Download Amino Acid Metabolism of NovikoÃ-FHepatoma

Amino Acid Metabolism of NovikoÃ-FHepatoma'
VICTORH. AUERBACH!ANDHARRYA. WAISMAN
(Sarah A. Workman Pediatrie Research Laboratory, Dept. of Pediatrics, Medical School,
University of Wisconsin, Madison, Wis.)
Numerous studies have been reported on the
metabolism of the transplantable rat "liver" tumor
first obtained by Novikoff (17). The evidence
presently available indicates that this very rapidly
growing neoplasm is deficient in many of the
enzyme systems which control the degradation of
some of the liver cell's most important metabolites.
Thus, Novikoff (17) has shown that the hepatoma
lacks uricase and contains less than 5 per cent of
the esterase and 13 per cent of the succinoxidase
of normal liver. Weber and Cantero (26) could
not demonstrate the presence of glucose 6-phosphatase in the tumor. De Lamirande and his
colleagues (1, 5, 6) have confirmed the finding that
Novikoff hepatoma lacks uricase and have further
demonstrated an absence of xanthine oxidase and
glutamic acid dehydrogenase, as well as a lower
content of nucleoside phosphorylase, 5'-nucleotidase, cathepsin, and guanine deaminase, in the
hepatoma as compared with normal liver. The
same group, however, has shown that the Novikoff
hepatoma contains more adenosine deaminase than
normal liver. Finally, Reynafarje and Potter (20)
have shown that Novikoff hepatoma tissue lacks
the enzymes DPN-TPN-transhydrogenase
and
TPN-cytochrome c reductase.
The present communication will report the pres
ence or absence of a number of enzymes concerned
with the metabolism of amino acids in Novikoff
hepatoma tissue.
MATERIALS AND METHODS
The Novikoff hepatoma was transplanted from
donor rats into healthy 200-gm. male, albino rats
obtained from the Holtzman Rat Company. The
donor tumor tissue was ground in a small hand
tissue grinder and diluted tenfold with isotonic
NaCl; suitable amounts (between 0.1 and 0.5 ml.
of the cell suspension) were injected intraperi* This investigation was supported in part by research grant
CY-3258 from the National Institute of Health and by an
institutional grant from the American Cancer Society.
t Present address: Dept. of Pediatrics, Temple University,
Philadelphia, Pa.
Received for publication November 14, 1957.
toneally (IP) into recipient rats. The animals were
used for experiments after a period of 5-7 days,
at which time the testes were cyanotic and the
tumor, together with a considerable amount of
serosanguineous fluid, could be palpated in the
abdomen.
Rats were killed by a sharp blow on the head and were
exsanguinated from the neck. The entire tumor, which formed
a discrete mass surrounding the greater omentum, was excised
immediately and chilled in cold running water. No efforts
were made to separate any necrotic areas which might have
been present inside some of the tumor masses. The liver from
the same animal was removed. After being weighed to the
nearest 0.1 gm., the tissues were homogenized in a chilled
stainless-steel micro-head Waring Blendor for 2 minutes with
2 volumes of ice-cold 0.14 MKC1 containing 0.0025 N NaOH.
A portion of the homogenate was further diluted with alkaline
KC1 until each part of liver was suspended in seven volumes
of KC1. The dilute homogenate was used for tryptophan
peroxidase-oxidase assays and to obtain dry weights. The
remainder of the dilute and concentrated homogenates were
centrifuged for 45 minutes at 25,000 X g at 0°C. in the high
speed attachment of the International Refrigerated Centrifuge,
Model PR-2. The supernatant fluids were used for the assay
of the other enzymes studied. Dry weights, corrected for the
amount of KC1 contained in the homogenizing fluid, were
obtained on 5-ml. samples of the 12.5 per cent homogenate
dried for 2 hours in tared aluminum moisture dishes at 110°
C.
Tryptophan peroxidase-oxidase assays were carried out by
the method of Knox and Auerbach (13) with 2 ml. of the
12.5 per cent homogenate. Tyrosine (a-ketoglutarate) transaminase and phenylalanine (pyruvate) transaminase assays
were carried out according to the borate-arsenate method of
Lin (16), in which 0.2 and 0.5 ml. of the supernatant fraction
of the 12.5 per cent homogenates were used, respectively. The
borate-arsenate method was also used to determine the activity
of p-hydroxyphenylpyruvic acid oxidase on 0.3 ml. of the 12.5
per cent homogenate supernatant fraction by measuring the
rate of decrease of the substrate.
Phenylalanine hydroxylase was determined by the method
of Udenfriend and Cooper (25), with 0.5 ml. of the supernatant
fraction of the 33 per cent homogenate. DPN was used,
although the enzyme is now known to be TPNH-dependent
(11).
Threonine dehydrase, serine dehydrase, and cysteine desulfhydrase activities were all measured according to the
methods described by Smythe (22). The assays were carried
out with 0.5 ml. of the 33 per cent homogenate supernatant
fraction. Six jug. of pyridoxal phosphate were added to the
incubation mixture in addition to the usual components. The
keto acids produced were estimated by the 2,4-dinitrophenylhydrazine method of Friedmann and Haugen (8).
Histidase activities were measured by the direct spectro-
543
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544
Vol. 18, June, 1958
Cancer Research
photometric method of Tabor and Mehler (24), with the use
of between 0.2 and 0.4 ml. of the supernatant fraction of the
12.5 per cent homogenate.
Arginase activity was determined at pH 9.1 in the presence
of 2.5 X 10"^ Mn"1"1"
ion and 0.5 ml. of the supernatant frac
tion of the 12.5 per cent homogenate. The urea produced
was determined by the use of isonitrosopropiophenone
according to Archibald (2).
Glutamine synthetase was determined according to Elliott
(7), except that tris (hydroxymethyl)aminomethane buffer was
substituted for the glyoxaline buffer recommended. In the
assay of this enzyme, 1.0 ml. of the supernatant fractions of
the 12.5 per cent and 33 per cent homogenates of the hepatoma
and of the liver were used, respectively.
Aspartic acid transcarbamylase, the last enzyme of this
series, was determined by conditions worked out by Kim and
Cohen (unpublished) for liver and neoplastic tissues. Carbamyl
aspartic acid formation was measured according to the method
of Marshall and Cohen (unpublished), which is a modification
of the method of Koritz and Cohen (14).
mg/100 gm rat), L-tyrosine (60 mg/100 gm rat),
or DL-threonine (48 mg/100 gm rat) by IP injec
tion of the suspension or the solution of the amino
acids. Five hours later the rats were killed, and
the tissues were assayed in the usual manner. The
conditions just described have been shown pre
viously to produce optimal adaptation of three
liver enzymes which catabolize these substrates
(12, 16, 21).
RESULTS
In a series of over twenty animals bearing
Novikoff hepatoma, the average body weight was
207 gm. (range, 180-225 gm.), the liver weight
was 6.7 gm. (range, 5.5-8.7 gm.), and the total
hepatoma weight, 8.6 gm. (range, 6.0-11.5 gm.).
TABLE 1
ENZYMEACTIVITIESIN NOVIKOFFHEPATOMA
ANDADJACENTLIVER
All enzyme activities are expressed as /¿moles
product formed (or substrate removed)/gm dry weight of tissue/hr at 38°C. under
optimal conditions of assay.
TISSCE
Adjacent199796411604340356021562699774
Novikoff hepatoma
ENZTUE
< 1* (6)t
Tryptophan peroxidase-oxidase
"
after tryptophan injection
< 1 (3)
<30
(6)
Tyrosine transaminase
"
after tyrosine injection
<30
(1)
Threonine dehydrase
"
after threonine injection
Phenylalanine transaminase
Phenylalanine hydroxylase
Cysteine desulfhydrase
p-Hydroxyphenylpyruvic acid oxidase
Histidase
103,000495(2)(3)
Arginase
Glutamine synthetase
681 (4
ITSliver.5(7)(3)(2)(1)(8)(2)(3)5(3)(3)(3)5(3)
Aspartic acid transcarbamylase
W
* Values following < sign indicate lowest extent of reaction that could be detected
by the methods used.
t Number of animals in each group.
All the enzymes measured are known to be
found in the soluble phase in normal liver tissue.
No attempts were made to test the possibility that
some activity might also be obtained from the
insoluble cell fractions, either from the liver or
from the hepatoma.
The enzyme activities are expressed in terms
of Rimólesof the product formed/gm dry weight
of liver or hepatoma/hour at 38°C., the tempera
ture used in all the reactions. Two exceptions to
these units of activities were that substrate dis
appearance and not product formation was meas
ured in the assay of p-hydroxyphenylpyruvic acid
oxidase and that threonine dehydrase activity was
expressed in terms of the color equivalent of a
pyruvic acid standard rather than in terms of the
actual amount of a-ketobutyric acid formed.
Some animals were given L-tryptophan (150
Of the enzymes investigated, no activity could
be demonstrated in the hepatoma for the following
systems: tryptophan peroxidase-oxidase, tyrosine
transaminase, phenylalanine hydroxylase, cysteine
desulfhydrase, serine dehydrase, threonine dehy
drase, histidase, and p-hydroxyphenylpyruvic acid
oxidase. The levels of phenylalanine transaminase,
arginase, and glutamine synthetase in the tumor
were much lower than the corresponding enzyme
levels of the livers of the same animals; while the
hepatomas were much richer in aspartic acid trans
carbamylase than were the livers. The data for
all these enzyme assays are presented in Table 1.
Of the enzymes measured, three (tryptophan
peroxidase-oxidase, tyrosine transaminase, and
threonine dehydrase) were induced by the prior
injection of their respective substrates. It was
thought that the unmeasurably low levels of these
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AuERBACH AND WAiSMAN—Amino Acid Metabolism of Novikoff Hepatoma
three enzymes in the hepatoma could be increased
by treatment of the animals with the three sub
strates. As is shown in Table 1, the hepatomas
of the animals treated in this manner were still
deficient in the three enzymes, while the level of
tryptophan peroxidase-oxidase in the liver showed
the usual adaptive response.
DISCUSSION
Novikoff hepatoma represents one of a class of
very rapidly proliferating tumors. After a period
of 1 week, the original injection material, amount
ing to about 20 mg., had grown into a tumor mass
weighing about 9 gm. It is not surprising, there
fore, in view of this rapid tissue growth, that the
tumor cells are relatively incapable of degrading
important metabolic intermediates. Instead, it
would seem that, given a source of energy, these
cells would utilize all the available pool of amino
acids to form new protein. The same picture ap
pears to pertain in the realm of purine and pyrimidine metabolism (1, 5, 6).
Of the amino acid-metabolizing enzymes shown
to exist in hepatoma cells, arginase was present
in extremely small amounts. Phenylalanine transaminase occurred at a level about of 10-20 per
cent of that found in liver (on a dry weight basis)
but in amounts comparable to the level found in
kidney, heart, and muscle (16).
Glutamine synthetase was also present in hepa
toma tissue at a much lower concentration than
normally found in liver. However, in view of recent
work on the essential nature of the incorporation
of glutamine into proteins in tissue culture (15),
it would be reasonable to assume that whatever
glutamine synthetase was present in the hepatoma
was concerned with the process of protein syn
thesis.
The large amount of aspartic acid transcarbamylase found in the tumor as compared with that
observed in liver is undoubtedly a reflection of
the increased synthesis of pyrimidines occurring
in the tumor cells. This observation is a confirma
tion of the findings of Calva (4) and Calva and
Cohen (unpublished) that primary hepatomas and
Ehrlich ascites tumor cells have a much higher
aspartic acid transcarbamylase level than adjacent
normal liver. It is currently believed that dihydroorotic acid, formed from carbamylaspartic acid,
is an intermediate on the pathway leading to
pyrimidine biosynthesis (10, 23).
The observed differences in liver enzyme levels
found between the livers of animals bearing the
hepatoma and the livers of normal animals (3)
can, in part, be ascribed to differences in age of
545
the rats and in part to the influence of the presence
of the hepatoma on the metabolism of the host.
A rational interpretation of the data available
would be to assume that all those enzymes
required for the continued rapid proliferation of
the tumor cells would be present in substantial
amounts, while degradative enzyme systems pres
ent would deplete the store of necessary synthetic
intermediates and would be entirely absent or
present in only small amounts. It must be remem
bered, however, that there exist many normal
tissues which are relatively deficient in catabolic
enzyme systems but are not undergoing rapid
proliferation. Knowledge of the precise mechanism
governing the rates of growth and division of
normal cells is still lacking.
Another approach to the problem is in terms
of what Potter et al. (18, 19) have called the
"deletion hypothesis." The tumor may be a tumor
simply because of the deletion of certain cytoplasmic particulates, enzyme-forming systems, or
enzyme systems which occurred during the growth
of normal tissue. The data presented herein could
tend to serve as an illustration of the results of
the working of the "deletion hypothesis."
Not only are some of the enzymes studied
absent in Novikoff hepatoma, but the correspond
ing enzyme-forming systems must also be absent,
as judged from the lack of production after sub
strate injection of the three specific substrateinducible systems: tryptophan peroxidase-oxidase,
tyrosine transaminase, and threonine dehydrase.
In the case of at least one of these systems, the
liver enzyme increased in the usual manner after
tryptophan injection. In the same animal, the
hepatoma gave no response. The small increase
in the liver tyrosine transaminase level following
tyrosine administration is not surprising, consider
ing that the amount of tyrosine transaminase
found prior to tyrosine injection was fairly high
initially. (The value for liver tyrosine transaminase
of normal rats is 405 /¿molesproduct formed/gm
dry weight/hr [3].) The reason for the lack of
change in liver threonine dehydrase activity after
threonine administration to animals bearing Novi
koff hepatomas is unknown.
One must also consider the possibility that the
Novikoff hepatoma is not derived from parenchymal liver cells but is an outgrowth of either biliary
duct epithelium or connective tissue cells. This
deduction could easily explain the absence of
many enzymes which are known to be largely
liver-specific in occurrence. However, the presence
of even a few enzymes such as phenylalanine
transaminase, arginase, glutamine synthetase, and
aspartic acid transcarbamylase known to occur in
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546
Cancer Research
liver, and thought to be located in the parenchymal cells in particular, makes one pause and
defer judgment. Furthermore, the work of Weiler
(27) with fluorescent antibodies prepared against
liver cell particulate fractions indicates that it is
the parenchymal liver cells which slowly lose the
ability to bind the antibody and then become
primary hepatomas when rats are treated with
4-dimethylaminoazobenzene. Novikoff hepatoma
is a transplantable cell type derived from such 4dimethylaminoazobenzene-induced
primary hepa
tomas. Weiler's work would imply that the Novi
koff hepatoma is indeed derived from parenchymal
liver cells and that the enzyme pattern found in
this tissue represents a deletion from liver rather
than a pattern characteristic of a different cell
Hughes el al. (9) have reproduced Weiler's
findings but state that the method as described
by Weiler does not produce an antiserum which
has any tissue specificity. Nevertheless, they, too,
observed zones in the livers of rats given 4dimethylaminoazobenzene
which did not stain
with the fluorescent antiserum but appeared to
be normal parenchymal cells, and also concluded
that these zones might represent microscopic can
cerous foci.
SUMMARY
1. A group of enzymes metabolizing amino
acids have been investigated in Novikoff hepatoma
tissue and in the livers of the same animals.
2. Novikoff hepatoma did not contain the fol
lowing enzyme systems: tryptophan peroxidaseoxidase, tyrosine transaminase, phenylalanine hydroxylase, threonine dehydrase, serine dehydrase
cystein desulfhydrase, histidase, and p-hydroxyphenylpyruvic acid oxidase.
3. Tryptophan
peroxidase-oxidase,
tyrosine
transaminase, and threonine dehydrase could not
be induced in these tumors by the administration
of the respective substrates.
4. Novikoff hepatoma contained less glutamine
synthetase and less arginase than did normal or
adjacent liver and about 4 times as much aspartic
acid transcarbamylase as did the adjacent liver.
5. These results are discussed in terms of the
metabolic characteristics of the tumor and in
terms of the "deletion hypothesis" of carcinogenesis.
ACKNOWLEDGMENTS
We wish to thank Dr. Van R. Potter for supplying us with
the original Novikoff hepatoma transplant material and Dr.
Philip P. Cohen for the use of his facilities for the measurement
of the aspartic acid transcarbamylase. Both have offered much
useful advice and given considerable encouragement as well
as material assistance
Vol. 18, June, 1958
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Amino Acid Metabolism of Novikoff Hepatoma
Victor H. Auerbach and Harry A. Waisman
Cancer Res 1958;18:543-547.
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