canavanine
(4, 17-22). These naturally
occurring
American Chemical Society, New York, N.Y., September 8-13,
1957.
1R. E. Neuman, this laboratory, unpublished data.
Received for publication September 30, 1957.
2Obtained from California Foundation for Biochemical
Research.
'Purity established by radioscan of paper strip chromatogram.
279
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Cancer Research
280
with ice-cold Earle's balanced salt solution, ex
tracted with 80 per cent alcohol, alcohol-ether
(1:1), cold 10 per cent trichloroacetic acid (TCA),
and hot 5 per cent TCA, according to the method
of Schneider¿(14).The protein residues were hydrolyzed in 6 N HC1 for 22 hours and evaporated
in vacuo to dryness. The evaporation was repeat
ed after addition of water, and the residues were
dissolved in 10 ml. of water.
Arginine radioassays were performed by pre
cipitant-carrier methods (7). All assays were made
in a windowless gas-flow sealer; glycine was dis
solved in aliquots of the cell extracts taken for
L-ARGININE-HCI
•
-0.2
rtiM
x —0.6 mM
°—
I.OmM
Vol. 18, April, 1958
the inhibition of growth of the tumor cells by
canavanine was of a competitive nature with
arginine.
The essentiality of arginine in the tissue culture
medium (8) was emphasized in the present study
by the failure of either ornithine or citrulline to
replace arginine, as shown in Table 1 (although
some alleviation of cell death was apparent at the
higher concentrations of citrulline). Analysis of the
citrulline by ion exchange and paper chromatography failed to reveal any arginine impurity. These
analytical methods were designed to detect as
little as 0.1 per cent arginine impurity. Therefore,
the slight response of citrulline was unexplained,
since a higher concentration of arginine impurity
would be necessary to yield the same alleviation of
cell death (8). For practical purposes, however, the
cells were incapable of utilizing either of the two
common precursors of arginine. The same results
were obtained when, in addition to citrulline,
TABLE 1
GROWTHRESPONSESTUDIESOF WALKERCAR
CINOSARCOMA
256 CELLSCUL
TUREDin Vitro*
Argininewas replacedby ornithine or citrulline.
SUPPLEMENT
CONCEKTHAT10N
(mu)
0
o
^
RATIO; L-CANAVANINE-H?S04/
L-ARGININE-HCI (mM) '
CHAKT1.—Growth response studies with arginine and
canavanine of Walker carcinosarcoma 256 cells cultured in
vitro. For culture conditions see text.
assay purposes in order to make all counts in the
3.4-4.3 mg/sq cm weight range (7). Assays of the
TCA extracts were made by first evaporating ali
quots to dryness with a few drops of l N HC1 in
glass counting containers.
RESULTS
In the 0.2-, 0.6-, and 1.0-mM arginine control
cultures with no canavanine, the cells increased
5.9-, 3.7-, and 4.3-fold, respectively. Chart 1 illus
trates the subsequent inhibitions found when ca
navanine was added. It can be seen that the degree
of inhibition was due primarily to the molar ratio
of antimetabolite to metabolite and was inde
pendent of the absolute concentrations of the
amino acids. The inhibition indices, selected as the
ratios which permitted only 50 per cent of the in
crease in cells in the controls, were found to be 1.2,
1.2, and 1.1. The constancy of these indicated that
PlR CENT DECREASE IN CELL COUNTsf
L-Ormthine-HCl
L-Citrulline
87
87
0.02
82
73
0.05
(Ì7
80
0.2
fiS
78
1.0
7i
65
5.0
74
43
10.0
74
19
* For culture conditions, see text,
t The decrease in cells divided by the initial inocu
lum, 500,000 cells/T-15 flask.
varying amounts of aspartic acid or NH4C1 and
ATP were included in the medium.
Subsequent studies of the mechanism of growth
inhibition by canavanine were confined solely to
the interference of the utilization of arginine by
the cells. These were performed with the use of
L-arginine-C14 with and without an equimolar
amount of L-canavanine. Table 2 shows that
canavanine inhibited the utilization of arginine
quite uniformly throughout the cell extracts and
in the cell protein. In both experiments, the total
of the extracts and protein radioactivities was 25
per cent of the amount of arginine utilized, and the
activity of each protein hydrolysate was found to
be due only to incorporated arginine. There was a
close correlation between the decrease in utiliza
tion of arginine (29 per cent) and the decrease in
the protein-bound arginine (25 per cent).
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
KRUSE AND McCoY—Effect of Canavanine
DISCUSSION
The severe toxicity of canavanine on tumor cell
growth was particularly interesting, since canava
nine is a naturally occurring amino acid. For the
purposes of discussion, however, comparisons will
be made with microbiological systems, since the
authors have been unable to find any studies con
cerning canavanine and its effect on tumor growth
in vitro. Horowitz and Srb studied the effect of
canavanine on three wild strains of Neurospora
(4). While the sensitivity and/or resistance to
canavanine appeared to be genetically determined,
an inhibition quotient of 1.2 (canavanine/arginine) was reported for the complete growth inhibi
tion of the most sensitive strain. In the present
study, an inhibition ratio of 1.2, which permitted
only 50 per cent of maximum growth, was also re
markably small, since Woolley has pointed out
that inhibition or antibacterial indices for most
competitive compounds have been calculated to
range from 100 to 10,000 (24).
Some interesting comparisons were made by
Volcani and Snell (21) of the effect of canavanine
on eight strains of bacteria differing in their ar
ginine requirements. In two strains which could
utilize only preformed arginine, L. mesenteroides
and S. faecalis, canavanine was without effect.
This was shown also by Suzuki, Muraoka, and
Konobu (18, 19). Other bacteria which could syn
thesize arginine from inorganic substances, ornithine, or citrulline were all inhibited by canava
nine. The situation in the present system is some
what the reverse. While the Walker 256 cells re
quired preformed arginine for growth, they were
markedly inhibited by canavanine.
The cause of resistance in S. faecalis has been
shown to be due to one major (5) and one minor
(6) canavanine-destroying enzyme. The former
catalyzed the reductive cleavage of canavanine to
guanidine and homoserine. Apparently, this en
zyme is not present in the Walker 256 cells. Fur
thermore, the present experimental data indicated
the absence of arginosuccinase (2), since the cells
could not utilize citrulline.
There is no doubt that the cause of canavanine
inhibition of the present system rested only with
its interference of the utilization (and not the
synthesis) of arginine. The experiments with arginine-C14 indicated that cell growth, arginine
utilization, and arginine incorporation were in
hibited to approximately the same extent as the
result of supplementation with canavanine. These
results, as well as the radioactivities noted in the
various cell extracts, inferred that canavanine ex
hibited a general inhibition of arginine metabolism
on Walker 256
281
and did not affect any specific area of its utiliza
tion.
In addition, the C14 studies have revealed an
other interesting facet of tumor metabolism. Only
25 per cent of the arginine utilized could be ac
counted for in the cellular components. This,
coupled with the fact that the total radioactivity
in the protein hydrolysates was due only to argi
nine, indicated that the carbons from arginine
were extensively converted to some substance(s)
other than protein-bound amino acids.
TABLE2
DISTRIBUTION
OFRADIOACTIVITY
IN CELLFRACTIONS
OFWALKER
CARCINOSARCOMA
256
The cells were grown in vitro* in the presence of L-arginineC14with and without L-canavanine.
At
C-At
Total
countfl/min
Total
counta/min
Medium:
Initial arginine radioactivity
1,000,000
1,000,000
Final arginine radioactivity
790,000
850,000
Arginine utilized
210,000
150,000
Cells:
80 per cent alcohol extract
2,600
1,700
Alcohol-ether (1:1) extract
1,000
50
Cold 10 per cent trichloroacetic
900
700
acid extract
Hot 5 per cent trichloroacetic
1,900
1,100
acid extract
Residual protein (as hydroly45,700
34,100
sate)
Growth:
Number of cells at 72 hours
12,900,000
11,700,000
Number of cells at 48 hours
6,200,000
6,200,000
Increase in number of cells
6,700,000
5,500,000
* For culture conditions see text; the cells were cultured on
unlabeled medium for 48 hours, followed by 24 hours in medi
um containing 1.0 millimolar L-arginine-C"-HCl, 350,000
counts/min/mg.
11.0 millimolar L-arginine-C'4-HCl.
11.0 millimolar L-arginine-C"-HCl and 1.0 millimolar L-ca
navanine •
BUSO4.
While canavanine has been found to inhibit the
multiplication of certain viruses (12, 13), negative
tumor regression data were reported with the use
of in vivo systems (3, 15, 16). These latter results
might be explained by known enzymes in the
mammalian liver and kidney, which catalyze urea
production (1) and transamidination to glycocyamine (10) and arginine (23) from canavanine.
SUMMARY
Canavanine was found to be a competitive an
tagonist of the utilization of arginine by Walker
carcinosarcoma 256 under tissue culture condi
tions, with an inhibition index of 1.2 for 50 per
cent of maximum growth. The tumor cells failed to
utilize either ornithine or citrulline in place of
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1958 American Association for Cancer Research.
282
Cancer Research
arginine. Carbon-14 studies have indicated that
the inhibition rests in a general interference with
the utilization of arginine. Some postulates have
been made concerning the absence of certain en
zyme activities in the Walker 256 cells.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Merle
Maxwell, Henry Carter, Sarah Seldin, and Nancy Hubbell.
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The Competitive Effect of Canavanine on Utilization of Arginine
in Growth of Walker Carcinosarcoma 256 Cells in Vitro
Paul F. Kruse, Jr. and Thomas A. McCoy
Cancer Res 1958;18:279-282.
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