BIOCHEMICAL
Vol. 168, No. 2, 1990 April 30, 1990
Primate's p53 inhibits Norimasa Miyamoto 192,
'Tsukuba
Life
Eiji
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 604-608
SV40 DNAreplication
Kiharalv2, Tetsuo Inada', Yasufumi Murakamil
Sci. Center, The Inst. of Koyadai, Tsulcuba, Ibaraki
2Lkpt. of Nadiat. Biol., Univ. of Tsukuba, l-l-l
zk tihz~ Shinji
Katsuralv3
Phys. and Chem. Res., 305, JAPAN
and
3-l-l
Inst. of Basic Med. Sci., Tennodai, Tsukuba, JApAN
3Dept. of Chem.Eng., Fat. of Eng., Univ. of Tokyo, 7-3-l Hongo, Tokyo 113, JAPAN Received
March
7, 1990
Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication 12 vj&oas well as k vYvowhile that from primate cells does not (l-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication ln v~z?zz p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication lj7 vitro. These results suggest that inhibition of SV40 DNA replication by ~53 should be regarded as general property of the protein and does not determine the mode of 1990Academic Press,Inc. species specific replication of SV40 DNA. CJ
Recent studies transformation It
demonstrated that
in assays carried
has also been shown that
and loss of the original
&
viw
demonstrated that
replication
L7 vl'tro.
DNA replication
of p53 is possibly playing (7).
p53 protein
(1, 2) as well
cells
as ok vitro
(3, 4).
of mouse p53 (3) as well with
baculo viral
From these results,
by p53 is
vector
line
(5, 6).
of human tumors an important role
species specific
hand, it
alpha-primase complex is required
that
604
v3~fY-oanalyses
as complete mouse p53 (4) inhibited
replication
a species specific
in the SV40 DNA replication
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
SV40 DNA
SV40 DNA
inhibition
of
and such a species specific
the other
was reported
In
they concluded that
determines the mode of species specific
Copyright All rights
suppress
forms a strong complex with
inhibition
0006-291X/90
to
Recent reports have shown that p53 inhibits
a part
produced in insect
embryonic cell
p53 is mutated in a wide variety
in a malignant transformation
replication
out with a rat
activity
SV40 T antigen (S-10).
p53 has an activity
of SV40.
Dn
DNA polymerase 1j7 tit.0
and
Vol.
BIOCHEMICAL
168, No. 2, 1990
such a species specific specificity
AND BIOPHYSICAL
requirement of the complex is a determinant of species
of SV40 and polyoma virus
DNA replication
reexamine the determinant of the species specific purified
p53-T antigen complex from cells
and characterized assayed & vi&z purified
RESEARCH COMMUNICATIONS
the
comlplex
with
li7 vi&-o (11,12).
DNA replication
in which SV40 replicates respect
to
the
from natural
such an inhibitory
host inhibited activity
SV40 DNA replication
is not a major determinant
of SV40, we efficiently
replication
Our results described below demonstrate
To
activity
that p53-T antigen oh vz'tro suggesting of species specific
replication
MATERIALSANDMETHODS Materials. CDS-1 cell and PAb419 cells were maintained under the conditions described previously (11, 13). Viral stock of SV40 ~~-1085 strain was prepared under the conditions previously described (11, 13). Purification of SV40 T antigen and T antigen-p53 complex. SV40 infected CDS-1 cell (in 20 roller bottles that has 85Ocm* of culture surface) extract were prepared by the procedure previously reported (11, 13). Cell extracts were applied onto PAb419 monoclonal antibody column (lml) (11, 13). The column was washed with a loading buffer (2OmMTris-HCl pH 8.0, 1mMdithiothreitol @'IT), 1mM EDTA, 1oOmM NaCl, 10% glycerol, 1% Nonidet P-40, 2OOug/ml phenylmethanesulfonyl fluoride) and then with 0.5M NaCl in the buffer A (2OmM Tris-HCl pH 8.5, 1mMDI'T, 1mMEDTA, 10% glycerol). Proteins were then eluted with 55% ethylene glycol in the buffer A. Fractions were analyzed by SDS-polyacrylamide gel and were dialyzed against T antigen dialysis buffer (20mMHepes-NaOHpH 7.5, 1mMDTT, 1mMEDTA, 1OOmM NaCl, 50% glycerol) and were strored at -2Op. All purification procedures were carried out at 4~. Assays. Protein concentrations were determined by the Bradford assay with a bovine serum albumin as a standard. Replication assays were carried out under the conditions described previously (11-13).
RESULTSANDDISCUSSION p53 purified
and analyzed previously
was only that
expressed in the insect
been purified
directly
from cell
of SV40 genome. We directly green monkey COS-1 cells.
buffer,
To purify
complex and free
differentially.
p53-T antigen
line
affinity-purified
Figure 1
shows an elution
from immunoaffinity
After
free
activity
to
profile
of
column with
from p53 were eluted
and subsequent western blot 605
african
washing the column with high
complex and T antigen
SDS-PAGEanalysis
replication
p53 from SV40 infected
T antigen
system
and primates p53 has never
~53, we took advantage of its
anti-SV40 T antigen monoclonal antibody. salt
cell
lines which permit an efficient
form a strong complex with T antigen. p53-T antigen
in the SV40 DNA replication
analysis
of
Vol.
BIOCHEMICAL
168, No. 2, 1990
JO." 0.15
-
0.05
-
0'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~55%
NaCl WASE-
E.G.
A nn
5 FRACTION
10
15 NUMBER
ELtiTION+
B
20
25
Figure 1. Purification of p53-T antigen complex from GOSS1 cells. of p53-T antigen complex and T antigen. (A) Immunoaffinity purification Elution profile of p53-T antigen complex and T antigen is shownin the figure. Closedcircles (0) indicate optical density at 280nmof the fraction. immunoaffinity
purified
shown in Figure contained
p53-T antigen
2A and Figure
p53 and T antigen
Replication
2B.
while
and T antigen
free
As shown in
the
fraction
assays with these fractions
figure,
B contained
only
were shown in Figure 3. aTAg
MAB
from p53 has been fraction T antigen. Plasmid DNA
ffp53
nn MABAB
31Figure Z.(A) SDS-PAGE analysis of purified fractions. Fractions obtained in the experiment shown in the figure 1 were analyzed bv sodium dodecvl sulphate-polyacrylamide gel electrophoresis (10% polyacryiamide). spb0is are; M: molecular weight marker, molecular massesare shownin the left side. A: 45Ongproteins of fraction A, B: 3OOngproteins of fraction B. Left panel showsprotein bandsvisualized by silver staining. (B) Western blot analysis of the purified fractions. On top of the right panel, antibodies used in the blot were indicated. Culture supernatant of PAb419was used as anti-SV40 T antigen antibody and purified antibody from PAbl801 hybridoma (16) culture supernatant was used as an anti-p53 antibody (purchasedfrom DncogeneScience Inc. USA). Symbolsare identical with (A).
606
A
Vol.
168,
which
No.
contained
antigen
free
contained mixing
replication from
characteristics (4).
respect
purified
known
lacked
(Figure
p53
HeLa cell
protein
extracts
0.25 SV40
assay
Various
vifro
was
same level
efficiently
for with 15)
specific
the virus. extracts and
these
We also
carried
extracts.
0
1.00
4
(pg)
~53.
these
prepared
from
extracts out
activity
an activity
If
experiments,
SV40
line
inactive
mixing
Mouse cell
we
cell
were
0.25
such au of SV40,
Friend
an
to
It has been
replication
Besides
assayed
as mouse p53
in COS-1 cells.
to species
in
experiment
extracts
0.50
0.75
TAg-p53
ADDED
had
1.00 (w)
Figure 3. Comparison of replication activity of purified proteins. Indicated amounts of fraction A and fraction B were assaved bv SV40 DNA reolication Closed circles (0) indicate replication achvity of system 12 ti&o (11-13). Incorporation fraction B and open squares ( 0 ) indicate that of fraction A. of dRlp into acid insoluble materials were determined as previously described (11-13). Figure 4. p53-T antigen complex inhibited SV40 DNA replication in tiz?o. Indicated amounts of fraction A were mixed with fraction B (0.4~ of protein per an assay). Incorporation of dTMP into acid insoluble materials were determined as previously described (11-13). 607
of
These
specific
cells'
a
amount
complex
of
indicate to rodent
amounts
inhibited.
that
results
out
With increasing
B.
T
A, which
carried
These
and mouse FM3A cell
TAg ADDED
4).
with
fraction
lj7 tifro
(14,
0.75
hand,
of SV40 DNA replication
not shown).
0.50
efficiently
of mouse p53-T antigen
had almost
of p53 contributed
(data
lh
COMMUNICATIONS
We then
amount of fraction
is not limited
not be a host
other
fractions
SV40 DNA replicates
assay
0
cells.
replication
On the
activity.
to that
replication
would
out
replication with
DNA
that
COS-1 cells
which
insect
effect
carried
similar
RESEARCH
SV40 replicated
replication
to the inhibition
SV40
inhibitory
DNA
BIOPHYSICAL
had little
Cur preparation
from
inhibit
SV40 were
previously
well
these
A were added to a fixed A,
of
B).
and ~53, with
AND
origin
p53 (fraction
experiment
fraction
with
BIOCHEMICAL
T antigen
fraction of
2, 1990
Vol.
168, No. 2, 1990
no inhibitory
activity
BIOCHEMICAL
in terms of replication
p53 were a major determinant
species specific should contain
mouse cell
extracts
would exhibit
an inhibitory
activity
conclude that p53 does not contribute
replication
of SV40 DNA,
From these results,
inhibition
replication
species inhibits
of the protein
As SV40 DNA replication
chromosomal DNA replication approach may clarify
SV40 DNA replication
we
of SV40
of SV40 DNA replication and should not be system 12 vitro
good model system for the analysis of chromosomal DNA replication, from various
If
enough amount of p53 and
to species specific
on p53 suggests that
to p53 of some species.
(data not shown).
in these assays.
should be regarded as a general character limited
activity
for
such inactive
genome. Our finding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2% vitro,
should be examined carefully.
is a
and as p53
effect Hopefully,
of p53 on such an
the mechanismhow p53 acts as a tumor repressor.
ACKNOWLEDGMENI'S This work is supported in part by Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and by The Naito Foundation. We are indebted to Ms. Harumi Aida for excellent technical assistance. REFERENCES 1) Braithwaite, A. W., Sturzbecher, H. W., Addison, C., Palmer, C., Rudge, K. and Jenkins, J. R. (1987) Nature 329, 458-460. 2) Sturzbecher, H-W., Braithwaite, A. W., Addison, C., Palmer, C., Rudge, If., Lynge-Hansen, D. and Jenkins, J. R. (1988) In &@r cells 6: Eukaryotic DNA replication. Cold Spring Harbor, NewYork. 159-163 . 3) Sturzbecher, H-W., Brain, R., Maimets, T., Addison, C., Rudge, K. and Jenkins, J. R. (1988) Dncogene 3, 405-413. 4) Wang, E. H.. Friedman, P. N. and Prives, C. (1989) Cell 57, 379-392. 5) Finlay, C. A., Hinds, P. W. and Levine, A. J. (1989) Cell 57, 1083-1093. 6) Eliyahu, D., Michalovitz, D., Eliyahu, S., Pinhasi-Kimhi, 0. and Oren, M. (1989) Proc. Natl. Acad, Sci.(USA) 86, 8763-8767. 7) Nigro, J. C., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R Cleary. K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., Glover, T 1: Collins, F. S., Weston, A., Modali, R., Harris, C. C. and Vogelstein, B. (1989) Nature 342, 705-708 8) Lane, D. P. and Crawford, L. V. (1979) Nature 278, 261-263. 9) McCormick, F., Clark, R., Harlow, E. and Tjan, R. (1981) Nature 292, 63-65.10) Reich, N. C. and Levine, A. J. (1982) Virology 117, 286-290. 11) Murakami, Y. Wobbe, C. R., Weissbach L., Dean, F. B. and Hurwitz, J. (1986) Proc. Natl. Acad. Sci. (USA) 83, 2869-2873. 12) Murakami. Y., Eki, T., Yamada,M., Prives, C. and Hurwitz, J. (1986) Proc. Natl. Acad. Sci. (USA) 83, 6347-6351. 13) Wobbe, C. R., Dean, F., Weissbach, L. and Hurwitz, J. (1985) Proc. Natl. Acad. Sci. (USA) 82, 5710-5714. 14) Mowat. M.. Cheng, A., Kimura, N., Bernstein, A. and Benchimol. S. (1985) Nature 314. 633-636. 15) Robinski, B., Munroe, D., Peacock, J, Mowat, M., Bernstein, A. and Benchimol S. (1987) Mol. Cell Biol. 7. 847-853. 16) Banks, L:, Matlashewski, G and Crawford, L. (1986) Eur. J. Biochem. 159, 529-534.
Vol. 168, No. 2, 1990 April 30, 1990
Primate's p53 inhibits Norimasa Miyamoto 192,
'Tsukuba
Life
Eiji
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 604-608
SV40 DNAreplication
Kiharalv2, Tetsuo Inada', Yasufumi Murakamil
Sci. Center, The Inst. of Koyadai, Tsulcuba, Ibaraki
2Lkpt. of Nadiat. Biol., Univ. of Tsukuba, l-l-l
zk tihz~ Shinji
Katsuralv3
Phys. and Chem. Res., 305, JAPAN
and
3-l-l
Inst. of Basic Med. Sci., Tennodai, Tsukuba, JApAN
3Dept. of Chem.Eng., Fat. of Eng., Univ. of Tokyo, 7-3-l Hongo, Tokyo 113, JAPAN Received
March
7, 1990
Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication 12 vj&oas well as k vYvowhile that from primate cells does not (l-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication ln v~z?zz p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication lj7 vitro. These results suggest that inhibition of SV40 DNA replication by ~53 should be regarded as general property of the protein and does not determine the mode of 1990Academic Press,Inc. species specific replication of SV40 DNA. CJ
Recent studies transformation It
demonstrated that
in assays carried
has also been shown that
and loss of the original
&
viw
demonstrated that
replication
L7 vl'tro.
DNA replication
of p53 is possibly playing (7).
p53 protein
(1, 2) as well
cells
as ok vitro
(3, 4).
of mouse p53 (3) as well with
baculo viral
From these results,
by p53 is
vector
line
(5, 6).
of human tumors an important role
species specific
hand, it
alpha-primase complex is required
that
604
v3~fY-oanalyses
as complete mouse p53 (4) inhibited
replication
a species specific
in the SV40 DNA replication
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
SV40 DNA
SV40 DNA
inhibition
of
and such a species specific
the other
was reported
In
they concluded that
determines the mode of species specific
Copyright All rights
suppress
forms a strong complex with
inhibition
0006-291X/90
to
Recent reports have shown that p53 inhibits
a part
produced in insect
embryonic cell
p53 is mutated in a wide variety
in a malignant transformation
replication
out with a rat
activity
SV40 T antigen (S-10).
p53 has an activity
of SV40.
Dn
DNA polymerase 1j7 tit.0
and
Vol.
BIOCHEMICAL
168, No. 2, 1990
such a species specific specificity
AND BIOPHYSICAL
requirement of the complex is a determinant of species
of SV40 and polyoma virus
DNA replication
reexamine the determinant of the species specific purified
p53-T antigen complex from cells
and characterized assayed & vi&z purified
RESEARCH COMMUNICATIONS
the
comlplex
with
li7 vi&-o (11,12).
DNA replication
in which SV40 replicates respect
to
the
from natural
such an inhibitory
host inhibited activity
SV40 DNA replication
is not a major determinant
of SV40, we efficiently
replication
Our results described below demonstrate
To
activity
that p53-T antigen oh vz'tro suggesting of species specific
replication
MATERIALSANDMETHODS Materials. CDS-1 cell and PAb419 cells were maintained under the conditions described previously (11, 13). Viral stock of SV40 ~~-1085 strain was prepared under the conditions previously described (11, 13). Purification of SV40 T antigen and T antigen-p53 complex. SV40 infected CDS-1 cell (in 20 roller bottles that has 85Ocm* of culture surface) extract were prepared by the procedure previously reported (11, 13). Cell extracts were applied onto PAb419 monoclonal antibody column (lml) (11, 13). The column was washed with a loading buffer (2OmMTris-HCl pH 8.0, 1mMdithiothreitol @'IT), 1mM EDTA, 1oOmM NaCl, 10% glycerol, 1% Nonidet P-40, 2OOug/ml phenylmethanesulfonyl fluoride) and then with 0.5M NaCl in the buffer A (2OmM Tris-HCl pH 8.5, 1mMDI'T, 1mMEDTA, 10% glycerol). Proteins were then eluted with 55% ethylene glycol in the buffer A. Fractions were analyzed by SDS-polyacrylamide gel and were dialyzed against T antigen dialysis buffer (20mMHepes-NaOHpH 7.5, 1mMDTT, 1mMEDTA, 1OOmM NaCl, 50% glycerol) and were strored at -2Op. All purification procedures were carried out at 4~. Assays. Protein concentrations were determined by the Bradford assay with a bovine serum albumin as a standard. Replication assays were carried out under the conditions described previously (11-13).
RESULTSANDDISCUSSION p53 purified
and analyzed previously
was only that
expressed in the insect
been purified
directly
from cell
of SV40 genome. We directly green monkey COS-1 cells.
buffer,
To purify
complex and free
differentially.
p53-T antigen
line
affinity-purified
Figure 1
shows an elution
from immunoaffinity
After
free
activity
to
profile
of
column with
from p53 were eluted
and subsequent western blot 605
african
washing the column with high
complex and T antigen
SDS-PAGEanalysis
replication
p53 from SV40 infected
T antigen
system
and primates p53 has never
~53, we took advantage of its
anti-SV40 T antigen monoclonal antibody. salt
cell
lines which permit an efficient
form a strong complex with T antigen. p53-T antigen
in the SV40 DNA replication
analysis
of
Vol.
BIOCHEMICAL
168, No. 2, 1990
JO." 0.15
-
0.05
-
0'
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~55%
NaCl WASE-
E.G.
A nn
5 FRACTION
10
15 NUMBER
ELtiTION+
B
20
25
Figure 1. Purification of p53-T antigen complex from GOSS1 cells. of p53-T antigen complex and T antigen. (A) Immunoaffinity purification Elution profile of p53-T antigen complex and T antigen is shownin the figure. Closedcircles (0) indicate optical density at 280nmof the fraction. immunoaffinity
purified
shown in Figure contained
p53-T antigen
2A and Figure
p53 and T antigen
Replication
2B.
while
and T antigen
free
As shown in
the
fraction
assays with these fractions
figure,
B contained
only
were shown in Figure 3. aTAg
MAB
from p53 has been fraction T antigen. Plasmid DNA
ffp53
nn MABAB
31Figure Z.(A) SDS-PAGE analysis of purified fractions. Fractions obtained in the experiment shown in the figure 1 were analyzed bv sodium dodecvl sulphate-polyacrylamide gel electrophoresis (10% polyacryiamide). spb0is are; M: molecular weight marker, molecular massesare shownin the left side. A: 45Ongproteins of fraction A, B: 3OOngproteins of fraction B. Left panel showsprotein bandsvisualized by silver staining. (B) Western blot analysis of the purified fractions. On top of the right panel, antibodies used in the blot were indicated. Culture supernatant of PAb419was used as anti-SV40 T antigen antibody and purified antibody from PAbl801 hybridoma (16) culture supernatant was used as an anti-p53 antibody (purchasedfrom DncogeneScience Inc. USA). Symbolsare identical with (A).
606
A
Vol.
168,
which
No.
contained
antigen
free
contained mixing
replication from
characteristics (4).
respect
purified
known
lacked
(Figure
p53
HeLa cell
protein
extracts
0.25 SV40
assay
Various
vifro
was
same level
efficiently
for with 15)
specific
the virus. extracts and
these
We also
carried
extracts.
0
1.00
4
(pg)
~53.
these
prepared
from
extracts out
activity
an activity
If
experiments,
SV40
line
inactive
mixing
Mouse cell
we
cell
were
0.25
such au of SV40,
Friend
an
to
It has been
replication
Besides
assayed
as mouse p53
in COS-1 cells.
to species
in
experiment
extracts
0.50
0.75
TAg-p53
ADDED
had
1.00 (w)
Figure 3. Comparison of replication activity of purified proteins. Indicated amounts of fraction A and fraction B were assaved bv SV40 DNA reolication Closed circles (0) indicate replication achvity of system 12 ti&o (11-13). Incorporation fraction B and open squares ( 0 ) indicate that of fraction A. of dRlp into acid insoluble materials were determined as previously described (11-13). Figure 4. p53-T antigen complex inhibited SV40 DNA replication in tiz?o. Indicated amounts of fraction A were mixed with fraction B (0.4~ of protein per an assay). Incorporation of dTMP into acid insoluble materials were determined as previously described (11-13). 607
of
These
specific
cells'
a
amount
complex
of
indicate to rodent
amounts
inhibited.
that
results
out
With increasing
B.
T
A, which
carried
These
and mouse FM3A cell
TAg ADDED
4).
with
fraction
lj7 tifro
(14,
0.75
hand,
of SV40 DNA replication
not shown).
0.50
efficiently
of mouse p53-T antigen
had almost
of p53 contributed
(data
lh
COMMUNICATIONS
We then
amount of fraction
is not limited
not be a host
other
fractions
SV40 DNA replicates
assay
0
cells.
replication
On the
activity.
to that
replication
would
out
replication with
DNA
that
COS-1 cells
which
insect
effect
carried
similar
RESEARCH
SV40 replicated
replication
to the inhibition
SV40
inhibitory
DNA
BIOPHYSICAL
had little
Cur preparation
from
inhibit
SV40 were
previously
well
these
A were added to a fixed A,
of
B).
and ~53, with
AND
origin
p53 (fraction
experiment
fraction
with
BIOCHEMICAL
T antigen
fraction of
2, 1990
Vol.
168, No. 2, 1990
no inhibitory
activity
BIOCHEMICAL
in terms of replication
p53 were a major determinant
species specific should contain
mouse cell
extracts
would exhibit
an inhibitory
activity
conclude that p53 does not contribute
replication
of SV40 DNA,
From these results,
inhibition
replication
species inhibits
of the protein
As SV40 DNA replication
chromosomal DNA replication approach may clarify
SV40 DNA replication
we
of SV40
of SV40 DNA replication and should not be system 12 vitro
good model system for the analysis of chromosomal DNA replication, from various
If
enough amount of p53 and
to species specific
on p53 suggests that
to p53 of some species.
(data not shown).
in these assays.
should be regarded as a general character limited
activity
for
such inactive
genome. Our finding
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2% vitro,
should be examined carefully.
is a
and as p53
effect Hopefully,
of p53 on such an
the mechanismhow p53 acts as a tumor repressor.
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