[CANCER RESEARCH 30, 123-126,
January 1970]
Adenyl Cyclase Activity in Morris Hepatomas 7777, 7794A, and
9618A1
Harry Darrow Brown, Swaraj K. Chattopadhyay,
Harold P. Morris,2 and Sam N. Pennington
Biochemistry Section, Cancer Research Center, Columbia, Missouri 65201
SUMMARY
Adenyl cyclase activity has been measured in Morris
hepatomas, moderately fast-growing 7777 (average genera
tion, 2 months) and slower-growing tumors 7794A (average
generation, 4 months) and 96ISA (average generation, 5
months). The activity levels of the tumors varied as a
function of their growth rate. Slower-growing tumors ex
hibited adenyl cyclase activity which was higher than the
normal level. The fastest-growing tumor (7777) had a much
higher level of adenyl cyclase activity than did normal liver.
All of the hepatomas had cyclase activity which was
irregular in response to epinephrine facilitation of cyclization, thought to be a primary element of organismal control
of cellular activity.
Adenyl cyclase activity of the liver of hepatomatous
animals differed from liver of healthy animals. The relation
ships were essentially the same as those of the several
hepatomas themselves to the normal liver. The fastestgrowing tumor had a higher adenyl cyclase activity; the
slower-growing tumor approached that of the normal. Like
the hepatomas themselves, the liver adenyl cyclase activity of
hepatomatous animals was affected less by epinephrine than
was normal liver.
INTRODUCTION
Epinephrine and other catecholamines play a role in the
control of a large number of metabolic reactions. It is
probable that the interaction
of metabolism-controlling
hormones with a cellular receptor involves the enzymatic
synthesis of cyclic adenylic acid. Cellular processes to an
important extent are influenced by the nucleotide product
of the cyclase reaction. For tabulation of this literature see
the reviews of Robison et al. (8) and of Sutherland et al.
(10).
Glycolysis is known to be affected by the level of cyclic
AMP3 in the cell. One may thus hypothesize that the
'This investigation was supported by USPHS Research Grants
CA08023 and CA10729 (H. P. M.).
2Present address: Howard University, School of Medicine, Washing
ton, D. C.
3The abbreviations used are: cyclic AMP, cyclic adenylic acid;
3',5'-AMP, cyclic 3',5'-adenylic acid.
Received June 17, 1968; accepted May 21, 1969.
cyclizing reaction is in fact the pivot at which the body
exerts its control of major energetic pathways. Schematic
representation of the interaction of agents, primarily the
biogenic amines, which appear to affect organismal control
over the cyclyzing have been published by Sutherland et al.
(10). Their scheme indicates product (cyclic AMP) influence
over a large number of enzymatically catalyzed reactions.
Glycolytic abnormalities, frequently reported to be associated
with tumor development may, it is hypothesized, reflect
a relationship to the cellular level of cyclic nucleotide
and hence in turn to the cyclyzing reaction and its control
by catecholamines. The tumors selected for study represent
members of a series of chemically induced, transplanted rat
liver tumors developed in the laboratories of one of us [H.
P. M. (5)].
These hepatomas are malignant neoplasms. They metastasize and ultimately kill their host. The primary Hepatoma
7777 was originally described as a well-differentiated trabecular carcinoma (H. Pitot, personal communication), but after
the 30th transfer most of the hepatoma cells were poorly
differentiated (D. R. Meranze, personal communication) and
the growth rate was more rapid (5). This tumor no longer
meets the criteria for a "minimal deviation" hepatoma (7). It
is known to have a high rate of glycolysis and a low rate of
aerobic respiration. The other tumor lines are well differ
entiated and slower growing. These tumors have a lower rate
of glycolysis and moderate to high rates of aerobic respira
tion (3).
MATERIALS
AND METHODS
Buffalo rats, with bilateral tumors growing intramuscularly
in the hind legs, and nontumor-bearing control animals from
identical normal stock were used. The animals with palpable
tumors were shipped by air from the Melpar Hepatoma
Contract Laboratories, Falls Church, Va., as needed and
authorized by H. P. M. Upon arrival they were kept in separate
cages in an isolated, temperature-controlled room. Commercial
chow and water were supplied ad libitum. The first group of
animals was used 1 week after the date of arrival, and the
others were used in sequence over a 3-month period.
Hepatomas of the Morris 7777 rapid-growing line, 7794A
medium-slow-growing line, and 9618A slower-growing line
were examined. Animals bearing fast-growing tumors (7777)
were used 2 to 3 months after tumor inoculation, and the
animals bearing the slowest-growing tumors were sacrificed
8.5 to 9 months after tumor implantation. Normal animals
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123
Brown, Chattopadhyay,
Morris, and Pennington
and tumor-bearing animals were studied simultaneously when
possible.
Animals bearing Tumor Line 7777, at the time of sacrifice,
exhibited considerable difficulty in walking because the
tumors were very large, averaging 7 g, wet weight, when
removed from the animal. Grossly, there was little difference
between these tumors and the 7794A tumor (5 g, average
wet weight). The slowest-gròwing tumors, type 96 ISA (3 g,
average wet weight), were small, and animals bearing these
tumors were distinguished from healthy animals only by
palpation. Characteristics of these types have been published
by Morris (4, 5) and by Nowell et al. (6).
Animals were stunned and decapitated immediately. Liver
and tumors were removed within 2 to 3 min. Muscle tissues
were removed from around the encapsulated tumor which
was then transferred to a vessel containing cold 0.1 M
Tris-0.25 M sucrose buffer, pH 7.2. Within 5 to 10 min, liver
or tumor was homogenized in 20 volumes Tris-sucrose buffer
in a Waring Blendor. The slurry was further homogenized in
a glass tissue mill with a Teflon pestle. By the use of an ice
jacket, the temperature was maintained through all steps of
enzyme preparation at 2—6°.
The slurry removed from the tissue mill was centrifuged at
600 X g for 20 min in a refrigerated centrifuge. This pellet
was rejected and the supernatant was dialyzed for 10 hr
against Tris-sucrose buffer with 5 mM disodium EDTA and
Tris-saturated
IRC-50 ion-exchange resin. The retained
materials were centrifuged at 20,000 X g each for 30 min
and the pellets were saved. Pellets thus obtained were
resuspended in 8 ml of the Tris-sucrose buffer and were
taken as the nuclear fraction. The supernatant from the final
centrifugation was used in some experiments.
Adenyl cyclase activity was measured by 2 techniques. The
first procedure followed essentially the method of Suther
land et al. (9). The reaction mixture contained 0.5 ml
enzyme and 4 ml substrate (15.00 mg disodium adenosine
triphosphate with 3.54 mg MgSO4, 2.08 mg NaF, 6.50 mg
caffeine in 0.05 M Tris-HCl buffer, pH 7.2) for 15 min. The
enzyme reaction was stopped by placing the tube containing
the reaction mixture into boiling water for 3 min and then
into an ice bath for 10 min. Clear supernatant was assayed
for cyclic 3',5'-AMP by ultraviolet absorption after ionexchange chromatography.
Columns for chromatography
of the nucleotides were
packed with charged Dowex 1-X4 ion-exchange resin, 200 to
400 mesh, as the formate form. Column heights were 29 to
30 cm. An aliquot of the supernatant solution containing
cyclic 3',5'-AMP as a reaction product was poured on to the
column. The column was then eluted with the use of a
buffer reservoir into which 0.5 M formic acid was dripped to
develop a gradient of eluting fluid which slowly decreased in
pH. Eluate fractions of 3 to 4 ml were collected mechani
cally. The tube contents were then read in turn for
ultraviolet absorbance at 260 my. Where an absorbance at
260 mn was observed, further readings were made at 275,
280, and 290 m/n. Identification of the absorbing material as
cyclic AMP was based upon the following criteria: (a) the
theoretical ratio (ultraviolet A275/260 and A280/260) of the
absorbing material. A value indicating the presence of the
124
adenosine moiety was further confirmed by obtaining an
ultraviolet spectrum (Gary Model 15 recording spectrophotometer) of the "unknown sample" which was compared
with a spectrum of a standard reagent and with published
curves, (b) Mobility (Rp) as compared with that of a
standard reagent (Sigma Chemical Company, St. Louis, Mo.)
on a similar Dowex 1-X4 column run from the same buffer
manifold was also a criterion. Further confirmation of the
identification of a separated product as cyclic AMP was
obtained by using the material as a "substrate" for 5'-nucleotidase. 5'-Nucleotidase (Sigma; from venom) was used as a
check against possible contamination
of 3',5'-cyclic AMP
with adenosine 5'-monophosphate.
5'-Nucleotidase catalyzes
liberation of inorganic phosphate from adenosine 5'-monophosphate.
Inorganic phasphate
was measured
colorimetrically using the method of Fiske and SubbaRow (1).
The nucleotidase procedure is a safeguard but essentially
redundant
since the cyclic AMP and adenosine monophosphate are separated by 12 to 15 ml in the eluate of the
Dowex 1-X4 column.
The data obtained for the enzyme activity by this
technique were duplicated by the radioisotopic procedure of
Krishna et al. (2).
RESULTS
Grossly, there was little difference in the appearance of
fast-growing and medium-fast-growing
tumors. However,
slow-growing tumors (9618 A) were small and sometimes the
host was hardly distinguishable from a normal animal.
Table 1 presents data regarding liver adenyl cyclase activity.
Enzymes obtained from the liver of host rats show a gradual
increase in activity corresponding to the growth rate of the
tumor, i.e., enzyme obtained from the liver of normal
nontumor-bearing rats has an average activity of 3.6 units,
while livers from slow-growing tumor-bearing animals have
average activity of 4.9 units. The liver adenyl cyclase from
hosts bearing medium-fast- and fast-growing tumors has average
activities of 5.8 and 6.8 units, respectively.
Tumor adenyl cyclase preparations have activity higher
than those from normal liver as well as of the host liver.
Fast-growing tumor enzyme preparations have higher rates of
activity than those from medium- and slow-growing tumors
(Table 2). However, the slow-growing and medium-fastgrowing tumor lines used did not show any substantial
change in activities. When average normal liver adenyl cyclase
activity was compared to that of slow-growing tumor, an
increase of 58% was found in the tumor. Similarly, the
medium-fast-growing
tumor has a moderately (80%) in
creased activity, but a much greater (155%) increase in
activity was noticed in fast-growing tumors.
Kinetics of the adenyl cyclase substrate inter-relationship
of liver and of Hepatoma 7777 have been considered. The
Km value for the liver preparation is 1.4 X IO"3 M; the Km
for Hepatoma 7777 is 1.9 X IO"3 M.
DISCUSSION
It is known that epinephrine activates the activity level of
liver adenyl cyclase and thus favors the accumulation of
CANCER RESEARCH VOL. 30
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Adenyl Cyclase Activity
Table 1
Adenyl cyclase activity of normal Buffalo rat liver and of liver of
Morris hepatoma-bearing animals
Liver of rat
bearing:Hepatoma
20,000pelletWith
Xg
4.4 X IO"5
in Hepatomas
Table 2
Adenyl cyclase activity of Morris hepatomas
pelletLiver
epinephrine7.06.87.56.86.06.86.46.8
epinephrine7.76.89.07.07.
bearing:Fast-growing
of rat
Activity" of 20,000 X g
4.4 X 10s
epinephrine12.012.411.210.17.47.77.08.56.19.2
epinephrine11.010.07.86.58.0
7777MeanMedium-slow-growing
Hepatoma*
7777MeanHepatoma
0.34.74.43.83.14.08.08.69.75.
±
±0.75.05.26.75.44.88.89.76.46.6
6
±2.04.7
7794AMeanHepatoma
7794AMeanSlow-growing
Hepatoma*
±1.25.0
5.05.16.86.412.15.16.54.94.75.67.012.95.86.6
±2.26.56.33.23.53.83.57.05.34.94.9
8
±1.44.45.82.83.23.03.48.24.26.54.6
1.75.23.96.46.86.25.55.84.26.47.45.
±
±1.95.03.95.85.55.66.
18AMeanNormal(nontumor-bearing
96
Hepatoma*9618AMeanWithout
±1.23.23.33.03.54.23.23.93.23.63.34.43.05.23.73.73.63.6+0.4of
±1.54.34.73.25.04.63.75.44.25.03.66.64.75.75.24.54.34.7
7 ±0.9With
"Activity is X 10~3 umole cyclic AMP/mg protein/15 min.
rats)MeanActivity0Without
''Trie control for these data is liver from nontumor-bearing rats (data
given in Table 1). Compare means 3.6 (without epinephrine) and 4.7
(with epinephrine) with those given above.
±0.5
"Activity is X 10 3 ornólecyclic AMP/mg protein/15 min.
cyclic 3',5'-AMP.
The cyclic nucleotide
±0.7
in turn acts as a
physiologically significant intermediate which serves to cause
increased phosphorylase
activity, an important
energyyielding phenomenon.
Our observation indicates that normal liver adenyl cyclase
is activated by epinephrine but that tumor tissue adenyl
cyclase is not. In nuclear preparations from tumor tissue,
epinephrine has little effect upon this catalytic activity. The
question of the basis of this difference has not been
approached experimentally; however, in view of reports (A.
White, personal communication) that NaF stimulates adenyl
cyclase maximally in some systems, the possible effect of
NaF must not be overlooked. An alternative thesis might be
based upon a hypothesized structural abnormality of the
enzyme molecule which results in increased activity of the
enzyme and nonreceptivity to epinephrine. The possibility
also exists that the variation in response to epinephrine is
related to the state of disruption of the enzyme-membrane
complex.
The increased adenyl cyclase activity found in liver enzyme
preparations of tumor-bearing animals can be speculatively
explained in terms of a tumor-synthesized agent borne by a
circulatory system to the liver. The lower level of adenyl
cyclase activity in tumor-host liver compared to that of
tumor tissue may indicate a smaller quantity of such an
agent. It is also possible, although improbable, that neo-
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125
Brown, Chattopadhyay,
Morris, and Pennington
plastic tissue might not have been transported through the
lymphatics to the host liver at the time of inoculation and
that these cells had produced a scattered neoplastic con
dition.
The finding of abnormality of this hormonally influenced
metabolic control point allows us to make certain extension
of a generalization made by Weber et al. (11). They have
noted that in the glycolysis of neoplastic cells it is the
rate-limiting, energetically expensive steps which appear to
be altered. The highly energonic transformation of adenosine
triphosphate to cyclic AMP appears to be another example
of such an altered control point. This may have a special
significance however because of the uniqueness of its direct
hormonal control.
5.
6.
7.
8.
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CANCER RESEARCH VOL. 30
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Adenyl Cyclase Activity in Morris Hepatomas 7777, 7794A, and
9618A
Harry Darrow Brown, Swaraj K. Chattopadhyay, Harold P. Morris, et al.
Cancer Res 1970;30:123-126.
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