Download Responsiveness of Two Urea Cycle Enzymes in

[CANCER RESEARCH 31, 12—18, January
1971]
Responsiveness of Two Urea Cycle Enzymes in Morris Hepatomas
to Metabolic Modulations'
Chung Wu, Jere M. Bauer, and Harold P. Morris
Departments ofBiological
Chemistry [C. W.J and Internal Medicine IC. W., .1.M. B.J , University ofMichigan
Medical School, Ann Arbor, Michigan
48104, and Department ofBiochemist,y, CollegeofMedicine, Howard University, Washington,D. C. 20001 fH. P. M.j
the hormone
SUMMARY
Arginase and argininosuccinate synthetase activities have
been assayed in some 14 hepatomas and the corresponding
host livers. The hepatomas
exhibited
an extremely
wide range
of activities of the two enzymes of the urea cycle. A good
correlation exists between the activities of the two enzymes in
the hepatomas. However, the activity of either enzyme -in the
hepatomas
bears no correlation
with
that in the host livers.
Moreover, there is no correlation between the enzyme activity
and the growth
rate or chromosome
number
of the hepatomas.
Cortisol raised arginase activity to a larger degree in Hep
atomas 7800 and 8999 than in the host livers, but the
hormone
enhanced
argininosuccinate
synthetase
activity more
in the host livers than in the two hepatomas. Thyroxine in
creased arginase activity in Hepatoma 7800 and the host liver
of intact
rats but decreased
it in adrenalectomized
rats. The
thyroid hormone also elevated the synthetase activity in
Hepatoma 7800 but not in the host liver. On the other hand,
thyroxine did not affect the activity of these two enzymes in
either Hepatoma 96 18A or the host liver.
While actinomycin D or cycloheximide blocked the hor
mone-induced increase in arginase activity of Hepatomas 7800
and 8999
and in the synthetase
activity
of Hepatoma
7800,
the antibiotics exerted no inhibitory effect on the enzymes in
the host livers. Increasing the protein content of the diet
elicited increases in the activity of the two enzymes in
Hepatoma
9618A,
as well as in the host
liver, but
not
manipulations
in the hepatomas
but not in the host livers (23),
we felt that we should take another look at the hormonal and
dietary effects on arginase and argininosuccinate synthetase in
hepatomas.
We observed a responsiveness of these 2 enzymes
in the hepatomas similar to that of glutamine synthetase under
comparable conditions. This greater responsiveness of these
enzymes in hepatomas than in host livers appears to be worthy
of further study and should aid our efforts to understand the
aberrant
regulatory
mechanisms
operating
in cancer.
paper will present the observations and implications
studies.
This
of our
MATERIALS AND METHODS
The tumor-bearing rats of the Buffalo and ACI strains used
in this study were sent by Dr. H. P. Morris to the University of
Michigan. They were fed a regular laboratory chow until the
tumor
had grown to about S g, at which time the animals were
used for experiments. When the dietary effect was studied, a
test diet with a basal composition similar to the one used
previously (22) replaced chow for 2 weeks before the experi
ment was completed.
Cortisol
suspended
in sesame
oil was given i.m., SO pg/g
body weight/day. L-Thyroxine dissolved in 0.9% NaCI solution
in
with minimal NaOH was injected s.c., 1 j.tg/g body weight/day,
for 7 days. Actinomycin
D was injected i.p., 0.2 j.ig/g body
by
by
weight/day, unless noted otherwise, for the number of days
specified in each experiment. Cycloheximide was injected i.p.,
the same enzymes in host livers. This overresponsiveness is
discussed as a manifestation of deranged control mechanisms
between 8 and 10 a.m., and the animals were killed 24 hr after
Hepatoma 8999.
These results together with others show an overresponse
enzymes in hepatomas, when compared with the response
in cancer.
Increase in arginase activity in rat liver following high
protein diets (1 , 6, 14) or large doses of corticosteroids (2 , 15)
is known. Earlier, we studied the effect of cortisol on 3 urea
cycle enzymes in both host livers and hepatomas and noted
that the enzymes
in the hepatomas
responded
This work was supported
in part by Grant
National
Institute
of Arthritis
and Metabolic
more readily to
AM-073 19 from the
Diseases and Grant
CA-10729 from the National Cancer Institute, USPHS.
Received May 27, 1970; accepted September 18, 1970.
12
5 ;ig/g body weight/day,
the last injection.
time.
INTRODUCTION
@
than did those in the host livers (24). Following
our demonstration that certain enzymes of glutamine metabo
lism, notably glutamine synthetase, responded to metabolic
Control
for 1 day. All injections were done
animals
were
killed
at the same
Total adrenalectomy and thyroidectomy of tumor-bearing
rats were done for us by Hormone Assay Laboratories,
Chicago,
Ill., 2 days after tumor inoculation.
For
adrenalectomized
rats, the drinking water contained 1%
NaCl. Hepatoma
7800 grew more slowly in adrenalectomized
rats than in intact rats.
Arginase and argininosuccinate synthetase activities were
assayed according to the method of Schimke (14). The
argininosuccinate
lyase used in the synthetase
assay was pre
pared from rat liver after the procedure of Ratner (13).
Arginase activity is expressed as mmoles urea formed/g tissue/
hr; argininosuccinate synthetase is expressed as j.zmoles urea
CANCER RESEARCH VOL. 31
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Responsiveness ofEnzymes
formed/g
tissue/hr.
The enzyme
activities
are expressed
on a
wet-weight basis. In the tables, the values are for means ±S.D.;
where the experiment used fewer than 3 rats, individual ob
servations
are presented.
The
1-digit number
in Hepatomas
if...... HOST'S
LIVER
in parentheses
a,
indicates the number of rats used.
U)
,//
0
E
RESULTS
E
Arginase. Table 1 shows arginase activity in 14 rat hep
atomas arranged in decreasing order of activity. The hep
U
0
atomas
w
display
an extremely
wide range
of activity:
>
from
U)
Hepatoma 7793 , which had an activity like that of normal
liver, to Novikoff and 2 other hepatomas, which had no
measurable activity. Hence, the highest activity differed from
the lowest by at least 3 orders of magnitude. Hepatoma
9618A, with a chromosome complement like that of normal
liver (9) and a very slow growth rate (about 8 months), had
much lower activity than many other hepatomas
normal chromosome
karyotypes and faster growth
with ab
rates. The
activity in the host livers also varied widely and appears irrele
vant to the activity in the hepatomas.
Following our earlier observation of the effect of cortisol on
arginase in certain hepatomas (24), we studied the change in
arginase activity
with days of cortisol
injection
in rats bearing
Hepatoma 7800, as seen in Chart 1. In the host liver, there was
no change in arginase activity
in the first 2 days of treatment.
The increase between the 2nd and 3rd days was sharp; after
the 3rd day, however, the activity gradually declined. On the
other hand, in the hepatoma
steady increase with number
the enzyme activity
of days of cortisol
showed a
treatment
until the 4th day, after which time the activity decreased.
Comparison of the 2 curves in Chart 1 shows that not only the
pattern of response but also the magnitude of response by
arginase in the host liver and hepatoma are different. Cortisol
increased arginase activity in the host liver by a mere 60%, but
it raised the activity
in Hepatoma
7800 by 440%. Hence, the
0
C
•0'
4
0
Days after cortisol
Chart 1. Effect of daily injections of cortisol (50 .ig/g body weight)
on arginase activity in Hepatoma 7800 (Generation 41) and in the liver
of the host. Each point represents the mean value from 3 to 5 rats;
vertical line, average deviation from the mean.
enzyme in the hepatoma showed a greater responsiveness than
the enzyme in the host liver.
To learn the nature of the increase in the enzyme activity in
hepatoma, we studied the effect of actinomycin D and cyclo
heximide on this increase. Table 2 shows the results. We found
that
in Hepatoma
8999
arginase
activity
increased
10-fold
after 2 days of cortisol treatment. This magnitude of increase
may be compared with the 3-fold increase observed in Hepa
toma 7800 (Chart
1) for the same period
oftreatment.
Hepa
toma 7800, which had higher basal activity of arginase than
Hepatoma 8999, showed smaller inducibiity by cortisol. The
cortisol-induced arginase in the hepatoma was sensitive to both
actinomycin D and cycloheximide. On the contrary, the
Table 1
Arginase activity in liver and hepatomas
(mmoles/g/hr)Rats
TumorActivity
liverHepatomaNo
bearing:generationHost
5bHepatoma7793(4)1871.7±
tumor (Buffalo) (6)a104
4Hepatoma
10.8Hepatoma
7794A (4)24106
16.0Hepatoma
9108
±
17.9116
± 975.9
± 4.968.1
± 0.957.1
(6)739.2
3.6Hepatoma
8995 (3)333.4
3.1Hepatoma
7800 (4)41,
4.1Hepatoma
9121 (4)2270.4
3.3Hepatoma
9633 (8)798.0
2.1Hepatoma
9618A (6)3,
1.3Hepatoma
9098 (4)449.2
0.2Hepatoma
7777 (4)483.0
0.19Novikoffhepatoma
8999 (5)6,
10.2<0.1Hepatoma (6)53.6
17.5<0.1Hepatoma
9618A2 (3)252.9
7288C (3)350.4
a@0 in parentheses, no. ofrats
4366.0
486.8
±14.013.9
±6.213.2
± 9.912.5
±6.912.4
±2.91
± 0.74.9
798.7
±21.60.65
±
±
±2.9<0.1
±
±
±
±
±
±
±
±
1.3 ±
±
±
used.
bValue for normal liver.
JANUARY
1971
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13
Chung Wu, Jere M. Bauer, and Harold P. Morris
Table 2
Effect ofcortisol and metabolic inhibitors on arginaseactivity in liver and hepatomas
Male rats bearing Hepatoma 8999 (Generation 6) and adrenalectomized rats bearing
Hepatoma 7800 (Generation 43) were used. Cortisol was given for the number of days shown.
In Experiment A, actinomycin D (1 @g/g
body weight) and cycloheximide were given for 1 day,
on the 2nd day of cortisol treatment. In Experiment B, actinomycin was given for 2 days
following the completion
of cortisol treatment.
(mmoles/g/hr)Host
liverHepatomaRats
TreatmentActivity
8999None([email protected]±21.60.65±
bearing Hepatoma
0.19Cortisol,
1.49Cortisol,
2 days (3)1
withactinomycin,
2 days,
0.21Cortisol,
1 day (3)117
withcycloheximide,
2 days,
0.89Rats
1 day (3)115
7800None(S)115
bearing Hepatoma
8.5Cortisol,
92.0)Cortisol,
4 days (4)157
15
±196.20
±
±101.75
±
±283.44
±
±3118.6
±1290.2
(89 8,
(118, 123)63.5
(40.6,
±
none,2 4 days +
76.4)Cortisol,
days (2)120
actinomycin,2days(3)140
4 days +
±14.1
±2935.0
a@40 in parentheses, no. of rats used.
Table 3
Effect of thyroxine on arginaseactivity in liver and hepatomas
Ihyroidectomized
and adrenalectomized
rats bearing
Hepatoma
7800 (Generations
45 and
43, respectively) and intact rats bearing Hepatoma 7800 (Generation 43) and Hepatoma 9618A
(Generation
6) were used. L-Ihyroxine
was given for 7 days; actinomycin
D was injected for 2
days, and cortisol was injected for 4 days, on the last 2 and 4 days, respectively,of thyroxine
treatment.RatsTreatmentActivity
(mmoles/g/hr)Host
liverHepatomaBearing
7800IntactNone
Hepatoma
(3)Ihyroidectomized
Thyroxine66.0
121
±14.0(5)@
±10 (3)13.9
±22
AdrenalectomizedNone
(77.7,95.8)Bearing
9618AIntactNone
Hepatoma
131
±45 (3)
None
115
±31
96.1 (88 2, 104)
Thyroxine
Thyroxine +
cortisol166
no. in parentheses,
139(137
similar observations on glutamine synthetase (23).
in Hepatoma
7800 grown in adrenalectomized
rats
(Table 2) showed slightly greater response to cortisol than the
14
141)47.3
±21
(3)
90.9 ±10.5 (4)
117
±3
(3)20.8
65.9± 19.0(3)
18.6 ± 8.5 (5)
9.1 (9.1,9.1)
86.8
±4.2 (3)
20.6 ±3.6 (4)
24.1 ±0.8 (3)
no. of rats used.
enzyme in the host liver was not affected by cortisol, nor was
it sensitive to the metabolic inhibitors. The difference between
the responsiveness of arginase in the hepatoma and the lack of
responsiveness in the host liver is striking. Recently, we made
Arginase
(5)
47.4 (46 8,47.9)
Thyroxine
Thyroxine +
actinomycin121
a One-digit
(3)
Thyroxine
Thyroxine+
actinomycin
± 3.1 (4)
22.2 ±6.9
(41.3, 53.2)
68.2 ±9.5 (3)
enzyme
in the same hepatoma
grown in intact hosts (Chart
1).
Apparently, high concentrations of cortisol are essential for
maintenance of the induced level of arginase activity, for in
the absence of continued injections of cortisol the activity
declined. Moreover, administration of actinomycin D for 2
days in the postcortisol period further reduced the enzyme
activity. On the other hand, the enzyme activity in the host
CANCER RESEARCH VOL. 31
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Responsiveness ofEnzymes
liver showed no appreciable changes with the cortisol or
actinomycin treatment. Hence, the responsiveness of arginase
in Hepatoma 7800 and the lack of responsiveness in the host
liver are similar to those in Hepatoma 8999-bearing rats.
The effect of thyroxine on arginase activity appears to be
related to that of corticosteroids (Table 3). In both intact and
thyroidectomized
rats bearing Hepatoma 7800, thyroxine
caused a slight increase in arginase activity in the hepatoma,
and the increase seemed unaffected by actinomycin D treat
ment for 2 days. In intact rats bearing Hepatoma 96l8A, the
enzyme
did not respond
to thyroxine
or actinomycin
in either
the host liver or the hepatoma. However, in adrenalectomized
rats bearing Hepatoma 7800, thyroxine lowered arginase
activity
by
Thyroxine
half
in both
the
host
liver
and
the
tumor.
also produced a moderate decline in the enzyme
Table 4
Effect ofprotein content in diet on arginaseactivity
in liver and hepatomas
activity
in the host liver of thyroidectomized
rats. If we can
disregard the generation difference of the hepatoma grown in
intact and in thyroidectomized rats, we can see that thyroidec
tomy elevated arginase activity in both the host liver and the
hepatoma.
Furthermore,
cortisol raised the enzyme activity
from a depressed
level following
thyroxine
treatment
of
adrenalectomized
rats to a level comparable with that attained
by administration
of cortisol without prior thyroxine
treat
ment (Table 2). Apparently, further experimentation
is needed
to clarify the role of thyroxine in arginase regulation. Never
theless, these results suggest that the thyroid hormone exerts a
repressive effect on arginase, which is lifted by thyroidectomy.
This repressive effect is at least partially offset by the induc
tive effect of cortisol. Thus, the 2 hormones appear to exercise
a check-and-balance influence on arginase.
Feeding of high-protein diets to the rat resulted in an in
crease in liver arginase activity (1 , 6, 14). The purpose of our
experiment
Male rats bearing Hepatomas 8999 (Generation 6) and 9618A
in Hepatomas
on the dietary
effect on this enzyme
was to show
whether the enzyme in the hepatoma responded differently
from that in the host liver. As the results in Table 4 show,
(Generation 5) were fed diets containing different percentages of casein
arginase
for 2 weeks. Variations in protein content of the diet were made at the
tein content of the diet, but the enzyme in Hepatoma 96l8A,
like that in liver, did.
Argininosuccinate Synthetase. This enzyme is believed to be
rate limiting in urea biosynthesis (5). Its activity in rat liver is
lower than arginase activity by 3 orders of magnitude. Since
arginase activity is in great excess, its substantial reduction in
many hepatomas will not restrict their capacity to synthesize
urea. On the other hand, any decrease in argininosuccinate
synthetase activity would curtail urea production. With the
expense of carbohydrate.
(mmoles/g/hr)Host
liverHepatomaRats
Protein in dietActivity
bearing Hepatoma 8999
5% casein(4)a
25%casein(5)
75%casein(3)
Rats bearing Hepatoma 96 18A
5% casein(4)
25%casein(4)
75%casein(4)99.8
a@0 in parentheses,
±20.9
132
168
±0.06
1.20±0.16
1.01±0.25
±25
±9
in Hepatoma
exception
78.4 ±6.0
96.0± 5.1
108 ±80.53
18.5 ±2.4
27.7 ±1.0
of Hepatoma
8999
did not respond
to the high pro
9 108 , all tested hepatomas
had lower
argininosuccinate synthetase activity than normal liver had
(Table 5). Although the precise order of hepatomas appearing
37.8 ±7.7
in this table
hepatomas
no. of rats used.
does not coincide
with the order
(of the same generations)
of the same
for arginase activity
Table 5
Argininosuccinate synthetaseactivity in liver and hepatomas
(@moles/g/hr)Rats
TumorActivity
liverHepatomaNo
bearing:generationHost
17bHepatoma
tumor (Buffalo) (6)a183
±
76Hepatoma7800(4)41,43115
9108 (6)742.1
± 8.4292
±
18Hepatoma
16Hepatoma
7793 (4)1854.7
8.7Hepatoma
8995 (3)3101
5.4Hepatoma9l2l(4)22129
7794A (4)24186
9.9Hepatoma
6.6Hepatoma
9098 (4)474.0
5.0Hepatoma
9633 (8)764.2
3.7Hepatoma
7777 (4)444.8
4.1Hepatoma
9618A (6)3,
2.9Novikoffhepatoma
7288C (3)3138
2.0Hepatoma
(9)101
3.0Hepatoma
8999 (5)6,
9618A2 (7)2138
±15117
±6.3109
±796.0
±3045.3
±
a@0
in parentheses,
±
±
±
± 529.7±
41
766.2
18
± 6.128.7
±10.526.3
±3.517.7
±1914.3
±2012.3
±1710.9
±4.210.4
±22<10
±
±
±
±
±
±
±
no. of rats used.
bValue for normal liver.
JANUARY 1971
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15
Chung Wu, Jere M. Bauer, and Harold P. Morris
shown in Table 1, we can see that the first 5 hepatomas listed
in Table 5 are also the first 5 listed in Table 1, the second 5
hepatomas listed in the two tables are also the same, and so are
the last 4 hepatomas.
between
Hence, there is a general correlation
these 2 enzyme activities in the hepatomas.
It appears
that the carcinogenic process has brought about coordinate
changes in these 2 enzymes
of the urea cycle. Presumably,
the
genes governing the synthesis of these 2 enzymes are so closely
placed in space that during carcinogenesis, if 1 gene is affected,
the other would be affected
similarly.
Table 6
Table 5 also shows the synthetase activity in the host livers.
In a majority of them, the activity was lower than in normal
liver.
The effect of cortisol on the raising of argininosuccinate
synthetase activity was less pronounced
in hepatoma than in
host liver. Table 6 shows the results with Hepatomas 8999 and
7800. In the host livers, cortisol significantly elevated the
enzyme activity. The elevation could not be sustained without
continued hormonal treatment (in Hepatoma 7800), and the
elevated enzyme activity was not subject to inhibition by
actinomycin. On the other hand, cortisol exerted little effect
on the enzyme
activity
in the 2 hepatomas.
This situation
Effect ofcortisol and actinomycin D on argininosuccinate
synthetaseactivity in liver and hepatomas
Male rats bearing Hepatoma 8999 (Generations 6 and 7) and
adrenalectomized rats bearing Hepatoma 7800 (Generation 43) were
used. Cortisol was injected for the number of days specified.
Actinomycin D was injected (1 j@g/gbody weight) for 1 day on the last
day of cortisol administration (for Hepatoma 8999) and for 2 days
stands in contrast to the responsiveness of arginase to cortisol
in these 2 hepatomas (Table 2).
following
increase in argininosuccinate synthetase activity in Hepatoma
7800 was similar in both intact and adrenalectomized rats,
the completion
of cortisol
administration
(for Hepatoma
7800).
indicating
(@imoles/g/hr)Host
actinomycin,
1 day
Cortisol,4days
the
effect
of thyroxine
increased
We found
7800 but
The small
is independent
of
hormones. The increase was greatest, however,
in thyroidectomized
rats receiving thyroxine,
where a 3-fold
increase
in activity
was observed.
Administration
of
bearing Hepatoma 8999
None
Rats bearing Hepatoma 7800
None
that
corticosteroid
liverHepatomaRats
TreatmentActivity
Cortisol, 2 days
Cortisol, 2 days, with
Freeland and Sodikoff (4) showed that thyroxine
argininosuccinate
synthetase activity in the rat liver.
that thyroxine
induced this enzyme in Hepatoma
not in the host liver. Table 7 shows the results.
±4.2 (5)a
±3.0 (6)
102
±12
(6)
16.7 ±5.4 (4)
103
±16
(6)
12.6 ±3.6 (3)
106 ±32 (5)
251
Cortisol, 4 days + none,
2 days
Cortisol, 4 days +
actinomycin, 2 days66.2
±24
(4)
163 (156, 170)
144 ±57 (5)
212 ±36 (4)
actinomycin D effectively blocked the enzyme induction in
the thyroxine-treated rat bearing Hepatoma 7800. Hence, the
increase in enzyme depends on continuous synthesis of RNA.
In contrast, the enzyme in the host liver did not respond to
thyroxine or to actinomycin D. These results serve as another
indication
hepatomas
101 (1 12, 88.9)
of the increased responsiveness
to metabolic modulations.
of enzymes
in some
On the contrary, in rats bearing Hepatoma 96l8A the effect
of thyroxine
22 (3)
178 ±
21 (3)10.4 180 ±
on argininosuccinate
synthetase
was apparent
in
the host liver, but its effect on the enzyme in the hepatoma
was insignificant.
a One-ii@it no. in parentheses,no. ofrats used.
Two points seem clear. First, the response by
Table 7
Induction ofargininosuccinate synthetase by thyroxine in liver and hepatomas
Male rats bearing Hepatomas 7800 (Generation 43) and 96 18A (Generation 6), male
thyroidectomized rats bearing Hepatoma 7800 (Generation 45), and male adrenalectomized
rats bearing Hepatoma 7800 (Generation 43) were used. L-Thyroxine was given for 7 days;
actinomycin D was given for 2 days on the 6th and 7th days of thyroxine treatment.
(@moles/g/hr)Host
RatsTreatmentActivity
liverHepatomaBearing
Hepatoma 7800
Intact
±15(4)a
Thyroidectomized
Adrenalectomized
Thyroxine
None
Thyroxine
Thyroxine +
actinomycin
D
129 ± 3 (3)
83.4 (75.9, 90.9)
79.1(73.9,
84.2)
None
106 ±32 (5)
Thyroxine
121(117,124)
68.7±20.5(3)
±18(4)
199 ±
25 (3)
101 ±23 (3)
312 ±13 (3)
168 ±12 (3)
144 ±57 (5)
212 (197,226)
Bearing Hepatoma 9618A
IntactNone
None
Thyroxine
Thyroxine +
actinomycinD115
a One-digit
16
no., in parentheses,
134
±20 (4)
20.4±2.1(3)
14.4 ±2.7 (4)
117
±19(3)117
17.0±1.6(3)
84.9 ±12.1 (3)
no. of rats used.
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Responsiveness ofEnzymes
Table 8
Effect
ofprotein
synthetaseactivity
content
ofdieton argininosuccinate
hepatomasMale
in liver and
8A(Generation
rats bearing Hepatomas 8999 (Generation 6) and 96 1
2weeks.Activity
5) received diets containing 5 , 25 , and 75% casein for
(j.zmoles/g/hr)Protein
HepatomaRatscontent
Host liver
2.575%
casein (5)
2.6Ratscasein (4)
38.9 ±8.8
10.7 ±
84.6 ±7.7
113 ±17
9.9 ±
10.6 ±
97.1 ±12.6
20.1 ±
9618A5%
bearing Hepatoma
1.025%
casein (4)
3.575%
casein (4)
167
±25
32.4 ±
casein (4)
268
±20
67.6 ±5.5
a@40in parentheses, no. of rats used.
an enzyme in the host liver depends on the hepatoma the host
bears. Second, different hepatomas can exhibit different re
sponses by a given enzyme
to a given set of conditions.
Like arginase, shown earlier (Table 4), argininosuccinate
synthetase in a hepatoma may or may not respond to the
protein content of the diet. The results in Table 8 show that
the enzyme activity in the host livers increased about 3-fold
when the protein content of the diet was raised from S to
75%. The enzyme
in Hepatoma
96 l8A exhibited
a similar in
crease, but the enzyme in Hepatoma 8999 was unresponsive to
changes in dietary protein. In this respect, argininosuccinate
synthetase and arginase in Hepatoma 96 18A are more like
these enzymes in liver than are those in Hepatoma 8999.
We have made a parallel study of the responsiveness of
arginase and argininosuccinate synthetase to hormonal, sur
gical, and dietary manipulations in Hepatomas 7800, 8999,
and 96l8A and in host livers. The responses of these 2 en
zymes in the hepatomas to a given set of experimental condi
tions are usually different. For instance, cortisol elicited large
increases in arginase activity in Hepatomas 7800 and 8999 but
did not evoke an appreciable increase in the synthetase
activity. While thyroxine significantly lowered arginase activity
in Hepatoma 7800 and the host liver of adrenalectomized rats,
it slightly raised the synthetase activity. Actinomycin D
blocked the thyroxine-induced
increase in the synthetase
activity in Hepatoma 7800 grown in thyroidectomized rats; it
did not block the small increase in arginase activity. These
contrasts indicate that, although the 2 enzymes are engaged in
the same metabolic cycle, they do not respond to metabolic
modulations in the same way.
In the course of this investigation, we have used more than
1 generation of several tumors and have found that the en
zyme activity in the host liver and in the tumor of I genera
tion may be different from that in those of another genera
tion. For this reason, the order of at least some of the
@
listed in Tables
1 and S depends,
to some extent,
on the generations of the tumors used. We have reported a
similar observation regarding glutamine synthetase (23).
JANUARY
the similarity
in vivo between
the aminoazo
protein conjugate and the fluorenyl-protein conjugate. All
Morris hepatomas have been induced by feeding carcinogens,
particularly fluorenylamine compounds, to the rat (7). Sorof
et al. (19) further demonstrated that certain Morris hepa
tomas,
notably
Hepatoma
7793 , had little or none of the pro
tein conjugating with the fluorenyl carcinogen. Since, as
shown in the present study, a majority of the Morris hepa
tomas had significant arginase activity and Hepatoma 7793
had an activity as high as that of the liver, evidently the
fluorenyl-conjugating protein that is low or lacking in Hepa
toma 7793 (19) is not arginase.
Although a majority of experimental results from various
laboratories (8, 10, 12, 16, 17, 21) have emphasized the lack
of responsiveness of enzymes in many hepatomas to metabolic
stimuli, we have gathered evidence to the contrary. Recently,
we demonstrated that glutamine synthetase and glutamine
aminotransferase
in the Morris hepatomas
responded
readily to
metabolic modulations, while these enzymes in the host livers
did not (23). In addition, this report shows that 2 enzymes of
the urea cycle in the hepatomas responded to hormones and
metabolic inhibitors, in contrast to the lack of response of
these enzymes in the host livers. Pitot (1 1) made a similar
observation on tyrosine aminotransferase in 2 Morris hepa
tomas.
DISCUSSION
hepatomas
Having recognized this anomaly, we find it necessary to
specify the generation number of every tumor used in the
present study. Therefore, in making any comparison of values
obtained with a hepatoma of different generations, we must
take this possible variation into consideration.
Arginase has been implicated as the protein that inhibits cell
proliferation in vitro (3, 20). Sorof(l8) has suggested that it is
the target protein of the aminoazo carcinogen in liver, and he
has emphasized
8999@%
bearing Hepatoma
1.025%
casem (4yZ
in Hepatomas
Hence,
this phenomenon
appears
to have wide occur
rence among hepatic tumors.
Careful consideration of the information at hand leads us to
conclude that lack of responsiveness of enzymes in tumors is
not the rule. Instead, certain enzymes in tumors can be over
responsive to metabolic modulations.
No doubt, both over
responsiveness and lack of responsiveness are manifestations
of
aberrations in control mechanisms in cancer. Any elucidation
of these aberrations at the molecular level must take into
account the fact that the 2 kinds of responsiveness
a neoplastic cell.
do exist in
ACKNOWLEDGMENTS
We thank Mrs. Shirley P. Johnson and Mr. Ronald C. Ling for their
technical assistance in assaying the activity of the enzymes and Mrs. C.
M. Jackson and Mrs. J. W. Lewis for their technical assistance in trans
planting the many hepatoma lines used in these experiments. We are
also grateful
to Dr. Clement
A. Stone,
Merck Institute
for Therapeutic
Research, for a gift of actinomycin D.
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CANCER RESEARCH VOL. 31
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Responsiveness of Two Urea Cycle Enzymes in Morris
Hepatomas to Metabolic Modulations
Chung Wu, Jere M. Bauer and Harold P. Morris
Cancer Res 1971;31:12-18.
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