Download Synthesis of a-Fetoprotein by Membrane-bound

[CANCER RESEARCH 34, 1813—1815,August 1974]
Synthesis of a-Fetoprotein by Membrane-bound Polysomes of Rat
Ascites Hepatoma Cells1
Koichi Kanai,2 Yasuo
Endo, Toshitsugu
Oda, and Naomi
Tanaka
The First Department of Medicine, Faculty of Medicine, University of Tokyo, Hongo 7-3-I, Bunkyo-ku, Tokyo, Japan
by hemolysis. Less than 5% of the cells were stainable by
trypan blue after hemolysis, and the tumor cells comprised
Free and membrane-bound polysomes prepared from rat about 98% of the total cell population.
Chemicals. Sodium deoxycholate and Freund's adjuvant
ascites hepatoma cells (AH66) incorporated leucine-14C
into a-fetoprotein in vitro. The amount of radioactivity were purchased from Difco Laboratories, Inc., Detroit,
incorporated into antiserum-precipitable a-fetoprotein frac Mich.; ATP, GTP, phosphoenolpyruvate, and PK were
tion by membrane-bound polysomes was 20 to 90 times from Boehringer Mannheim, Inc., Mannheim, Germany;
and EDTA and leucine-14C (240 mCi/mmole, uniformly
higher than that by free polysomes, indicating that a-feto
protein is mainly synthesized on membrane-bound poly labeled) were the products of Daiichi Pure Chemicals Co.,
Tokyo, Japan. The 2,5-bis[2-(5-/er/-butylbenzoxazolyl)]somes in rat ascites hepatoma cells.
thiopene was obtained from Packard Instrument Co.,
Downers Grove, 111.
INTRODUCTION
Antisera. Antiserum against rat AFP was prepared by
immunizing rabbits with purified rat AFP (a gift of Dr.
Since Abelev et al. (2) reported its appearance in sera of
of Hokkaido University) emulsified in complete
hepatoma-bearing mice, detection of AFP3 has become an Watabe
Freund's adjuvant. Four injections were made at 2-week
important aid to the diagnosis of primary liver cancer (1,3, intervals. The total amount of rat AFP used for immuniza
5, 19). Although there are several reports concerning the tion was 0.5 mg. Rabbits were bled 10 days after the last
production of AFP in hepatoma cells (2, 6, 8, 13, 15, 20), immunization.
little is known about the precise mode of AFP synthesis, its
Anti-rat AFP serum contained no detectable antibodies
regulation mechanism, or the biological function of AFP.
to adult rat serum by double diffusion in agar and gave a
In mammalian cells there are 2 types of polysomes, free single precipitation line against fetal rat serum and amand membrane-bound. Although the functional differences nionic fluid, as well as purified rat AFP. Immunoelectrobetween these 2 polysomes are not yet completely eluci phoresis compared with the precipitation in an adjacent
dated, increasing evidence suggests that free polysomes trough produced by an antiserum against rat serum revealed
mainly synthesize proteins that are utilized within the cells;
the antiserum reacted with a protein that had the
membrane-bound polysomes, on the other hand, synthesize that
mobility of an a-globulin.
proteins for export (7, 9, 12, 14, 16, 17, 18, 21).
Antisera against rat serum was prepared by the same
In the present study, AFP-synthesizing ability of free and
method,
normal rat serum as an antigen. Normal
membrane-bound polysomes of rat ascites hepatoma AH66 rabbit IgGwith
was prepared by salt fractionation and DEAEwas investigated in an attempt to determine the intracellular cellulose column chromatography (11). Goat antiserum
site of AFP synthesis and to obtain some information as to against rabbit IgG was purchased from Dainabot Laborato
its biological function.
ries, Inc., Tokyo, Japan.
To determine the maximum precipitation between rat
AFP and its antiserum, 10 to 200 /ig of rat AFP were mixed
MATERIALS AND METHODS
with increasing amount of antiserum. After the mixture was
Animals and Hepatoma Cells. AH-66 rat ascites hepatoma left overnight at 4°,the amount of precipitate was mea
cells that produce AFP were transplanted i.p. to male sured. The maximum precipitation was obtained when 0.4
Donryu rats weighing 100 to 150 g. Ascites cells were ml of antiserum was added to 50 /ig of rat AFP.
Preparation of Polysomes. Free and membrane-bound
collected by peritoneal puncture 7 days after inoculation of
polysomes
were prepared from the tumor cells as follows.
the tumor cells. Contaminating erythrocytes were removed
Ten to 15 g of packed cells obtained from 20 tumor-bearing
1This work was partly supported by a Grant-in-Aid from the Ministry rats were homogenized with 3 volumes of Medium A
containing 50 mM Tris-HCl (pH 7.6), 50 mM KC1, 5 mM
of Education, Japan.
2To whom requests for reprints and communications should be
MgCl2, and 5 mM mercaptoethanol in 0.25 Msucrose. After
addressed.
centrifugation
at 10,000 x g for 15 min, 2 ml of the
3The abbreviations used are: AFP, a-fetoprotein; PK, pyruvate kinase;
postmitochondrial supernatant were layered on a top of
TCA, trichloroacetic acid.
Received October 2, 1973; accepted March 6, 1974.
discontinuous sucrose gradient consisting of 1.5 ml of 2 M
SUMMARY
AUGUST
1974
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1813
K. Kanai el al.
sucrose in Medium A and 1.5 ml of 0.5 M sucrose in
Medium A in a 6-ml centrifuge tube. Centrifugation was
performed in a Hitachi Model 55 centrifuge at 160,000 x g
for 3.5 hr at 4°.Free polysomes were precipitated as clear
pellets at the bottom of the tubes, and membrane-bound
polysomes were sedimented as a turbid zone between 2 and
0.5 M sucrose at the end of centrifugation. The fraction
containing membrane-bound polysomes was collected and
5% sodium deoxycholate was added to the fraction to a final
concentration of 1.3% to remove membrane fraction from
the polysomes. Four ml of the fraction treated with sodium
deoxycholate were placed on 1 ml of l Msucrose in Medium
A, and the 2nd centrifugation at 160,000 x g for 60 min was
carried out to precipitate bound polysomes.
Free polysomes were used without further purification.
The A260/A280 ratio of these 2 polysome preparations
ranged between 1.75 and 1.82. The supernatant fraction was
prepared from normal rat liver according to the method of
Kashiwagi et al. (IO).
Cell-free Protein-synthesizing System. Free and mem
brane-bound polysomes were tested for their protein-synthe
sizing ability in a cell-free system containing 1 mM ATP,
0.25 mM GTP, 10 mM phosphoenolpyruvate, 100 ¡igof PK,
6 mg of supernatant proteins, 2.5 ¿¿Ci
of leucine-14C, and 1
to 2 mg of polysomes in Medium A. The total volume of the
reaction mixture was 2 ml. Incubation was carried out at
37°for the period as indicated. In some experiments,
EDTA was added to the reaction mixture after incubation,
and the incubation was continued for an additional 10 min.
The reaction mixture was then centrifuged at 160,000 x g
for 60 min and the supernatant was analyzed for radio
activity.
Measurement of Radioactivity. Nonspecific precipitates
were first removed from the supernatant 3 times with rabbit
IgG and its antiserum. The final supernatant was divided
into 2 equal parts to which were added, to Part A, 50 ßg
of
rat AFP and 0.4 ml of its antiserum and, to Part B, 50 jig of
rabbit IgG and 0.4 ml of its antiserum. They were left
overnight at 4°after 30 min of incubation at 37°.The
amount of precipitates generated in these 2 parts was about
the same. The precipitated proteins were washed 5 times
with cold 0.9% NaCl solution and dissolved in 0.3 ml of 88%
formic acid. After adding 10 ml of scintillation fluid
containing 0.5% 2,5-bis[2-(5-fm-butylbenzoxazolyl)]thiopene in 50% toluene-50% methylcellusolve, radioactivity
was measured or? a Packard Tri-Carb Model 3320 liquid
scintillation counter. To calculate the radioactivity of AFP,
the count, Part A, was subtracted from the count, Part B, in
order to exclude the possible contamination by nonspecific
coprecipitation, expressed as the radioactivity of AFP.
For the determination of total-protein radioactivity,
proteins were precipitated from the supernatant by 10%
TCA, heated at 95°for 20 min, then washed 5 times with
5% TCA. The precipitates were dissolved in 0.3 ml of formic
acid, and radioactivity was counted as described above.
RESULTS
AND DISCUSSION
The incorporation time of leucine-14C into total proteins
of the tumor cells is presented in Chart 1. Radioactivity
1814
increased rapidly for the 1st 20 min and reached a plateau at
about 40 min in both free and membrane-bound polysomes.
It was also observed that the activity of free polysomes
which were prepared without sodium deoxycholate treat
ment was apparently higher than that of membrane-bound
polysomes treated with sodium deoxycholate in agreement
with the results of other investigators (4, 6). When EDTA
was added, the radioactivity of the supernatant increased by
50% in both free and bound polysomes. The relative amount
of AFP released from the polysomes was not altered by
EDTA.
It was also confirmed, in our preliminary experiment of a
similar line, that membrane-bound polysomes prepared
from rat liver incorporated leucine-14C into serum albumin,
whereas free polysomes did not (unpublished observation).
Synthesis of AFP by hepatoma cells has been reported (1,
15, 20), while the presence of AFP in hepatoma cells has
also been confirmed morphologically (6, 8, 13). According
to Nishioka et al. (13), AFP was most commonly found in
the cytoplasm of tumor cells. However, the intracellular site
r
E
lulu'
20
40
60 mm
Chart 1. Incorporation of leucine-"C into proteins by free and mem
brane-bound polysomes of rat ascites hepatoma cells. Two mg of free (O)
or bound (•)polysomes were incubated in a cell-free system containing 1
mMATP, 0.25 m.MGTP, 10 mMphosphoenolpyruvate, 100jig of PK, 6 mg
of supernatant proteins, and 2.5 /iCi of leucine-"C at 37°.Reaction was
stopped by 5% TCA, and the radioactivity of the precipitated proteins was
counted as described in "Materials and Methods."
Table 1
Incorporation ofleucine-"C into AFP by free and bound polysomes of rat
ascites hepatoma cells
One to 2 mg of free or bound polysomes were incubated at 37°for 60
min in complete reaction mixture with 2.5 nCt of leucine-"C. After
incubation, EDTA was added to a final concentration of 15 mM. The
reaction mixture was centrifuged at 160,000 x g for 60 min, and the
supernatant was analyzed for radioactivity of total proteins and AFP as
described in "Materials and Methods."
Radioactivity (cpm/mg rRNA)
Experiment123PolysomesFree
protein
(%)0.032.770.08
proteins11,452
4,38412,571 12210
BoundFreeBoundFreeBoundTotal
9,82515,898
7,813AFP3
1527
1.540.04
101AFP/total 1.29
CANCER RESEARCH
VOL. 34
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AFP Synthesis by Bound Poly somes of Rat Hepatoma
of AFP synthesis has not yet been elucidated.
As shown in Table 1, incorporation of leucine-14C into the
AFP fraction by membrane-bound polysomes was about 20
to 90 times higher than that by free polysomes. These results
indicate clearly that AFP is synthesized mainly on mem
brane-bound polysomes in rat ascites hepatoma cells,
suggesting that AFP in hepatomas belongs to "excretory
proteins" such as serum albumin.
We do not know, however, how the synthesis of AFP is
regulated in hepatoma cells and this, as well as its biological
function, should be the focus of further studies.
ACKNOWLEDGMENTS
The authors are grateful to Professor T. Higashi, Department of
Biochemistry, Showa University, for helpful discussions during the course
of the study; to Dr. H. Watabe, Department of Biochemistry, Hokkaido
University, for supplying us with purified rat AFP, as well as AH-66
hepatoma cells; and to M. Tamoto for excellent technical assistance.
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AUGUST
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1974
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1815
Synthesis of α-Fetoprotein by Membrane-bound Polysomes of
Rat Ascites Hepatoma Cells
Koichi Kanai, Yasuo Endo, Toshitsugu Oda, et al.
Cancer Res 1974;34:1813-1815.
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