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Oncogene (2002) 21, 176 ± 189
2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00
www.nature.com/onc
Hetero-oligomerization does not compromise `gain of function' of
tumor-derived p53 mutants
Debabrita Deb1,2,3, Mariano Scian1,2,3, Katherine E Roth1,2, Wei Li1,2, Jane Keiger1,2,
Abhay Sankar Chakraborti1,2, Swati Palit Deb1,2 and Sumitra Deb*,1,2
1
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia, VA 23298, USA; 2Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University,
Richmond, Virginia, VA 23298, USA
Tumor-derived p53 mutants activate transcription from
promoters of various growth-related genes. We tested
whether this transactivation function of the mutant protein
is sucient to induce tumorigenesis (`gain of function').
Tumor-derived mutant p53-281G transactivates the
promoters of human epidermal growth factor receptor
(EGFR) and human multiple drug resistance gene (MDR1). To determine whether the C-terminal domain functions
only as an oligomerization domain in mutant p53mediated transactivation, we have replaced the tetramerization domain of p53 by a heterologous tetramerization
domain; although this mutant protein formed tetramers in
solution, it failed to transactivate signi®cantly. Therefore,
for successful mutant p53-mediated transactivation,
sequences near the C-terminus of mutant p53 are required
to perform functions in addition to tetramerization. We
also demonstrate that co-expression of a deletion mutant
of p53 (p53 del 1-293), which retains the p53 oligomerization domain, inhibits this transactivation. p53 del 1-293
co-immunoprecipitates with p53-281G suggesting that
hetero-oligomers of p53-281G and p53 del 1-293 are
defective in transactivation. We also show that a cell line
stably transfected with p53-281G expresses higher levels
of endogenous NF-kB and proliferating cell nuclear
antigen (PCNA) compared to that transfected with vector
alone. On co-expression, p53 del 1-293 lowered the levels
of NF-kB and PCNA in p53-281G-expressing cells.
However, on co-expression, p53 del 1-293 did not inhibit
the tumorigenicity and colony forming ability of p53-281G
expressing cells. Our earlier work showed that a deletion
of the C-terminal sequences of p53-281G overlapping the
oligomerization domain obliterates `gain of function'.
Taken together, the above information suggests that the
C-terminal sequences have some critical role in `gain of
function' in addition to transactivation.
Oncogene (2002) 21, 176 ± 189. DOI: 10.1038/sj/onc/
1205035
Keywords: p53; transcriptional activation; repression;
oligomerization
*Correspondence: S Deb; E-mail: [email protected]
3
These two authors contributed equally to the project
Received 2 April 2001; revised 14 September 2001; accepted 9
October 2001
Introduction
Wild-type p53 is a tumor-suppressor and is found to be
mutated in a large number of human cancers (Donehower
and Bradley, 1993; Greenblatt et al., 1994; Ko and Prives,
1996; Lane, 1994; Levine, 1993, 1997; Lowe, 1999; Oren,
1999; Ozbun and Butel, 1995; Prives and Hall, 1999;
Sheikh and Fornace, 2000; Soussi et al., 2000; Vogelstein
et al., 2000). Under normal situations wild-type p53 has a
short half-life. However, under stress such as that caused
by DNA damage p53 is stabilized and its intracellular
level rises due predominantly to post-transcriptional
modi®cations; p53 then induces G1-S arrest or apoptosis
such that the damaged cell cannot perpetuate.
The majority of p53 mutations found in human cancer
are missense in nature (Greenblatt et al., 1994; Levine et
al., 1991; Soussi et al., 2000). Presumably, tumor-derived
p53 mutants cannot induce the degradation pathway
activated by wild-type p53 through induction of MDM2.
The resulting mutant protein is, thus, usually present at a
relatively high level inside the cell (Haupt et al., 1997;
Kubbutat et al., 1997; Prives and Hall, 1999). Wild-type
p53 acts as a sequence-speci®c transcriptional activator
for a series of genes, including some that are involved in
cell cycle control and apoptosis (Levine, 1997; Oren,
1999; Prives and Hall, 1999; Vogelstein et al., 2000).
Tumor-derived p53 mutants are defective in this
sequence-speci®c transactivation. The wild-type protein
can also repress promoters of a number of cellular and
viral genes (Deb et al., 1992; Ginsberg et al., 1991;
Lechner et al., 1992; Santhanam et al., 1991; Seto et al.,
1992; Subler et al., 1992, 1994). It has been shown to
inhibit the endogenous expression of a number of genes
(Lee et al., 1999; Murphy et al., 1996, 1999; Zhao et al.,
2000). Although the molecular mechanism behind the
transcriptional repression is not completely clear, that
tumor-derived p53 mutants have in general lost this
function (Subler et al., 1992) is suggestive of its
functional importance.
Wild-type p53 has a well-de®ned domain structure
(Ko and Prives, 1996). Near the N-terminus it has two
transactivation domains (amino acid 1 ± 42, 43 ± 63)
(Candau et al., 1997; Subler et al., 1994; Zhu et al.,
1998), followed by a proline rich segment (amino acids
64 ± 91) (Sakamuro et al., 1997; Venot et al., 1998;
Walker and Levine, 1996). At the center is the DNA-
Mutant p53-mediated transactivation
D Deb et al
binding domain (amino acids 100 ± 300) (Prives, 1994);
this is followed by the presumptive nuclear localization
signal (amino acids 316 ± 325) (Ko and Prives, 1996;
Levine et al., 1991) and tetramerization domain (amino
acids 320 ± 360); (Sturzbecher et al., 1992; Subler et al.,
1994; Wang et al., 1993) and ®nally near the Cterminus is the basic domain (amino acids 360 ± 393)
(Ko and Prives, 1996; Levine et al., 1991). The Cterminal segment can take part in DNA strand transfer
and reassociation (Bakalkin et al., 1994; Brain and
Jenkins, 1994). It can also bind to DNA di€erent
structural complexities, including DNA damaged by
ionizing radiation or chemicals (Lee et al., 1995, 1997;
Reed et al., 1995). The C-terminal region can bind to
DNA non-sequence-speci®cally; it is also the site for
modi®cations such as phosphorylation, acetylation or
binding to proteins such as 14-3-3 protein. These
modi®cations seem to regulate p53's transcriptional
and DNA-binding activities (Abarzua et al., 1995;
Baudier et al., 1992; Fourie et al., 1997; Halazonetis et
al., 1993; Hansen et al., 1996, Hupp et al., 1992,
Takenaka et al., 1995; Waterman et al., 1998).
A number of tumor-derived p53 mutants that are
defective in transactivating genes normally induced by
wild-type p53 can transactivate the promoters of
growth-related genes such as, human proliferating cell
nuclear antigen, EGFR, MDR-1, vascular endothelial
growth factor, human interleukin 6, basic ®broblast
growth factor, human heat shock protein 70, c-fos,
insulin-like growth factor II, BAG-1, 15-lipoxygenenase
and c-myc (Chin et al., 1992; Frazier et al., 1998;
Kelavkar and Badr, 1999; Kieser et al., 1994; Margulies
and Sehgal, 1993; Preuss et al., 2000, Tsutsumi-Ishii et
al., 1995; Ueba et al., 1994; Yang et al., 1999). Lin et al.
(1995) showed that amino acids at positions 22 and 23
in the N-terminal transactivation domain of the mutant
protein p53 (Arg to Gly) [p53-281G] are required for
transactivation of the MDR-1 promoter. They also
showed that although murine 10(3) cells (free of
endogenous p53) expressing p53-281G are tumorigenic
in nude mice, cells expressing p53-281G with substitutions at amino acids 22 and 23 are not. This suggests a
direct relationship between the transactivation ability of
mutant p53 and the `gain of function' where mutant p53
procures a dominant oncogenic role.
More recently, using similar assay procedures as
described above, we showed that the `gain of function'
phenotype of tumor-derived p53 mutants also requires
the C-terminally located oligomerization/nucleic acidbinding domain (Lanyi et al., 1998). Whether the Cterminal region is only required to perform oligomerization or it has other function(s) necessary for mutant
p53-mediated transactivation and/or `gain of function'
is not known. Results of experiments with wild-type p53
in which the tetramerization domain has been replaced
by a heterologous dimerization or tetramerization
domain have shown that the heterologous oligomerization domain preserves wild-type p53-mediated transactivation and growth suppression (including G1-S
inhibition) functions (Attardi et al., 1996a,b; Ishizaka
et al., 1995; Pietenpol et al., 1994; Reed et al., 1993;
Waterman et al., 1996). However, no information is
available regarding the consequence of replacement of
the oligomerization domain of tumor-derived p53
mutants by heterologous oligomerization domains.
Here, we determine whether transactivation by
tumor-derived mutant p53 is responsible for its `gain
of function' phenotype and describe our investigation
of the importance of oligomerization in the tumorderived p53-mediated transcriptional activation and
`gain of function'. We have also replaced the
tetramerization domain of p53-281G by a coiled-coil
dimerization motif or a modi®ed coiled-coil structure
that codes for a tetramerization domain. Although the
modi®ed heterologous coiled-coil tetramerization domain induced tetramerization of the mutant p53, it
could not successfully substitute the domain in mutant
p53-mediated transactivation of the EGFR promoter.
This suggests that sequences near the C-terminus of
mutant p53 are required to perform functions in
addition to tetramerization for successful mutant p53mediated transactivation. Since wild-type p53 with its
tetramerization domain replaced by a heterologous
dimerization/tetramerization motif retains transcriptional functions (Attardi et al., 1996a,b; Ishizaka et
al., 1995; Pietenpol et al., 1994; Reed et al., 1993;
Waterman et al., 1996) our work suggests a mechanistic di€erence between transactivation mediated by
wild-type p53 and its tumor-derived mutants. Heterooligomerization of wild-type p53 by a mini-protein
with the oligomerization domain can eciently inhibit
wild-type p53's biological functions leading to transformation of rat embryo ®broblasts (Reed et al., 1993;
Shaulian et al., 1992). We, therefore, tested whether
hetero-oligomerization between p53 del 1-293 and p53281G would lead to inhibition of tumorigenicity (`gain
of function') by p53-281G, and found that coexpression did not signi®cantly impact tumorigenesis.
We ®nd that hetero-oligomerization between full-length
mutant p53-281G and a p53 deletion mutant preserving the oligomerization domain inhibits transactivation of the EGFR and MDR-1 promoters by tumorderived mutant p53. We also show that a cell line
stably transfected with p53-281G expresses higher
levels of endogenous NF-kB2 and PCNA compared
to that transfected with vector alone. On co-expression,
p53 del 1-293 lowered the levels of NF-kB2 and PCNA
in p53-281-expressing cells. Since sequences from 393327 are required for tumorigenicity (Lanyi et al., 1998),
the C-terminal sequences must have a crucial role in
`gain of function' in addition to transactivation.
177
Results
Replacement of the tetramerization domain of mutant p53
by a heterologous dimerization or tetramerization domain
has a strong inhibitor effect on mutant p53-mediated
transactivation
Earlier, we have shown that several tumor-derived p53
mutants could transactivate promoters of a number of
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Mutant p53-mediated transactivation
D Deb et al
178
Oncogene
growth-related genes including EGFR (Deb et al.,
1992; Ludes-Meyers et al., 1996). However, the
deletion mutant p53-281G del 393-327 that eliminates
the oligomerization domain failed to transactivate
(Lanyi et al., 1998; Ludes-Meyers et al., 1996)
suggesting that proper oligomerization of a tumorderived p53 mutant is required for the transactivation
function of the mutant protein. We, therefore, checked
whether the C-terminally located oligomerization/nonsequence-speci®c nucleic acid binding domain is
required only for oligomerization. It was earlier shown
that replacement of the tetramerization domain of
wild-type p53 by the dimerization domain consisting of
the coiled-coil structure from the yeast GCN4 protein
(or a derivative of the coiled-coil structure that results
in a tetramerization domain) could e€ectively sustain
wild-type p53's transactivation and growth suppression
functions (Attardi et al., 1996a; Pietenpol et al., 1994;
Waterman et al., 1996). Performing similar experiments
we wanted to determine whether a p53-281G derivative
with such a replacement of the tetramerization domain
would retain the transactivation function. Therefore,
we studied the e€ect of expression of p53-281G, p53281G 343cc (the coiled-coil structure from GCN4 is
laced after the amino acid 343) (Pietenpol et al., 1994)
and p53-281G TZ334NR (a modi®ed GCN4 coiled-coil
structure is placed after amino acid 334 such that it
becomes a tetramerization domain) (Waterman et al.,
1996) on the activity of the EGFR promoter.
Transfections were carried out in Saos-2 cells.
Luciferase assay results shown in Figure 1 demonstrate that p53-281G-mediated transactivation is not
preserved by replacing its tetramerization domain by a
heterologous dimerization/tetramerization motif suggesting that this region performs some vital function(s)
required for mutant p53-mediated transactivation that
cannot be substituted by a heterologous dimerization/
tetramerization domain. Amino acid residues 320 ± 360
of p53 code for the tetramerization domain (Sturzbecher et al., 1992; Subler et al., 1994; Wang et al.,
1993); besides substitution of this domain (starting
from amino acid 334), C-terminal amino acids past the
oligomerization domain up to 393 are missing in p53281G TZ334NR, and this segment may also play an
important role in mutant p53-mediated transactivation.
Therefore, we have tested one more mutant p53-281G
TZ334NRI352, which has the sequences 352 ± 393
inserted after an Isoleucine at amino acid 352 following
the TZ moiety. This mutant transactivated the best
among all the domain replacement mutants. The
Western blot analysis shown with the luciferase assay
®gure demonstrated equivalent amounts of expression
by p53-281G and p53-281G TZ334NR, suggesting that
expression de®ciency cannot explain the inability of
p53-281G TZ334NR to transactivate. Thus, the results
of our experiments suggest that the C-terminally
located amino acids 352 ± 393 perform a vital function
for mutant p53-mediated transactivation. Earlier, a
similar conclusion was reached by Frazier et al. (1998)
demonstrating a requirement for amino acids 370 ± 380
for transactivation the c-myc promoter by p53-281G.
Replacement of the tetramerization domain of mutant p53
by a heterologous tetramerization domain preserves
tetramerization of the protein
One possible reason for the failure of p53-281G
TZ334NR and p53-281G TZ334NRI352 to transactivate the EGFR promoter eciently can be a defect in
oligomerization in the context of mutant p53. We,
therefore, tested whether p53-281G TZ334NR and p53281G TZ334NRI352 exist predominantly as tetramers
in solution. For this purpose we have used glutaraldehyde cross-linking assays as described earlier
(Subler et al., 1994).
We have synthesized 35S-labeled p53-281G, p53-281G
TZ334NR, p53-281G TZ334NRI352 and p53-281G del
393-327 using an in vivo transcription-translation system
as described in Materials and methods. Synthetic
proteins were incubated with glutaraldehyde at varying
®nal concentrations (Figure 2). After cross-linking,
proteins were analysed by gradient (4 ± 20%) sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The data shown in Figure 2 indicate that p53-281G,
p53-281G TZ334NRI352 and p53-281G TZ334NR
oligomerized, while p53-281G del 393-327 could not,
although there may be some variations in tetramerization among p53-281G, p53-281G TZ334NRI352 and
p53-281G TZ334NR. Since p53-281G TZ334NRI352
and p53-281G TZ334NR are capable of oligomerization
into tetramers, the results of our experiments shown in
Figure 1 demonstrate that for p53-281G-mediated
transactivation, the C-terminally located oligomerization/nucleic acid binding domain is required for
functions in addition to oligomerization.
Transactivation of the EGFR and MDR-1 promoters by
the tumor-derived mutant p53-281G was inhibited by
co-expression of p53 del 1-293
Hetero-oligomerization of wild-type p53 with miniproteins containing its C-terminal oligomerization domain
results in inactivation of wild-type p53's function
(Shaulian et al., 1992). Therefore, to investigate the
role of oligomerization further we tested whether such
hetero-oligomerization of a tumor-derived mutant p53281G would inactivate its transactivation potential.
Saos-2 cells were co-transfected with EGFR.Luc
(Ludes-Meyers et al., 1996; Deb et al., 2001) or MDR1.CAT (Chin et al., 1992; Lin et al., 1995), the
expression plasmid for p53-281G (or expression vector
alone) and the expression plasmid for p53 del 1-293 (or
expression vector alone). Transfections and CAT (or
luciferase) assays were carried out as described in
Materials and methods. Figure 3a,b shows the CAT
(and luciferase) assay data, it is apparent that although
the tumor-derived p53-mutant transactivated the
EGFR and MDR-1 promoters, the transactivation
ability was reduced signi®cantly by the co-expression of
p53 del 1-293. Western blot analyses examining
expression of p53-281G and p53 del 1-293 for a
representative experiment shown in the ®gure demonstrate that proteins were adequately expressed; thus,
Mutant p53-mediated transactivation
D Deb et al
179
Figure 1 Replacement of the tetramerization domain of mutant p53 by a heterologous dimerization or tetramerization domain has
a drastic e€ect on mutant p53-mediated transactivation. (a) Schematic representation of p53-281G derivatives used in the domain
exchange experiments. The tetramerization domain of p53 amino acid 320 ± 360 is shown by a dark box. p53-281G TZ334NR has
sequences from amino acids 334 to 393 replaced by a modi®ed coiled-coil structural domain (shown by a stippled box) that makes it
a tetramerization domain, p53-281G TZ334NRI352 has the sequences 352 ± 393 inserted after an Isoleucine at amino acid 352
following the TZ moiety, p53-281G 343cc has sequences from amino acids 344 to 393 replaced by the coiled-coil structural domain
(shown by a shaded box, coding for a dimerization motif) from yeast GCN4 protein. (b) Transactivation of the EGFR promoter by
p53-281G derivatives. Saos-2 cells were co-transfected with EGFR.Luc and the expression plasmid for tumor-derived mutant p53281G or one of its derivatives (or expression vector alone) in a 24 well plate by Lipofectamine 2000 as described in Materials and
methods. After transfection, cells were treated as described, and luciferase assays were performed. The left panel shows luciferase
assays and the right panel shows Western blot analysis carried out with equal amounts of protein from each luciferase assay extract
from one representative experiment using a monoclonal anti-p53 antibody (PAb421)
Figure 2 Replacement of the tetramerization domain of mutant p53 by a heterologous tetramerization domain preserves
tetramerization of the protein. In vivo translated p53-281G and its derivatives (p53-281G TZ334NR, p53-281G TZ334NRI352 and
p53-281G del 393-327) were incubated with the indicated concentrations of glutaraldehyde as described (Subler et al., 1994).
Samples were loaded onto a gradient (4 ± 20%) SDS polyacrylamide gel after cross-linking, electrophoresed, dried and
autoradiographed. Positions of presumptive monomers, dimers, trimers and tetramers of p53 derivatives are indicated
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Mutant p53-mediated transactivation
D Deb et al
180
Figure 3 Transactivation of the EGFR and MDR-1 promoters by the tumor-derived mutant p53-281G is inhibited by coexpression of p53 del 1-293. Saos-2 cells were transfected with (a) EGFR.Luc or (b) MDR-1 CAT. The experiments with EGFR.Luc
were done using 24 well plates. Each well had 170 ng of the EGFR-Luc, 170 ng of pCMVBam p53-281G (or expression vector
alone) and 510 ng of pCMVBam p53 del 1-293 (or expression vector alone). The experiments with MDR1-CAT were done in 10 cm
plate with 2.5 mg of MDR1.CAT, 2.5 mg of pCMVBam p53-281G (or expression vector alone) and 7.5 mg of pCMVBam p53 del 1293 (or expression vector alone). After transfection, cells were treated as described in Materials and methods and CAT (or
luciferase) assays were performed. The upper panels show the results of the CAT (or luciferase) assay and the lower panels show
Western blot analyses carried out with equal amounts of protein from each system of one representative experiment. The positions
of the p53-281G (detected by monoclonal antibody Pab1801) and p53 del 1-293 (detected by monoclonal PAb421) bands are shown
by arrowheads. Migration of the molecular weight markers are shown on the right
lack of transactivation by p53-281G in the presence of
p53 del 1-293 cannot be explained by any protein
expression problem. It is possible that hetero-oligomers
between p53-281G and p53 del 1-293 are inactive in
transactivation.
p53 del 1-293 forms complexes with p53-281G in vivo
Next, we checked whether p53-281G would form heterooligomeric complexes with p53 del 1-293 in vivo. We cotransfected expression plasmids for p53-281G (or
expression vector alone) and p53 del 1-293 (or expression
vector alone) into Saos-2 cells. Twenty-four hours after
transfection, cells were labeled in vivo with 35Smethionine as described in Materials and methods. Cell
extracts were used for immunoprecipitation analysis
(Figure 4). Although DO1 could not immunoprecipitate
p53 del 1-293 alone (lane 4), it could precipitate both
p53-281G and p53 del 1-293 when both were present
(lane 3). This shows that p53 del 1-293 binds with p53281G in vivo forming a hetero-oligomeric complex. Thus,
results of these experiments (Figures 3 and 4) suggest
that proper oligomeric forms of p53-281G are necessary
for its transactivation function.
Effect of co-expression of p53 del 1-293 on the
endogenous transactivation ability of p53-281G
Hetero-oligomerization of wild-type p53 with tumorderived p53 mutants (or polypeptides covering the
oligomerization domain of p53) inhibit wild-type p53's
Oncogene
biological functions leading to immortalization and
transformation of rat embryo ®broblasts (Shaulian et
al., 1992). Since co-expression of p53 del 1-293 and p53
(wild-type or mutant) inhibits p53's transactivation
(Deb et al., 1999; this communication), it was
important to test whether hetero-oligomerization
between p53 del 1-293 and p53-281G would lead to
inhibition of transactivation of endogenous genes by
p53-281G.
To identify genes that are endogenously transactivated by p53-281G, we have used murine 10(3) cells
that are devoid of endogenous p53 and have
compared gene expression pro®les of cells stably
transfected with p53-281G with that of cells stably
transfected with vector (pCMVBamNeo, Hinds et al.,
1990) alone. Microarray analysis done by Incyte
Genomics (to be reported separately) indicated a
number of genes that are expressed at a higher level
in cells expressing p53-281G than in cells stably
transfected with vector alone. We chose two such
genes, PCNA and NF-kB2, to see the e€ect of coexpression of p53 del 1-293. We have used quantitative real-time PCR analysis of cDNA, made from
total RNA, to determine the level of expression of
PCNA and NF-kB2 RNAs. Figure 5 shows that the
levels of both the genes are considerably higher in
10(3) cells expressing p53-281G compared to that in
cells stably transfected with vector alone. Co-expression of p53 del 1-293 lowered the level of the RNAs
to some extent, suggesting that hetero-oligomerization
indeed inhibits transactivation by mutant p53. In-
Mutant p53-mediated transactivation
D Deb et al
explicably, 10(3) cells expressing p53-del 1-293 expressed a little more of the two RNAs compared to
vector-transfected cells.
Figure 4 p53 del 1-293 co-immunoprecipitates with p53-281G by
DO1 that does not bind to p53 del 1-293. Saos-2 cells were
transfected in 10 cm dishes with 3.2 mg of the expression plasmid for
p53-281G (or expression vector alone) and 9.6 mg of an expression
plasmid for p53 del 1-293 (or expression vector alone). Twenty-four
hours after transfection, cellular proteins were labeled in vivo by 35Smethionine as described in Materials and methods. The cellular
extracts were then immunoprecipitated with monoclonal antibodies
as depicted in the ®gure. The immunoprecipitates were resolved in a
15% SDS-polyacrylamide gel followed by autoradiography. The
positions of the p53-281G and p53 del 1-293 bands are shown by
arrowheads. Migration of the molecular weight markers is shown
on the right. The upper panel shows the binding sites for
monoclonal antibodies PAb421 and DO1 on p53-281G
181
Effect of co-expression of p53 del 1-293 on the `gain of
function' property of p53-281G as determined by
tumorigenicity of 10(3) cells expressing the mutant
protein
Since co-expression of p53 del 1-293 and p53 (wild-type
or mutant) inhibits p53's transactivation (Deb et al.,
1999; this communication), it was important to test
whether hetero-oligomerization between p53 del 1-293
and p53-281G would lead to inhibition of tumorigenicity of mutant p53 expressing cells.
10(3) cells are murine cells that are devoid of
endogenous p53 and are non-tumorigenic if injected
subcutaneously in nude mice (Dittmer et al., 1993). We
have shown that although 10(3) cells expressing p53281G form tumors, those stably expressing p53-281G
del 393-327 (defective in oligomerization) could not
(Lanyi et al., 1998). This suggested a role for
oligomerization in the `gain of function'. We, therefore,
tested whether co-expression of p53 del 1-293, which
inhibits p53-281G's transactivation ability, would also
disrupt its tumorigenicity. Stable cell lines were
generated from 10(3) cells (1) expressing p53-281G,
(2) expressing p53 del 1-293, (3) expressing p53-281G
and p53 del 1-293 and (4) stably transfected with
vector alone [10(3) V4] (see Figure 6). Two di€erent
clones from each variety were injected subcutaneously
into two or three di€erent nude mice (two sites on each
mouse) as described in Materials and methods. The
mice were then observed for several weeks for
appearance of tumors. As shown in Table 1, 10(3)
cells stably transfected with pCMVBam vector alone or
stably expressing p53 del 1-293 did not show any
tumor formation even after 5 months. For 10(3) cells
expressing p53-281G and p53 del 1-293 tumors
appeared within 1 ± 2 weeks of injection; similar is the
situation with 10(3) cells expressing p53-281G alone.
Since co-expression of p53 del 1-293 and p53-281G did
not reduce tumorigenicity, hetero-oligomer formation
may not interfere with mutant p53-mediated tumorigenesis.
Figure 5 E€ect of co-expression of p53 del 1-293 on the endogenous transactivation ability of p53-281G. Total RNA was prepared
from 10(3) cells stably transfected with vector alone, p53-281G, p53 del 1-293, or p53-281G and p53 del 1-293 as described in the
text. Quantitative real-time PCR was done as described in Materials and methods to determine the relative amounts of mRNA for
NF-kB2 and PCNA genes. Histograms show the relative levels of (a) PCNA mRNAs and (b) NF-kB2 mRNAs (normalized to
GAPDH levels in cells) in di€erent stable transfectants of 10(3) cells
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Mutant p53-mediated transactivation
D Deb et al
182
Figure 6 Hetero-oligomers are produced between p53-281G and p53 del 1-293 in 10(3) cells stably expressing the two proteins.
10(3) cell lines stably transfected with vector alone, p53-281G, p53 del 1-293, or p53-281G and p53 del 1-293 were constructed and
maintained as described in Materials and methods. Protein extracts from the respective cells were obtained by using the luciferase
assay extraction procedure (see Materials and methods). Equal amounts of protein were loaded in each well of 10% SDS
polyacrylamide gel, electrophoresed and the proteins transferred to a nitrocellulose membrane. The blot was developed using the
monoclonal antibody PAb421. Positions of molecular weight markers are shown. The ®rst four lanes show the Western blot
analysis. Cell extracts from 10(3) cells expressing p53 del 1-293 alone and 10(3) cells expressing p53-281G and p53 del 1-293 were
prepared and immunoprecipitation reactions were performed (see Materials and methods) with DO1 an antibody that binds to the
N-terminal region of p53. The immunoprecipitates were then separated on an SDS polyacrylamide gel followed by Western blot
analysis. The blot was developed with PAab421, an antibody that recognizes a motif near the C-terminus and binds to both p53281G and p53 del 1-293. Antibody DO1 was run and analysed by Western blot to depict the positions of light and heavy chains of
IgG. The last ®ve lanes show this Western analysis
Hetero-oligomers are produced between p53-281G and
p53 del 1-293 in 10(3) cells stably expressing p53-281G
and p53 del 1-293
Since co-expression of p53-281G and p53 del 1-293 in
10(3) cells did not interfere with tumorigenicity, we
needed to verify in vivo hetero-oligomer formation
between p53-281G and p53 del 1-293. Therefore, we
prepared extracts from 10(3) cells expressing p53 del 1293 alone and 10(3) cells expressing p53-281G and p53
del 1-293, and performed immunoprecipitation reactions
(see Materials and methods) with DO1, an antibody that
binds to the N-terminal region of p53. The immunoprecipitates were separated on an SDS polyacrylamide gel,
and proteins transferred to a nitrocellulose membrane.
The blot was then developed with PAb421 an antibody
that recognizes a motif near the C-terminus and binds to
Oncogene
both p53-281G and p53 del 1-293. The data shown in
Figure 6 demonstrate that DO1 immunoprecipitates
both p53-281G and p53 del 1-293 indicating heterooligomer formation between p53-281G and p53 del 1293; as expected, DO1 did not interact with p53 del 1293. Taken together, the results presented in Figures 5
and 6 suggest that hetero-oligomerization of p53-281G
does not inhibit the tumorigenicity.
p53 del 1-293 did not inhibit colony formation by
p53-281G
Expression of p53-281G gives growth advantage to
cells expressing the mutant protein (Dittmer et al.,
1993); we wanted to check whether p53 del 1-293
expression could in¯uence the cell growth advantage.
We have used the colony formation assay as described
Mutant p53-mediated transactivation
D Deb et al
in Materials and methods to assay for cell proliferation. We used 10(3) cells stably transfected with the
expression vector (pCMVBamneo) alone or the expression plasmid for p53-281G as starting materials and
transfected them with the following expression plasmids: (1) pCMVBam and pHyg, (2) pCMVBam p53
(wild-type) and pHyg, (3) pCMVBam FLAG p73 a
and pHyg, (4) pCMVBam p53 del 1-293 and pHyg.
The data given in Table 2 indicate p53-281G expressing
cells have a small but discernable growth advantage
over vector-transfected cells. We can see that the
systems with wild-type p53 and p73 a had substantial
e€ects on the number of colonies formed on p53-281Gexpressing cells compared to vector-transfected cells.
p53 del 1-293 had little e€ect, if any (compared to
vector alone), on the growth properties of p53-281Gexpressing cells. Thus, once again we ®nd that del 1293 could not signi®cantly change mutant p53's growth
enhancing function, suggesting that hetero-oligomerization may not be enough to inhibit `gain of function'.
Earlier, we showed that a deletion of the C-terminal
sequences of p53-281G overlapping the oligomerization
domain disrupts `gain of function', and here we
demonstrate that hetero-oligomerization disables transactivation by p53-281G without eliminating `gain of
function' (tumorigenicity). Taken together, this information suggests that the C-terminal sequences have a
separate and critical role in `gain of function' in
addition to that in transactivation.
Table 1 Tumorigenicity of 10(3) cells expressing p53 derivatives.
E€ect of overexpression of p53 derivatives on the tumorigenic
potential of p53 null 10(3) murine ®broblast cells
Cells stably
transfected with
Number of mice/
number of sites
injected
Number of tumors
generated/
number of sites
injected
2/4
2/4
2/4
3/6
3/6
2/4
3/6
0/4
0/4
0/4
0/6
6/6
4/4
6/6
3/6
6/6
Vector alone, clone 2
Vector alone, clone 21
p53 del 1-293, clone 17
p53 del 1-293, clone 19
p53-281G, clone 13
p53-281G, clone 4
p53-281G, clone 4, p53 del
1-293 clone 22
p53-281G, clone 4, p53 del
1-293, clone 10
Table 2
183
Discussion
Since the C-terminal region of wild-type p53 harbors
the oligomerization domain, and the oligomerization
domain is necessary for the biological functions of
wild-type p53 (Attardi et al., 1996a; Deb et al., 1999;
Ishioka et al., 1997; Ozbun and Butel, 1995, Pietenpol
et al., 1994; Reed et al., 1993; Sang et al., 1994; Subler
et al., 1994; Waterman et al., 1996), it was hypothesized that the C-terminal sequences for mutant p53
would be required for oligomerization, and the
oligomerization may be critical for `gain of function'
(Lanyi et al., 1998). We have tested this hypothesis
using di€erent approaches. An N-terminal deletion
mutant of wild-type p53 that deletes amino acids 1
through 293 has been used as a tool to perform heterooligomerization studies. This mutant retains the entire
oligomerization domain but dispenses o€ the transactivation domain and a large portion of the sequencespeci®c DNA-binding domain inactivating the transactivation and DNA-binding activities of wild-type p53
(Subler et al., 1994). By co-transfection experiments
followed by immunoprecipitation analysis we show
that p53 del 1-293 forms hetero-oligomeric complexes
with p53-281G (Figure 4). p53 del 1-293 inhibits p53281G-mediated transactivation of the EGFR and
MDR-1 promoters (Figure 3) suggesting that heterooligomerization inactivates transcriptional functions of
mutant p53. We have also determined that coexpression of p53 del 1-293 and p53-281G reduced
transactivation potential of p53-281G in stably transfected 10(3) murine cells. Although 10(3) cells stably
transfected with p53-281G express 5 ± 6-fold more
PCNA and NF-kB2 mRNA compared to the cells
transfected with vector alone, co-expression of p53 del
1-293 reduced this transactivation considerably (Figure
5). Our data, therefore, support the idea that proper
oligomeric forms of mutant p53 are required for its
transactivation function.
We have used another approach to investigate the
importance of oligomerization in mutant p53-mediated
transactivation, and swapped the tetramerization domain of p53-281G with a coiled-coil dimerization
domain from the yeast GCN4 protein (Pietenpol et
al., 1994) or a modi®ed coiled-coil domain that acts as a
tetramerization motif (Waterman et al., 1996). Transcriptional data generated with these heterologous
Colony formation in 10(3) cells stably transfected with vector (pCMVBamNeo) or p53-281G after transfection
with di€erent expression plasmids
Cells stably transfected with
Expression plasmids used for transfection
Vector alone, clone
p53-281G, clone 4
Vector alone, clone
p53-281G, clone 4
Vector alone, clone
p53-281G, clone 4
Vector alone, clone
p53-281G, clone 4
pHyg
pHyg
pHyg
pHyg
pHyg
pHyg
pHyg
pHyg
4
4
4
4
and
and
and
and
and
and
and
and
pCMVBam
pCMVBam
pCMVBamp53 wt
pCMVBamp53 wt
pCMVBamp73a
pCMVBamp73a
pCMVBamp53 del 1-293
pCMVBamp53 del 1-293
Number of colonies (1 mm or more)
formed after 3 weeks of selection
0,0,2; avg. 0.67
137, 117, 132; avg. 129
4,0,0; avg. 1.33
4,21,5; avg. 10
3,8,38; avg. 16.33
0,8,18; avg. 8.67
1,4,0; avg. 1.67
113,125,107; avg. 115
Oncogene
Mutant p53-mediated transactivation
D Deb et al
184
Oncogene
constructs showed that mutant p53-281G requires the
C-terminally located oligomerization/non-sequence-speci®c nucleic acid-binding domain for its transactivation
properties (Figure 1). This requirement is sequencespeci®c and not met by merely allowing the protein to
oligomerize, as we demonstrated also that p53-281G
with its tetramerization domain replaced by a heterologous tetramerization domain preserves the tetrameric
structure of the protein (see Figure 2), but disrupts the
transactivation function (Figure 1). However, replacement of the tetramerization domain of wild-type p53 by
the dimerization domain, consisting of the coiled-coil
structure from the yeast GCN4 protein (p53 343cc),
could e€ectively sustain wild-type p53's transactivation
and growth suppression functions (data not shown)
(Attardi et al., 1996a; Ishioka et al., 1997; Pietenpol et
al., 1994; Reed et al., 1993; Waterman et al., 1996).
Interestingly, in the context of wild-type p53 the
replacement of its tetramerization domain by a
derivative of the coiled-coil structure that results in a
tetramerization domain could not activate transcription
from the human p21 promoter as well as p53 343cc
(data not shown). The reason for this apparent
discrepancy is not known at the present time, but could
be related to the DNA concentrations used in the
di€erent transfections (Subler et al., 1994). Also, the
wild-type p53 derivatives with heterologous dimerization/tetramerization domain inhibited the CMV immediate-early promoter signi®cantly; the extent of
inhibition is comparable to that mediated by wild-type
p53 (data not shown). Thus, the functions of wild-type
p53 are preserved when a heterologous oligomerization
domain replaces the oligomerization domain of p53 as
shown earlier (Attardi et al., 1996a; Ishioka et al., 1997;
Pietenpol et al., 1994; Reed et al., 1993; Waterman et
al., 1996). Taken together, these data strongly suggest
that the C-terminal region of wild-type p53 mainly
functions in oligomerization in transient-transfection
transcription assays, whereas mutant p53 requires an
additional function. Thus, there is a clear distinction in
the requirement of the C-terminal domain for wild-type
and mutant p53-mediated transcriptional functions.
For mutant p53-mediated transactivation, sequence
requirements at the C-terminus are stringent, suggesting
that the region is needed perhaps for sequence-speci®c
functions in addition to oligomerization. A number of
possible explanations for this requirement can be o€ered:
(1) p53 has a nucleic acid binding activity associated with
its C-terminal region (Ko and Prives, 1996; Reed et al.,
1995). It is possible that under appropriate conditions
mutant p53 utilizes this activity to interact with the
promoter to activate transcription. (2) A number of posttranslational modi®cations, e.g. phosphorylation and
acetylation (Prives, 1998; Prives and Hall, 1999;
Vogelstein et al., 2000) of wild-type p53 have been
reported to occur in speci®c sites located near the Cterminus. Perhaps mutant p53 requires these modi®cations for its transactivation function. (3) The C-terminal
region could be a site for protein ± protein interaction
[e.g., interaction needed with c-abl (Yuan et al., 1996)]
needed for mutant p53-mediated transactivation; speci®c
interactions may occur between a transcription factor
and the tetrameric C-terminal region of mutant p53. It is
possible that overexpression of p53 del 1-293 would also
compete for such modi®cations or functions with the
intact protein causing an inhibition of transactivation.
Analyses involving more speci®c mutations near the Cterminus along with functional assays are required before
we can get a comprehensive idea about the function of
the C-terminus in mutant p53-mediated transactivation.
We also investigated whether a homo-tetrameric
structure of the mutant p53 protein is necessary for its
`gain of function' by studying the in¯uence of heterooligomerization on the `gain of function' as determined
by tumorigenicity and colony forming ability of cells in
which the mutant is expressed. We found that coexpression of p53 del 1-293 and p53-281G caused
inhibition of transactivation of p53-281G, and earlier,
we and others have shown that transactivation is directly
related to `gain of function' (Lanyi et al., 1998; Lin et al.,
1995). Therefore, a likely scenario would be that coexpression of p53 del 1-293 would hinder p53-281G's
`gain of function' property as determined by the
tumorigenicity assay. Our results shown in Table 1,
however, indicate that co-expression of p53 del 1-293
does not inhibit tumorigenicity of 10(3) cells expressing
p53-281G. Results from our co-immunoprecipitation
experiments (Figure 6) demonstrate that p53-281G
forms hetero-oligomers with p53 del 1-293 in 10(3) cells
stably expressing both proteins. This observation argues
that hetero-oligomerization although disrupts transactivation capacity of mutant p53 cannot eliminate its `gain
of function' (tumorigenicity) property noticeably. The
colony formation assay also indicates that p53 del 1-293
had no signi®cant impact on growth of p53-281G
expressing cells; it behaved very similarly to the vector
control. In these assays, however, wild-type p53 and p73
a reduced colony numbers signi®cantly for cells expressing p53-281G, showing that the growth advantage
induced by p53-281G was countered better by the growth
suppressors. Thus, hetero-oligomerization between p53281G and p53 del 1-293 may not inhibit p53-281G's
growth enhancing properties, although it does inhibit its
transactivation property. This observation goes against
the notion that the transactivation property of the
tumor-derived p53 mutants may be responsible for the
`gain of function'. We must add though that before we
draw such a conclusion it must be proven that all the
mutant p53 protein molecules are complexed with p53
del 1-293, because it is possible that any free mutant p53
molecule could induce the growth enhancement.
Although we have shown that p53 del 1-293 and p53281G forms hetero-oligomeric complexes in 10(3) cells
expressing the two proteins, it is possible that some p53281G molecules are still free. Because of extended
number of passages that the cells had to pass through,
it is also possible that the 10(3) cells expressing p53-281G
became spontaneously transformed independent of the
involvement of p53-281G; in that case neutralization of
mutant p53 would not reduce its oncogenicity. 10(3) cells
indeed have a tendency to become spontaneously
tumorigenic (data not shown).
Mutant p53-mediated transactivation
D Deb et al
Interestingly, expression of `gain of function' mutants
of p53 has also been shown to enhance drug resistance,
interfere with p53-independent apoptosis, and induce
centrosome abnormalities in cells expressing them
(Blandino et al., 1999; Li et al., 1998; Murphy et al.,
2000; Sigal and Rotter, 2000; Wang et al., 1998). It is not
known whether mutant p53-mediated transactivation is
involved as the etiologic agent for these phenotypes.
Gualberto et al. (1998) reported that some mutant p53
confer a dominant `gain of function' phenotype that
disrupts the spindle checkpoint control. They also
showed that for that function the mutant protein does
not require transactivation. Thus, it is possible that some
`gain of function' is achievable even in the absence of the
transactivation function of mutant p53.
How mutant p53 induces `gain of function' is still an
open question. It is possible that the newly found family
members of p53, such as p73 and p63, may act as
surrogate p53 in p53's absence and all p53 mutants do is
to neutralize their activity, perhaps by hetero-oligomerization, and some such evidence have been presented (Di
Como et al., 1999; Gaiddon et al., 2001; Marin et al.,
2000; Strano et al., 2000). It has also been shown that for
its neutralizing and hetero-oligomerization activity
a€ecting p73 or p63 the mutant p53 protein requires
only its central `DNA-binding domain'. However, earlier
work (Lanyi et al., 1998; Lin et al., 1995) indicate that the
transactivation domain and C-terminally located region
overlapping the oligomerization and non-sequencespeci®c nucleic acid-binding domains are required for
`gain of function'. This shows that sequences outside the
region needed for neutralizing p73 are needed for `gain of
function'. Therefore, the `gain of function' cannot be
fully explained by p73-mutant p53 interactions. Also,
our attempts to identify endogenous p73-p53-281G
complexes in 10(3) cells expressing p53-281G failed so
far (data not shown). This, however, suggests that even if
p73-p53-281G complexes are formed, they are low in
number. The work presented here shows that heterooligomerization inhibited but not obliterated transactivation, and Dittmer et al. (1993) reported that the tumorderived p53 mutants that di€er in transactivation did not
di€er in their tumorigenicity suggesting that any remnant
transactivation might be enough to induce `gain of
function'. Thus, a small fraction of free mutant p53 may
induce oncogenic transformation by transactivating
expression of critical growth-related genes. At this point,
it is not clear whether mutant p53-mediated transactivation is the cause of `gain of function'. Thus, it is necessary
to identify target genes that are a€ected by `gain of
function' directly, and to determine whether transactivation of these genes lead to increased oncogenesis (`gain of
function').
Materials and methods
DNA
The p53 and p73 expression plasmids contained the human
p53 and p73 cDNAs under the regulation of the CMV
immediate-early promoter in the pCMVBam expression
vector (Hinds et al., 1990; Subler et al., 1992). The Nterminal deletion derivative p53 del 1-293 was generated in
the context of the human wild-type p53 cDNA as described
earlier (Subler et al., 1994). p73 a and p73 b cDNAs have
been cloned in such a way that they have a FLAG epitope at
the N-terminus when converted into the respective proteins
(Deb et al., 2001).
p53 mutants, p53 343cc and p53 TZ334NR, have been
described earlier (Pietenpol et al., 1994; Waterman et al.,
1996). p53 343cc had sequences from amino acids 344 to 393
replaced with the coiled-coil structural domain from the
yeast GCN4 protein, while p53 TZ334NR had sequences
from amino acids 334 to 393 replaced by a modi®ed coiledcoil structure de®ning a tetramerization domain. p53-281G
343cc and p53-281G TZ334NR contained the amino acid
substitution at the position 281 from Arg to Gly; these
clones were constructed by standard sub-cloning techniques
by replacing a restriction fragment of the wild-type p53
cDNA overlapping the sequence coding for amino acid 281
with the corresponding fragment from p53-281G cDNA. All
the coding sequences were cloned in the pCMVBam vector
in the correct orientation downstream of the CMV
immediate-early promoter. pHyg (Invitrogen) contained the
hygromycin resistance gene cloned under the herpes simplex
virus thymidine kinase promoter (Clontech).
The chloramphenicol acetyltransferase (CAT) plasmids
utilized the Escherichia coli CAT gene under the transcriptional control of the MDR-1 (Lanyi et al., 1998) promoter.
The luciferase plasmids used the ®re¯y luciferase (luc) gene
under the control of the human EGFR and p21 promoters
(Deb et al., 2001; el-Deiry et al., 1993).
185
Cell culture and transfection
Human osteosarcoma (Saos-2) cells were cultured and
transfected by the calcium phosphate-DNA co-precipitation
method, as described previously (Subler et al., 1992). In a
typical experiment 36106 cells in a 10 cm dish were
transfected with indicated amounts of plasmids. Some
transfections were done using Lipofectamine 2000 (Life
Technologies) using 24-well plates following manufacturer's
protocol. For 24-well plates each well was transfected with
1 mg total DNA. In general, the promoter luciferase construct
and the e€ector plasmid are added at 170 ng per well, and
the rest of the 1 mg DNA came from pUC 19 DNA.
Chloramphenicol acetyltransferase assay
Cells were harvested 36 ± 40 h post-transfection and lysed by
three successive cycles of freezing and thawing. Extracts were
normalized for protein concentration and assayed for CAT
enzyme activity as described earlier (Ludes-Meyers et al.,
1996). Multiple independent experiments (three or more) were
done to determine the standard deviations. CAT activity was
detected by thin-layer chromatographic separation of [14C]chloramphenicol from its acetylated derivatives followed by
autoradiography. Quantitation was done using a Phosphorimager (Molecular Dynamics).
Luciferase assay
Luciferase assays were carried out using a commercial kit
from Promega as suggested by the vendor, and luminescence
was measured in a Luminometer made by Turner Designs.
Cells were harvested 36 ± 40 h after transfection, and extracts
were prepared as suggested. As described for the CAT assay,
Oncogene
Mutant p53-mediated transactivation
D Deb et al
186
equal amounts of protein from each extract were used for the
luciferase assay. Relative luciferase activity was plotted on
the Y-axis and the value of the vector control was set to
100%.
In vivo labeling of cellular proteins and immunoprecipitation
Saos-2 cells were transfected with p53 expression plasmids (or
expression vector alone) as described in the text. Twenty-four
hour post-transfection labeling of cellular proteins was carried
out with 35S-methionine for 2 h, after which cells were
harvested and cell extracts prepared as described earlier
(Subler et al., 1992). Immunoprecipitations were carried out
for 14 h at 48C using indicated monoclonal antibodies as
described earlier (Subler et al., 1992). PAb421 (Lane and
Crawford, 1979; Vojtesek et al., 1992) was used to bind p53
proteins with an intact C-terminus (intact p53-281G as well as
p53 del 1-293) and DO1 (Lane and Crawford, 1979; Vojtesek
et al., 1992) was used to detect p53 proteins with an intact
N-terminus (p53-281G but not p53 del 1-293).
Establishment of stable 10(3) cell lines expressing p53 del 1-293
(or p53-281G and p53 del 1-293)
Untransfected 10(3) cells (Dittmer et al., 1993) or 10(3) cells
stably expressing p53-281G (Lanyi et al., 1998) were
transfected with pCMVBam p53 del 1-293 and pCMVBam
neo (Hinds et al., 1990) or pCMVBam p53 del 1-293 and
pHyg (Clontech), respectively, using the calcium phosphate
precipitation technique (Subler et al., 1992). Forty-eight
hours after transfection, cells were washed, trypsinized,
subcultured at 1 : 4, and plated into selective media
supplemented with 200 mg/ml G-418 (active concentration)
for previously untransfected 10(3) cells and 200 mg/ml G-418
and 200 mg/ml hygromycin for p53-281G-expressing cells.
Cells were maintained in the selective media with twice
weekly changes of media. After 14 ± 21 days in selective
media, cells from individual antibiotic-resistant colonies were
isolated and expanded into cell lines. Western blot analyses
were used to detect p53 del 1-293 or p53 del 1-293 and p53281G expressed in these cell lines.
Immunoprecipitation followed by Western blot analysis to
determine in vivo complex formation between p53-281G
and p53 del 1-293
In vivo complex formation between p53-281G and p53 del 1293 in 10(3) cells stably transfected to express p53-281G and
p53 del 1-293 was done by immunoprecipitation of p53-281G
with an antibody (DO1) that recognizes a motif on the Nterminal part of the protein and is absent in p53 del 1-293.
We carried out the procedure following the method described
by Leng et al. (1995). Brie¯y, cells from a 10 cm dish were
harvested in 4 ml of 10 s bu€er (containing 250 mM HEPES,
pH 7.2, 1.5% NP40, 0.5% Triton X-100, 0.025% SDS,
50 mM sodium phosphate bu€er, pH 7.0, 5 mM NaF, 0.5 mM
phenyl methyl sulphonyl ¯uoride and 2.5 mM dithiothreitol)
as described. One ml of the extract was then incubated with 1
or 2 mg of p53 antibody DO1 and 16 ml of protein A agarose
(Calbiochem) and shaken overnight (head-to-tail) at 48C. The
following morning, the immunoprecipitate was washed three
times with 10 s bu€er and once with a bu€er containing
10 mM Tris pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate
and 0.5% Triton X-100. The pellet was then suspended in
20 ml of 26 Laemmlli loading bu€er, boiled and analysed by
15% SDS polyacrylamide gel electrophoresis, followed by
Western blot analysis using PAb421 as the probe.
Oncogene
Tumorigenicity assay
The tumorigenic potential of 10(3) cells expressing p53
derivatives was tested using nude mice. Cells (56106 per site)
were subcutaneously injected into ¯anks of athymic nude mice
(National Cancer Institute) and tumor development was
monitored as described (Dittmer et al., 1993; Lanyi et al., 1998).
Colony formation assay
10(3) cells stably transfected with the vector pCMVBam Neo
(Hinds et al., 1990) or stably expressing p53-281G (Lanyi et
al., 1998) in each 10 cm dish were transfected with 1 mg of
pHyg and 5 mg (1) pCMVBam (Subler et al., 1992), (2)
pCMVBam wild-type p53 (Subler et al., 1992), (3)
pCMVBam FLAG p73 a (Deb et al., 2001), (4) pCMVBam
FLAG p73 b Deb (Deb et al., 2001), or (5) pCMVBam p53
del 1-293 (Subler et al., 1994). Transfections were done by
calcium phosphate precipitation technique as described earlier
(Lanyi et al., 1998). After 48 h of transfection cells were
subcultured 1 : 4; hygromycin was added next day at 200 mg/
ml to start the selection process. Neomycin (G418 at 200 mg/
ml) was also added to those cells selected earlier for neomycin
resistance as described before (Lanyi et al., 1998). Every 2 ± 3
days media was changed. After 3 weeks, cells were ®xed with
methanol and stained with methylene blue.
Western blot analysis
Western blot analysis was carried out after SDS polyacrylamide gel electrophoresis using monoclonal antibodies against
p53 or FLAG and a commercial kit (Promega) for labeling
with an alkaline phosphatase-conjugated secondary antibody.
Manufacturer's protocol was followed for this procedure.
Oligomerization assays with in vitro-synthesized intact
p53-281G and its derivatives
Analyses for the presence of oligomeric forms of p53-281G
and its derivatives were carried out as described (Subler et al.,
1994). Brie¯y, human p53-281G and its derivatives were
generated by a coupled in vivo transcription-translation system
(Promega) and were incubated with glutaraldehyde at varying
®nal concentrations at 378C for 15 min. After cross-linking,
proteins were boiled in 26 Laemmli loading dye (Subler et al.,
1994), subjected to gradient (4 ± 20%) SDS-polyacrylamide gel
electrophoresis, and visualized by autoradiography.
RNA isolation and microarray hybridization analysis
Total RNA was isolated from 10(3) cells stably transfected
with vector alone [pCMVBamNeo (Lanyi et al., 1998)],
pCMVBamNeo and expression plasmid for p53-281G,
pCMVBamNeo and expression plasmid for p53 del 1-293,
and pCMVBamNeo and expression plasmids for p53-2981G
and p53 del 1-293 using Trizol (Gibco) reagent following
manufacturer's protocol. Quality of the RNAs was checked
by 1.2% agarose Tris-borate-EDTA gel electrophoresis.
Microarray analysis from RNA from 10(3) cells stably
transfected with (1) vector alone [pCMVBamNeo (Lanyi et
al., 1998)] and (2) pCMVBamNeo and expression plasmid
for p53-281G was done by Incyte Genomics, Inc. (Palo
Alto, CA, USA) starting from the whole RNA that we
sent. Brie¯y, polyadenylated RNA was isolated with the
OligoTex (Qiagen) RNA kit. Polyadenylated RNA was
reverse transcribed to generate Cy3- and Cy5-labeled cDNA
probes, respectively. cDNA probes were competitively
Mutant p53-mediated transactivation
D Deb et al
hybridized to a mouse Unigene 1 cDNA microarray (Incyte
Genomics, Inc.) containing immobilized cDNA fragments
(average cDNA length, 500 ± 5000 base pairs). Cy3 and Cy5
¯uorescence were imaged individually, and the normalized
ratios of Cy3/Cy5 ¯uorescence at a given spot on the
microarray were used to calculate di€erential gene expression.
Quantitative real-time PCR
Relative quantitation of gene expression was performed using
quantitative real-time PCR (QPCR). The relative amount of
each sample was calculated from a standard curve after
employing the Fit Points algorithm for quanti®cation. This
expression value was normalized to a housekeeping gene,
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
(Krause et al., 2000). A relative gene expression was
determined by assigning 10(3) V4 [10(3) cells stably
transfected with the vector pCMVBam Neo (Hinds et al.,
1990)] a relative value of 1.0, with all other values relative to
10(3)V4. This method is described in detail by Johnson et al.
(2000); Tanguay et al. (2000). Following is a brief description
of the entire process.
(1) cDNA synthesis Total RNA was isolated from 56106
mouse 10(3) cells stably transfected to express p53 281G,
clone 4, p53D1.291, clone 10, p53 281GD1-293, clone 22, and
vector (V4) alone using Trizol reagent (Gibco BRL,
Gaithersburg, MD, USA) following the manufacturer's
instructions. Ten micrograms of total RNA was treated with
RA1 Rnase-free DNase (Promega, Madison, WI, USA),
followed by phenol extraction, chloroform extraction and
ethanol precipitation. cDNA was synthesized with random
hexamer using the Thermoscript RT ± PCR System (Gibco
BRL) according to the manufacturer's protocol. cDNA
volume was increased twofold to decrease pipetting error.
(2) Primers Primers were designed using Oligo 5.0 Software
(National Biosciences, Inc, Plymouth, MN, USA) and were
synthesized by Integrated DNA Technologies. Primer
optimization was performed to determine the optimal
concentration of MgCl2 and annealing temperature for each
primer set.
(3) Relative standard curves Relative standard curves were
constructed for each target gene using previously generated
PCR products of the target. Serial dilutions were made, and
were given arbitrary values corresponding to the dilutions.
187
(4) Quantitative real-time PCR Quantitative real-time PCR
was performed using a LightCycler (Roche). Each reaction
(20 ml) contained 2 ml of the respective diluted cDNA,
primers (0.5 mM), and 4 mM MgCl2, and FastStart Reaction
Mix SYBR Green I, (Roche) which includes FastStart Taq
DNA polymerase, reaction bu€er, dNTPs, and SYBR Green
I dye. A negative control was included in each QPCR run.
Except for the composition of the standard curves, each
reaction was performed in triplicate. All samples were
treated with an initial denaturation step at 958C for
10 min to activate the enzyme and denature the cDNA.
Samples investigated with GAPD primers (F) 5'CCAGCCTCGTCCCGTAGACA-3'
and
(R)
5'GCCTCACCCCATTTGATGTTAGTG-3' underwent 40
cycles of the following: 958C for 15 s, 568C for 5 s, and
708C for 12 s. Samples checked with PCNA primers (F)
5'-GATAAAGAAGAGGAGGCGGTAA-3'
and
(R)
5'-ACACGCTGGCATCTCAGGAGCA-3' underwent 45
cycles of 958C for 15 s, 518C for 5 s, 728C for 12 s. NFkB2 samples (F) 5'-TGGCCCCTATCTGGTGATTGT-3'
and (R) 5'-ACACTCCCAACTCTGAACACTGCT-3' underwent 50 cycles of 958C for 15 s, 568C for 5 s, and 728C for
10 s. The presence of SYBR Green I, a non-speci®c dye
which binds to double-stranded DNA, can be detected by
¯uorescence at the end of each extension step. In this way
the synthesis of DNA is determined in real-time. Agarose gel
electrophoresis and melting curve analysis were used to
demonstrate the presence of a speci®c product.
Acknowledgments
This work was supported by grants from NIH to Sumitra
Deb (CA70712) and to Swati Palit Deb (CA74172). Part of
the work for this manuscript was done in the Department
of Microbiology, University of Texas Health Science
Center at San Antonio, San Antonio, Texas and Department of Cancer Biology, Wake Forest University School of
Medicine, Winston-Salem, North Carolina. We thank
Arnold Levine, Bert Vogelstein, Glenn Merlino, and
Thanos Halazonetis for providing us with plasmids and
cells.
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