Tài liệu Báo cáo khoa học: DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner pptx

DNA modification with cisplatin affects sequence-specific
DNA binding of p53 and p73 proteins in a target
site-dependent manner
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Hana Pivonkova1, Petr Pecinka1, Pavla Ceskova2 and Miroslav Fojta1
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
2 Masaryk Memorial Cancer Institute, Brno, Czech Republic

Keywords
cisplatin; DNA damage; protein p73;
sequence-specific DNA recognition; tumor
suppressor protein p53
Correspondence
M. Fojta, Institute of Biophysics, Academy
of Sciences of the Czech Republic,
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Kralovopolska 135, CZ-612 65 Brno,
Czech Republic
Fax: +420 541211293
Tel: +420 541517197
E-mail:
(Received 25 April 2006, revised 18 July
2006, accepted 17 August 2006)
doi:10.1111/j.1742-4658.2006.05472.x

Proteins p53 and p73 act as transcription factors in cell cycle control, regulation of cell development and ⁄ or in apoptotic pathways. Both proteins

bind to response elements (p53 DNA-binding sites), typically consisting of
two copies of a motif RRRCWWGYYY. It has been demonstrated previously that DNA modification with the antitumor drug cisplatin inhibits
p53 binding to a synthetic p53 DNA-binding site. Here we demonstrate
that the effects of global DNA modification with cisplatin on binding of
the p53 or p73 proteins to various p53 DNA-binding sites differed significantly, depending on the nucleotide sequence of the given target site. The
relative sensitivities of protein–DNA binding to cisplatin DNA treatment
correlated with the occurrence of sequence motifs forming stable bifunctional adducts with the drug (namely, GG and AG doublets) within the
target sites. Binding of both proteins to mutated p53 DNA-binding sites
from which these motifs had been eliminated was only negligibly affected
by cisplatin treatment, suggesting that formation of the cisplatin adducts
within the target sites was primarily responsible for inhibition of the p53 or
p73 sequence-specific DNA binding. Distinct effects of cisplatin DNA
modification on the recognition of different response elements by the p53
family proteins may have impacts on regulation pathways in cisplatintreated cells.

The tumor suppressor protein p53 is known as a transcription factor involved in cell cycle control [1–3]. It
plays a crucial role in preventing malignant transformation of a cell via induction of cell cycle arrest or
programmed cell death in response to stress conditions
(e.g. DNA damage). The functions of p53 are closely
related to sequence-specific recognition of response elements [p53 DNA-binding sites (p53DBSs)] in promoters of downstream genes such as p21WAF1 ⁄ CIP1
(involved in cell cycle arrest), Bax (apoptosis), and
mdm2 (negative feedback regulation of p53) [1–3].
Using chromatin immunoprecipitation combined with

a paired-end ditag DNA sequencing strategy, Wei
et al. have recently established a global map of p53binding sites encompassing over 540 loci in the human
genome [4]. A typical p53DBS consists of two tandem
copies of the motif RRRCWWGYYY (where R ¼ A
or G, Y ¼ C or T, and W ¼ A or T), which may be
separated by one or more base pairs [4,5]. Natural p53

response elements exhibit surprisingly high sequence
variability and may contain one or several nucleotides
not fitting the above formula [6,7]. The p53 protein
binds to the response elements as a tetramer via its
core domain. The importance of p53 sequence-specific

Abbreviations
cisPt-DNA, cisplatin-modified DNA; CTDBS, C-terminal DNA-binding site; EMSA, electrophoretic mobility shift assay; fl, full length;
IAC, intrastrand crosslink; oligo, oligonucleotide; p53DBS, p53 DNA-binding site.

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DNA binding is underlined by the fact that most of
the cancer-related point mutations of p53 are located
in its core domain and the mutants are typically
unable to recognize the p53 response elements [1,8,9].
Besides the nucleotide sequence, binding of p53 to
the p53DBSs appears to depend on conformational
features of its target sites. It has been proposed that
intrinsic bending of the p53DBSs contributes significantly to the stability of the p53–DNA complexes [10].
In addition, interactions of the p53 protein with certain response elements can be controlled by changes in

DNA topology inducing formation of non-B DNA
structures within the binding sites [11,12]. Interactions
of p53 with DNA are regulated mainly via post-translational modifications (phosphorylation, acetylation)
within the protein C-terminal domain [3,13,14]. Truncated forms of p53 lacking a negative-regulating segment at the protein C-terminus (residues 369–383 [15])
are constitutively active for sequence-specific DNA
binding [7,16]. On the other hand, the C-terminus of
p53 was shown to be critical for its conformationselective DNA binding [11,12,17,18] and to favor p53
interactions with p53DBSs within long DNA molecules
[19,20].
The p73 protein has been identified as a p53 homolog exhibiting 63% amino acid sequence identity in the
DNA-binding domain [21–23]. In agreement with this
homology, the p73 protein can recognize the same
response elements as the p53 protein and activate an
analogous set of downstream genes. Multiple splice
isoforms of the p73 protein have been found that differ
in the structure of their N-terminal and ⁄ or C-terminal
domains [21,22]. Although it was originally supposed
that the p53 homologs have redundant functions in the
regulation of gene expression, more recent data suggest
that p73 and p63 proteins do not act as ‘classic’ tumor
suppressors, but rather play important roles in the
regulation of cell development and differentiation
[21,23]. Nevertheless, some observations suggest that
p73 is involved in the cellular response to DNA damage and in apoptosis control [24,25].
Cisplatin [cis-diamminedichloroplatinum(II)] is a
clinically used anticancer agent [26,27]. The drug binds
covalently to DNA, forming several kinds of adduct,
among which the most abundant are intrastrand crosslinks (IACs) between neighboring purine residues.
The spectrum of cisplatin adducts identified in globally
modified chromosomal DNA comprises about 50%

of 1,2-GG IACs, 25% of 1,2-AG IACs, 10% of
1,3-GNG IACs and interstrand crosslinks, and another
2–3% of monofunctional adducts. It has been found
that cisplatin cytotoxicity is related mainly to the IACs
that induce significant changes in the DNA
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conformation, including bending and unwinding of the
DNA double helix [26,28]. The lesions are selectively
bound by a variety of nuclear proteins, and it was proposed that these interactions are important for the
anticancer activity of the drug [26,29,30].
Interactions of the p53 protein with cisplatin-modified DNA (cisPt-DNA) have recently been studied [31–
36]. In the absence of the p53DBS, enhancement of
p53 sequence-nonspecific DNA binding due to DNA
cis-platination was observed [33–36]. On the other
hand, the same DNA treatment resulted in inhibition
of p53 sequence-specific binding [31,32]. An analogous
inhibitory effect was observed with the anticancer trinuclear platinum complex BBR3464 but not with the
clinically ineffective transplatin. Quite recently, it has
been shown that DNA modification with a transplatin
analog,
trans-[PtCl2NH3(4-hydroxymethylpyridine)],
inhibits p53 binding to the same p53DBS similarly as
does cisplatin [37]. It has been proposed that the inhibitory effects of the anticancer platinum complexes are
due to the formation of platinum adducts within the
p53DBS [31,32]. To our knowledge, no analogous
studies of the p73 protein interactions with chemically
damaged DNA have been reported yet.
In this work, we investigated the effects of global
DNA modification with cisplatin on sequence-specific

binding of p53 and p73 proteins to different target
sites. We demonstrated that the sensitivity of the protein–DNA interactions to cisplatin DNA treatment
correlated with the occurrence of sequence motifs
forming the cisplatin IACs (namely GG and AG doublets) within the given p53DBS. Binding of both proteins to mutated target sites not containing these
motifs was not significantly affected by the DNA cisplatination. Formation of the cisplatin adducts outside
the p53DBSs did not apparently influence p53
sequence-specific DNA binding.

Results
To analyze the sequence-specific DNA binding of p53
and p73 proteins, we designed 50-mer oligonucleotide
substrates bearing various p53DBSs (Fig. 1). In most
experiments, we used a C-terminally truncated, constitutively active p53(1–363) to eliminate the sequencenonspecific p53 interactions with the cis-platinated
DNA, which have been shown to be mediated primarily by the p53 C-terminal DNA-binding site
(CTDBS) [34]. In the presence of competitor nonspecific DNA, sequence-specific binding of the p53(1–
363) protein to the 32P-labeled 50-mer targets resulted
in the appearance of a distinct retarded band R53 in
the polyacrylamide gel (Fig. 2). Binding of the p73b

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Fig. 1. Scheme of DNA substrates used in this work. All p53 DNA-binding sites (p53DBSs) were placed in the center of 50-mer oligonucleotides (oligos), being flanked with the sequences shown on the top (the same stretches flank the p53DBSs in the pPGM1 and pPGM4 plasmids). The left part of the scheme shows two p53DBSs derived from natural p53 response elements in p21 (5¢-promoter) and mdm2
promoters, as well as the synthetic p53DBS PGM1. Motifs forming bifunctional adducts with cisplatin are highlighted (GG doublets are in

bold and underlined, AG doublets are in bold, and GNG triplets are marked by brackets). The p53DBSs shown on the right are derivatives of
p21 (p21a and p21b) or pPGM1 (pPGM4). In the latter targets, the incidence of the cisplatin-reactive sites was reduced or eliminated. Bases
not fitting the ‘canonical’ p53DBS [5] are denoted by lower-case letters.

A

B

Fig. 2. Electrophoretic mobility shift assay of sequence-specific binding of p53 or p73 proteins to a 50-mer oligonucleotide (oligo) involving
the p53 DNA-binding sites (p53DBSs). (A) The 32P-labeled p21 target was incubated with the given protein in presence of competitor calf
thymus DNA, and this was followed by electrophoresis on 5% polyacrylamide gel. Lane 1 contains only DNA without any protein; lanes 2, 3
and 4 correspond to DNA complexes with p53(1–363), p73d and p73b, yielding retarded bands R53, R73d and R73b, respectively. In lanes 5–7,
the protein–DNA complexes are supershifted with monoclonal antibodies DO-1 (p53) or anti-HA (both p73 isoforms; the respective supershifted bands are denoted as SR53, SR73d and SR73b; the presence of two supershifted bands in each of the lanes 5–7 corresponds to two
possible stoichiometries of the antibody–protein complexes). (B) Sections of an autoradiogram showing retarded bands due to binding of
p53(1–363) or p73d proteins to 50-mer target oligos containing PGM1, PGM4, mdm2, p21, p21a and p21b sites. Other details as in (A), lanes
2 and 3.

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and p73d proteins to the DNA targets caused the formation of analogous retarded bands (denoted as R73b
or R73d, respectively; lanes 3 and 4 in Fig. 2A) whose

mobilities reflected different molecular weights of the
p73 isoforms. To verify the specificity of the band
shifts for DNA complexes with the proteins studied,
we used the band supershift assay with antibodies
against the p53 or p73 proteins. Addition of the DO1 antibody [17,38,39] mapping to the N-terminus of
the p53 protein resulted in further retardation of the
specific p53–DNA complexes (lane 5 in Fig. 2A),
producing two supershifted bands (SR53; Fig. 2A).
Formation of the two bands corresponded to two
possible stoichiometries of the antibody–p53 complex,
involving either one or two antibody molecules bound
per p53 tetramer [16,39]. For supershifting of DNA
complexes with the p73 constructs, which were tagged
with hemagglutinin (HA), we used antibody to HA
and obtained analogous band patterns to those
obtained with p53 (Fig. 2; lanes 6–7, bands SR73b
and SR73d), confirming the specificity of the observed
protein–DNA complexes. All 50-mer substrates used
in this work were efficiently bound by the p53 and
p73 proteins [shown in Fig. 2B for p53(1–363) and
p73d], although their affinities for the proteins differed to some extent (which was manifested by different intensities of the R bands). To eliminate these
differences, the effects of DNA cis-platination on the
protein–DNA interactions were always normalized
with the intensity of the retarded band resulting from
protein binding to the same but unmodified p53DBS.
Effects of cisplatin DNA modification on
sequence-specific binding of the p53 protein
Previously, it has been shown [31,32] that DNA modification with cisplatin causes dose-dependent inhibition
of the full-length (fl) p53 sequence-specific DNA binding to the synthetic target site PGM1 (Fig. 1). Here,
we studied the effects of DNA treatment with cisplatin

on p53(1–363) binding to the p53DBSs PGM1, p21
and mdm2 (Fig. 1) within the 50-mer oligonucleotides
(oligos) (Fig. 3A). All targets were treated with the
drug in excess of nonspecific calf thymus DNA. Interaction of the protein with any of these targets was significantly affected by the cisplatin treatment, but the
levels of inhibition observed with individual p53DBSs
at the same degree of global DNA cis-platination differed significantly. The steepest decrease in p53–DNA
binding with degree of DNA modification was exhibited by the mdm2 target. The R53 band due to the
p53–mdm2 complex exhibited only 10% intensity for
rb ¼ 0.02, compared to the R53 band due to protein
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binding to the same but unmodified substrate (the rb
value refers to the number of platinum atoms per total
DNA nucleotide). In contrast, the PGM1 and p21 targets retained 75% and 53% of the p53-binding capacity at rb ¼ 0.02, respectively (Fig. 3A). Increasing the
DNA modification degree to rb ¼ 0.04 resulted in a
decrease of p53–p21 binding to 42%, whereas the
PGM1 site bound only 16% of the protein, compared
to the same but unmodified p53DBS. At rb ¼ 0.06, all
mdm2, PGM1 and p21 targets exhibited very weak
p53 binding (about 4% for mdm2 and PGM1 and
10% for p21).
Sensitivity of the sequence-specific p53 DNA
binding to DNA cis-platination depends on the
incidence of cisplatin-reactive motifs within the
p53DBSs
The mdm2, PGM1 and p21 target sites (Fig. 1) differ
significantly in the occurrence of sequence motifs
known to form the cisplatin IACs [26,27]. The p21 site,
showing the weakest sensitivity of p53 binding to cisplatin treatment, contains only one GGG triplet within
the p53DBS. The PGM1 site possesses two AGG triplets in one strand and two AG steps in the other. The

mdm2 target, whose interaction with p53 was most
strongly affected by DNA cis-platination, contains
GG, GGG and AG motifs in one strand and GG and
GTG motifs in the other, thus offering not only the
highest total number of reactive motifs among the
p53DBSs tested, but also the highest number of sites
known to be modified most frequently (i.e. the GG
doublets).
For the subsequent experiments, we designed
mutated p53 target sites from which the cisplatin-reactive motifs were eliminated. Two p53DBSs were derived
from the p21 target site (Fig. 1); in p21a, the GGG
triplet in the bottom strand was mutated into GAG.
This exchange resulted in elimination of the most
reactive GG doublets and the introduction of less
reactive AG and ⁄ or GNG motifs [26]. In p21b, the
GGG triplet in the bottom strand was replaced by
GAA, which contains neither RG nor GNG motifs
(Fig. 1); owing to this mutation, all sites suitable for
formation of the bifunctional cisplatin adducts were
removed from the p53DBS. In addition, we derived
another ‘unreactive’ p53DBS from the PGM1 target
(PGM4; Fig. 1) by replacing all guanine residues,
except for those at the strictly conserved positions
[4,5], by adenines. All of these mutated p53DBSs
(when cisplatin-unmodified) exhibited sequence-specific
p53 binding comparable to that of the parent targets
(Fig. 2B).

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A

Cisplatin effects on p53 ⁄ p73 DNA recognition

B

Fig. 3. Effects of DNA modification with cisplatin on p53(1–363) binding to various target sites: (A), natural p53 DNA-binding sites (p53DBSs)
mdm2 and p21, and the synthetic PGM1 sequence; (B) mutated p53DBSs PGM4, p21a and p21b (Fig. 1). The top panels show sections of
autoradiograms showing the R53 bands corresponding to complexes of p53 with the 50-mer target oligonucleotides (oligos) (Fig. 2). The
extents of DNA modification with cisplatin (rb) are indicated. Other details are as in Fig. 2. The graphs show the dependence of relative p53
binding to the targets on the degree of DNA modification (data obtained from densitometric tracing of the autoradiograms; for each target
site, the intensity of the R53 band resulting from p53 binding to unmodified DNA was taken as 1.0, and the intensities of bands corresponding to p53 binding to the same but cisplatin-treated substrate were normalized to this).

We studied how the cisplatin treatment influences
interaction of the p53(1–363) protein with the
mutated target sites. The 50-mer oligos containing
sequences p21a, p21b or PGM4 were treated with
cisplatin as above. DNA modification to rb ¼ 0.02
resulted in a decrease of p53 binding to the p21a target by about 15%, which represented weaker inhibition than observed with the p21 target (25% decrease;
Fig. 3). More conspicuous differences between the
p21a and p21 targets appeared at rb ¼ 0.04 (35% or
58% inhibition, respectively). At rb ¼ 0.06, the p21a
target retained 45% of the p53 binding, thus exhibiting at least four times higher binding capacity than
the natural p21 p53DBS treated in the same way.
Binding of p53 to the mutated target p21b exhibited

even more remarkable resistance to the cisplatin treatment. For rb values of 0.02, 0.04 or 0.06, 100%, 91%
or 85% of the p21b target was bound by the protein,
respectively, when compared to the untreated p21b.

The behavior of the PGM4 site was similar to that of
p21b, showing practically no inhibition of p53–PGM4
binding for rb ¼ 0.02 or 0.04 and about 10% inhibition for rb ¼ 0.06. The PGM4 site also exhibited
practically no loss of its p53-binding capacity due to
the DNA cis-platination when located within a
474 base pair fragment of the pPGM4 plasmid (not
shown), in contrast to the behavior of the analogous
pPGM1 fragment [31]. These data revealed a clear
correlation between the sensitivity of the p53
sequence-specific DNA binding to DNA treatment
with cisplatin and the ability of the particular p53
target site to accommodate the cisplatin IACs. The
higher the probability of formation of the cisplatin
IACs within the p53DBSs due to the occurrence of
the GG, AG and ⁄ or GNG motifs, the stronger the
inhibition of the p53 sequence-specific DNA binding
to these targets caused by the DNA treatment with
cisplatin.

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We also performed parallel experiments with fl p53
(expressed in insect cells). Like p53(1–363), the fl protein was able to recognize sequence specifically all of
the targets tested (not shown). The effects of the
degree of DNA cis-platination on recognition of the
particular target site by the fl p53 were analogous to
those observed with the C-terminally truncated p53(1–
363) (shown in Fig. 4 shown for the mdm2, PGM1
and PGM4 targets).
Effects of DNA cis-platination on sequencespecific DNA binding of p73 proteins
We tested the influence of DNA modification with
cisplatin on the binding of two p73 isoforms, p73d and
p73b, to the p21 target site (Fig. 5). The intensity of
the resulting R73d band decreased almost linearly with
increase in the cis-platination level; for rb ¼ 0.06,
about 90% inhibition of p73d–p21 binding was
observed. Almost the same results were obtained when
interaction of the p73b isoform with the p21 target
was examined (Fig. 5). In contrast, modification of
p21b to rb ¼ 0.02 or 0.04 had no significant effect on
its interaction with either of the p73 isoforms; at rb ¼
0.06, only slight (10–15%) inhibition of binding was
detected. Thus, the p73d and p73b proteins exhibited
behavior upon binding to cisplatin-treated p21 and
p21b target sites that was very close to that of the p53
protein. Analogous results were obtained with the
PGM1 and PGM4 target sites (not shown).

Competition experiments
We studied the influence of cisplatin DNA modification on the competition between two p53 target sites
for the protein (Fig. 6). The 474 base pair fragments

Fig. 4. Effects of DNA modification with cisplatin on full-length p53
binding to the mdm2, PGM1 and PGM4 targets. For more details,
see Figs 2 and 3.

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of plasmids pPGM1 or pPGM4 were used as the
sequence-specific competitors, and changes in p53(1–
363) binding to the 32P-labeled 50-mer targets were followed. We first tested the effect of the presence of the
competitor fragments (unmodified or treated with cisplatin) on p53 binding to the unmodified PGM1 probe
(Fig. 6A). Addition of either of the unmodified fragments (70 ng per sample) resulted in a partial decrease
(by 35–45%) of the R53 band intensities due to binding
of a portion of the p53 molecule to the competitor
p53DBS. Modification of the pPGM1 fragment with
cisplatin caused a reduction of its competitiveness,
which was manifested by increasing relative intensity
of the R53 band yielded by the p53 complex with the
radiolabeled PGM1 probe. When the pPGM1 fragment was cis-platinated to rb ¼ 0.04 or 0.06, its presence had practically no effect on the R53 band
intensity, suggesting that the modified pPGM1 fragment had lost its ability to compete for the protein
(Fig. 6A). In contrast, the competition ability of the
pPGM4 fragment was not significantly influenced by
its cis-platination. This observation was in agreement
with the resistance of p53 binding to the PGM4 target
site to the cisplatin DNA treatment (see above).
In addition, we modified with cisplatin equimolar
mixtures of the pPGM1 fragment with the 32P-labeled

50-mer targets p21 or p21b (in the presence of nonspecific competitor DNA), and performed a p53-binding
assay (Fig. 6B). In the unmodified DNA, the competitor pPGM1 fragment caused about 70% inhibition of
p53(1–363) binding to either of the two targets. Modification of the p21 ⁄ pPGM1 mixture resulted in
rb-dependent inhibition of p53 binding to the p21 target, but in contrast to the results shown in Fig. 3
(where only the p21 target and nonspecific competitor
DNA were present in the sample), the cisplatin inhibition effect was detectable only at rb ¼ 0.04 and 0.06.
The apparent lack of the cisplatin effect at rb ¼ 0.02
can be attributed to partial loss of the competitiveness
of the pPGM1 fragment due to its modification, which
compensated for inhibition of p53 binding to the (relatively less reactive) p21 target. When the mixture of
the p21b target with the pPGM1 competitor fragment
was treated with cisplatin in the same way, the intensity of the R53 band on the autoradiogram increased
with the degree of DNA modification. The increase
was already significant at rb ¼ 0.02. At rb ¼ 0.04 or
0.06, the relative intensity of the R53 band reached
about 90% of the value observed with unmodified
DNA (Fig. 6B). Such behavior reflected inhibition of
p53 binding to the competitor pPGM1 fragment due
to its cis-platination, whereas interaction of the protein
with the p21b target remained practically unaffected

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of the protein than for its ‘activated’ forms [33].
Recently, it has been reported that accessibility of the
p53 CTDBS is critical for (sequence-nonspecific) cisPtDNA recognition [34]. On the other hand, sequencespecific binding of p53 to the synthetic p53DBS PGM1
was inhibited in cisplatin-treated DNA [31,32]. As the
PGM1 site contains several sequence motifs known to
form the most abundant cisplatin adducts (see Fig. 1),
the cisplatin inhibitory effects could be explained by
DNA damage within the p53DBS. It is known that the
cisplatin IACs induce considerable DNA bending and
untwisting as well as perturbation of hydrogen bonding within the base pairs [26–28]. Cisplatin adducts
occurring within p53DBS can therefore be expected to
cause severe deformations of the binding site with concomitant destabilization of the p53–DNA interaction
(or even prevention of target recognition by the protein).
DNA binding of the C-terminally truncated
p53(1–363) protein

Fig. 5. The effects of DNA modification with cisplatin on binding of
the p73 proteins to the p21 and p21b target sites. In the graph,
squares correspond to p73b and triangles to p73d. For other details,
see Figs 2 and 3.

under the same conditions. The results of these model
competition experiments suggest that global modification of DNA with cisplatin may shift the distribution
of the p53 protein among different target sites, depending on the susceptibility of the particular p53DBSs to
modification with the drug.

Discussion
It has been demonstrated previously that interactions
of the tumor suppressor protein p53 with DNA are
influenced by covalent modification of the DNA by

antitumor platinum complexes [31–36]. Sequence-nonspecific DNA binding (in the absence of the p53DBS)
of the p53 protein was significantly enhanced by DNA
modification with cisplatin [31,33,34]. The ability of
p53 to recognize the cisPt-DNA was more pronounced
for the post-translationally unmodified (‘latent’) form

In this work, we studied the effects of cisplatin treatment of various p53DBSs on the sequence-specific
binding of a truncated tetrameric p53 construct lacking
the C-terminal DNA-binding site, p53(1–363) [18,34].
This variant of the protein is known to be constitutively active for sequence-specific DNA binding [16].
Models of p53 latency considering the (post-translationally unmodified) p53 C-terminus solely as a negative regulator of sequence-specific DNA binding [40,41]
have recently been questioned [42–44]. Instead, the p53
CTDBS has been proposed to cooperate with the core
domain in complex p53–DNA interactions. The
CTDBS has been shown to be essential for p53 binding to target sites adopting non-B conformations (such
as stem–loop or cruciform structures) [11,12,45–47].
On the other hand, p53 constructs lacking the CTDBS
are capable of efficient binding to short linear model
DNA targets in which the p53DBS is present in its
double-helical B-form. Moreover, deletion of the
CTDBS (amino acids 363–382) makes it possible to
separate sequence-specific p53 DNA binding from
other modes of p53–DNA interaction that are mediated by the protein C-terminus, particularly the
sequence-nonspecific binding of p53 preferentially to
cisPt-DNA [33,34]. Another CTDBS-lacking tetrameric
p53 construct, p53CT (spanning amino acids 94–360),
has recently been used by Weinberg et al. for evaluation of the protein-binding affinities for 20 natural p53
recognition elements [7]. A comparative study involving four of them showed practically the same cooperative binding of the fl p53 as exhibited by p53CT [7].

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A

B

Likewise, our parallel experiments with fl p53 yielded
results that were very similar to those obtained with
p53(1–363) (Figs 3 and 4). Hence, the C-terminally
truncated constructs are suitable models for comparative studies of p53 sequence-specific DNA binding to
various and ⁄ or variously modified target sites.
Inhibition of p53 sequence-specific DNA binding
is linked to cisplatin adduct formation within the
p53DBSs but not outside these target sites
The 50-mer target DNA substrates were treated with
the drug in the presence of an excess of nonspecific
competitor calf thymus DNA mimicking randomsequence natural genetic material that can accommodate the cisplatin adducts regardless of the reactivity
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Fig. 6. Competition between two different
p53 target sites in globally cisplatin-modified
DNA for the p53(1–363) protein. In (A),

32
P-labeled, unmodified PGM1 50-mer was
mixed with cisplatin-treated competitor fragments of plasmids pPGM1 or pPGM4 (and
with unmodified calf thymus DNA) prior to
addition of the p53 protein. When the competitor fragment was unmodified, the p53
protein was distributed between it and the
labeled probe target (a). Upon cis-platination
of the pPGM1 competitor fragment (b), its
affinity for the protein was decreased due
to formation of cisplatin adducts within the
p53 DNA-binding site (p53DBS), resulting in
increased p53 binding to the labeled probe.
The pPGM4 fragment (c) contains cisplatinresistant p53DBS, and its cis-platination did
not change its competitiveness for p53(1–
363). In (B), the 32P-labeled targets p21 (i)
and p21b (ii) were treated with cisplatin
together with the competitor pPGM1 fragment, and this was followed by the p53binding assay. Such treatment resulted in a
decrease of p53 binding to the p21 target
[in agreement with formation of the adducts
within both p21 and pPGM1 p53DBSs; see
(i)]. In contrast, apparent p53 binding to the
p21b target increased under the same conditions [because the cisplatin adducts were
formed within p53DBS of the competitor
but not within the p21b target; see (ii)]. The
graphs show the relative binding of p53 to
the radiolabeled targets as a function of rb;
the intensities of the R53 bands observed
for the unmodified targets in the absence of
the competitor fragments (first samples of
each set) were taken as 1. For other details,

see Figs 2 and 3.

of the particular p53DBS. The frequency of DNA
modification within the p53DBSs could thus be expected to reflect the known distribution of cisplatin
adducts in globally modified chromosomal (genomic)
DNA [26]. Provided that the cisplatin inhibitory effect
on p53 sequence-specific DNA binding is linked primarily to the IACs formed within the target sites, the
susceptibility of different targets to the drug treatment
should correlate markedly with the incidence of the
cisplatin-reactive motifs in the p53DBSs. Such a correlation was indeed found: the sensitivity of the target
sites to treatment with the drug followed the trend
mdm2 > PGM1 > p21 > p21a > p21b  PGM4, in
accordance with the number and kind of motifs suitable for formation of the IACs inside the p53DBSs
(Fig. 1).

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H. Pivonkova et al.

In the p21 50-mer target and its derivatives p21a
and p21b, the 5¢-neighboring guanines in the ‘top’
strand form another GG doublet with the first guanine
of the p53DBS (Fig. 1). Interestingly, the presence of
this reactive motif had no conspicuous effect on p53–
p21b binding in the cisplatin-treated DNA, as there
were no significant differences between the behavior of
p21b and that of PGM4 (lacking this boundary GG

doublet; Fig. 1). The results presented in this article do
not make it possible to decide whether a single cisplatin IAC, wherever it is within the p53DBS, can fully
abrogate p53 sequence-specific DNA binding, or whether the protein can recognize such a cis-platinated
site, albeit with lower affinity. Nevertheless, our data
show clearly that a single reactive motif located within
the 20 base pair recognition element (e.g. in p21;
Fig. 1) caused significant sensitivity of p53–p53DBS
binding to the DNA treatment with cisplatin, whereas
the presence of an overlapping GG doublet formed by
one guanine inside and the other outside the p53DBS
was practically without effect.
Under the conditions used in this work, the apparent sensitivity of p53 (or p73) DNA binding to cis-platination was influenced primarily by the probability of
adduct formation within the target sites, regardless of
the positions of the cisplatin adducts. The adduct positioning may be nevertheless be important with respect
to the stereochemistry of the protein–DNA recognition
(cis-platination induces significant bending and torsional deformations of the DNA double helix [28]) and
the availability of functional groups ensuring the essential protein–DNA contacts. For example, formation of
the cisplatin crosslinks within the CWWG box (which
represents an area where the p53 Arg248 residue interacts with the DNA via a minor groove [48]) might be
particularly critical. The mdm2 site is the only
p53DBS analyzed in this work that involves an AG
doublet within the CWWG tetramer (Fig. 1), which
may contribute to its high sensitivity to the cisplatin
treatment. We tested this possibility using another
p53DBS containing a single AG doublet (and no other
reactive motif) derived from PGM4 by inverting the
TA pair at position 6. Inhibition of p53(1–363) binding
to this site due to its treatment with cisplatin did not
exceed the effect observed with the p21a site (also
involving a single AG motif but outside the CWWG

box), suggesting that the highest sensitivity of the
mdm2 site towards cis-platination was connected with
the abundance of the highly reactive GG motifs rather
than with the location of the AG doublet within the
CWWG tetranucleotide. On the other hand, our preliminary results (M. Fojta et al., unpublished data)
suggest that the behavior of cisplatin-treated target

Cisplatin effects on p53 ⁄ p73 DNA recognition

sites possessing a single GG motif at various positions
may differ significantly (more details will be published
elsewhere).
Altered sequence-nonspecific interactions of the p53
protein with DNA due to its cis-platination outside the
p53DBSs might, in principle, influence recognition of
the target sites by the protein. Nevertheless, control
tests of binding of the p53(1–363) protein to unmodified PGM1, PGM4, p21 and p21b targets in the presence of unmodified or cis-platinated (rb ¼ 0.06) calf
thymus competitor DNA revealed no apparent effect
of the competitor modification. This observation was
in agreement with the recently reported lack of ability
of p53(1–363) to recognize the nonspecific cisPt-DNA
[34]. Furthermore, we were interested in whether the
presence of cisplatin adducts within DNA stretches
flanking the p53DBSs affects the ability of p53 to bind
the specific sequence. The flanking segments in all 50mer substrates used in this work (Fig. 1) contain three
motifs expected to form the 1,2-IACs (one GG and
two AGs). Another two sets of 50-mer substrates, in
which the PGM1 or PGM4 sites were flanked by segments either totally lacking the cisplatin-reactive motifs
or containing multiple guanine doublets and ⁄ or triplets
(Fig. 7), were used to check the influence of cisplatin

adducts in the vicinity of p53DBS. Again, the effects
of DNA cis-platination on p53(1–363) binding to these
substrates were dependent on the presence of cisplatinreactive motifs within the p53DBS but not within the
flanking stretches (Fig. 7), suggesting that cisplatin
adducts outside the binding site (albeit close to it) do
not significantly affect sequence-specific DNA recognition. However, it should be emphasized that such
conclusions need not be applicable to the posttranslationally unmodified form of fl p53, which
exhibits apparently weaker binding to p53DBS but significant sequence-nonspecific preferential binding to
globally cis-platinated DNA [31,33,34].
Binding of p73 proteins to the recognition
elements is affected by DNA cis-platination in a
similar way to p53 binding
In agreement with the considerable homology between
the p53 and p73 DNA-binding (core) domains, the p73
protein can bind to the p53 response elements [21,22].
Among the known p73 splice isoforms [21,23], p73d
(coded by exons 2–10 of the p73 gene) is most similar
to the p53 protein with regard to the protein domain
structure as well as molecular size. The p73b isoform
differs from p73d in its C-terminal domain, which,
in p73b, is extended by a stretch coded by exons 11
and 12. In neither of the p73 isoforms has another

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H. Pivonkova et al.

Cisplatin effects on p53 ⁄ p73 DNA recognition

Fig. 7. The effect of DNA modification with
cisplatin on p53(1–363) binding to p53 DNAbinding sites (p53DBSs) flanked by stretches either totally lacking sites reactive to
cisplatin (PGM1-AT, PGM4-AT) or involving
multiple reactive motifs (PGM1-GC,
PGM4-GC). The flanking stretches are
shown at the top; for the p53DBS,s see
Fig. 1. The experimental conditions are as in
Figs 2 and 3.

DNA-binding site (besides the core domain) analogous
to the p53 CTDBS been identified. Our results showed
that both p73d and p73b bound efficiently to all
(unmodified) p53DBSs used in this work, and that
cisplatin treatment of p21, p21b (Fig. 5), PGM1 and
PGM4 (not shown) affected the p73 sequence-specific
DNA binding basically in the same manner as
observed with p53.
Possible impacts on gene expression
in cisplatin-treated cells
It has been well established that modification of DNA
with cisplatin affects fundamental processes such as
DNA synthesis and transcription [26]. The bifunctional
cisplatin DNA adducts slow down or block DNA or
RNA polymerization and can hamper the initiation of
DNA transcription [49]. Strong differential inhibition
of marker gene expression was observed in cells treated

with cisplatin [50]. Interestingly, expression of genes
with stronger promoters was strongly inhibited,
whereas some genes possessing weaker promoters were
induced. It was proposed that the strong promoters
were associated with accessible chromatin and therefore more easily modified by the drug [50]. However,
to our knowledge, no systematic study of the sensitivity of various promoters (and particularly those controlled by the p53 family proteins), differing in the
occurrence of the cisplatin-reactive nucleotide sequence
motifs, to cisplatin treatment has been conducted to
date.
In response to genotoxic stress, the wild-type p53
can activate two different response pathways with
quite different impacts on the fate of the cell. The first
4702

involves cell cycle arrest via p21WAF1 ⁄ CIP1 induction
and activation of DNA repair processes that, in general, confer chemoresistance to cancer cells. The other
pathway leads to programmed cell death through activation of proapoptotic genes such as Bax, PUMA and
Noxa [1–3]. The apoptosis trigger is the desired event
in cancer therapy. Despite considerable recent progress
in understanding the functions of p53 and its homologs, it has not yet been clarified how the checkpoint
proteins decide which pathway to activate. Particularly, no unambiguous correlation between wild-type
p53 expression and cancer cell susceptibility to cisplatin-induced apoptosis has been established. Although
some authors reported a clear p53-dependent apoptotic response to cisplatin [51–54], other investigations
revealed a less distinct link between p53 status and cell
sensitivity to cisplatin, or even suggested opposite
effects [55–57]. Several observations suggest that apoptosis in cisplatin-treated cells may be regulated via
p53- and ⁄ or p73-dependent or -independent pathways
[24,56,58]. Hence, the response of a cancer cell to cisplatin seems to be rather complex, and its relationship
to the status of the p53 family proteins does not
appear to be straightforward.

The results of our in vitro binding experiments suggest that the expression of various p53 downstream
genes might be differentially affected in the cisplatintreated cells, due to different susceptibilities of the
p53 response elements to modification with the drug.
The natural p53DBSs [6,7] differ significantly in this
respect. Among the 20 response elements recently
characterized by Weinberg et al. [7], GADD45 (a gene
taking part in DNA repair ) no GG, three AG
doublets) and the p21 5¢-site (a single GGG triplet)

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H. Pivonkova et al.

Cisplatin effects on p53 ⁄ p73 DNA recognition

appear to be the most ‘cisplatin-resistant’, whereas
the most ‘reactive’ are the two apoptosis-related
response elements Noxa and PUMA BS2 (four GG
and two AG, or five GG and one AG, respectively).
Global DNA cis-platination may shift the distribution
of p53 protein molecules bound to various p53DBSs
towards those less susceptible to the modification
(Fig. 6). However, promoters of functionally related
genes (arrest ⁄ repair or apoptosis) do not generally
tend to cluster along the scale of potential reactivity
to cisplatin [6,7]. Expression of the p53 downstream
effectors in cisplatin-treated cells may thus be entirely

unbalanced rather than shifted towards one of the
response pathways. The generally unpredictable cellular response to cisplatin treatment can be connected
with the facts that modification of particular sites in
the genome of living cells is naturally stochastic on
the one hand, and influenced by the actual structural
and functional state of the given chromatin domain
on the other. Moreover, some of the p53 downstream
genes possess more than one p53 response element,
and these may differ in their susceptibilities to cis-platination (for example, the second p21 site involves
one GG and four AG internal motifs [7]). Cellular
response pathways involve many factors whose functions are differentially affected by DNA damage, and
regulation of the tumor suppressor protein activity
itself can also be influenced by genomic DNA cis-platination, due to preferential binding of the post-translationally unmodified (‘latent’) form of p53 to the
cisPt-DNA in the absence of the p53DBS [33–35].
Inhibition of sequence-specific p53 (or p73) protein
DNA binding due to formation of the cisplatin
adducts within its response elements could thus represent an important, but not the only, factor affecting
cellular regulation pathways in cells exposed to the
drug.

Genomics and nucleotide triphosphates by Sigma (St Louis,
MO, USA).

Experimental procedures

DNA-binding assays

DNA samples
Synthetic 50-mer oligonucleotides containing different
p53DBSs (Fig. 1) were supplied by VBC Genomics

(Vienna, Austria). Plasmids pPGM1 and pPGM4 [derivatives of pBSK(+) vector containing the PGM1 and PGM4
sites; Fig. 1] were prepared as previously described [17,33].
The 474 bp fragments of pPGM1 and pPGM4 (delimited
by PvuII restriction sites) were prepared using PCR and
purified with the Qiagen PCR Purification kit (Qiagen, Hilden, Germany). Restriction endonucleases were supplied by
Takara (Otsu, Japan), thermostable Pfu DNA polymerase
by Promega (Madison, WI, USA), PCR primers by VBC

DNA modification with cisplatin
DNA samples were incubated with cisplatin (Sigma) in
10 mm NaClO4 at 37 °C for 48 h in the dark. Radioactively
(32P) labeled 50-mer substrates (10 lgỈmL)1 in the reaction
mixture) were modified in the presence of an excess of nonspecific calf thymus DNA (400 lgỈmL)1) with 27, 54 or
81 lm cisplatin. The competitor fragments of plasmids
pPGM1 or pPGM4 (35 lgỈmL)1) were treated in the
absence of the calf thymus DNA (the fragments themselves
contain random-sequence stretches representing major parts
of the DNA molecules), and cisplatin concentrations of 2.3,
4.6 or 6.9 lm were applied to maintain the cisplatin ⁄ nucleotide ratios and thus the levels of global DNA modification.
The number of cisplatin moieties bound per DNA nucleotide (rb) under controlled conditions was previously determined by flameless atomic absorption spectroscopy or
polarographically [31,59]. The rb values attained under the
conditions used in this work were 0.02, 0.04 or 0.06.

Preparation of p53 and p73 proteins
C-terminally truncated p53(1–363) and fl p53 were
expressed in bacterial Escherichia coli BL21 ⁄ DE3 cells or in
insect Sf9 cells, respectively, purified and characterized as
described previously [18,33]. Proteins p73b and p73d were
prepared using the TNTÒ Quick Coupled Transcription ⁄ Translation System (Promega). Plasmids pcDNA3HA-p73b or pcDNA3-HA-p73d (1 lg) coding the respective
p73 isoforms (both HA-tagged at their N-termini) were

mixed with 1 lL of 1 mm methionine, 40 lL of TNTÒ T7
Quick Master Mix and nuclease-free water to a final volume of 50 lL. Samples were incubated at 30 °C for 90 min.
Protein concentrations were determined densitometrically
from Coomassie blue G-250-stained polyacrylamide gels.

In all experiments, the p53 or p73 proteins were mixed with
the DNA substrates in 2 mm dithiothreitol, 50 mm KCl,
5 mm Tris (pH 7.6) and 0.01% Triton X-100 (total volume
20 lL) and incubated on ice for 30 min. The protein ⁄ DNA
target site molar ratio (i.e. protein tetramers per radiolabeled 50-mer probe) was 5 ⁄ 1. The reaction mixture contained
50 ng of the 32P-labeled oligo and 2 lg of nonspecific competitor calf thymus DNA. After the incubation period, the
protein–DNA complexes were analyzed by electrophoretic
mobility shift assay (EMSA) in 5% native polyacrylamide
gel containing 30 mm Tris, 30 mm H3BO3, 0.7 mm EDTA
buffer (pH 8.0) at 4 °C and 120 V for 3 h. The specificity
of the protein–DNA complexes was checked by a band

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Cisplatin effects on p53 ⁄ p73 DNA recognition

supershifting assay with antibodies DO-1 [38] or anti-HA
(Sigma). Gels were dried and autoradiographed using

Phosphorimager Storm. Band intensities on the gels were
quantified with image-quant software. Average values and
standard errors shown in the graphs were calculated from
three experiments.

Acknowledgements
ˇ
The authors thank Dr Borˇ ek Vojtesˇ ek for providing the
´
´
monoclonal antibodies, Dr Marie Brazdova for her help
with preparation of the p53 proteins, and Professor G.
Melino for donation of plasmids pcDNA3-HA-p73b
and pcDNA3-HA-p73d. This work was supported by
IGA MH CR grant No. NC ⁄ 7574-3 and partly by
GACR grant 301 ⁄ 05 ⁄ 0416. Personnel costs were partly
covered from the research plan No. AVOZ50040507.

References
1 Vousden KH & Prives C (2005) P53 and prognosis: new
insights and further complexity. Cell 120, 7–10.
2 El-Deiry WS (2003) The role of p53 in chemosensitivity
and radiosensitivity. Oncogene 22, 7486–7495.
3 Appella E & Anderson CW (2001) Post-translational
modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268, 2764–2772.
4 Wei CL, Wu Q, Vega VB, Chiu KP, Ng P, Zhang T,
Shahab A, Yong HC, Fu Y, Weng Z et al. (2006) A
global map of p53 transcription-factor binding sites in
the human genome. Cell 124, 207–219.
5 El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW &

Vogelstein B (1992) Definition of a consensus binding
site for p53. Nat Genet 1, 45–49.
6 Qian H, Wang T, Naumovski L, Lopez CD &
Brachmann RK (2002) Groups of p53 target genes
involved in specific p53 downstream effects cluster into
different classes of DNA binding sites. Oncogene 21,
7901–7911.
7 Weinberg RL, Veprintsev DB, Bycroft M & Fersht AR
(2005) Comparative binding of p53 to its promoter and
DNA recognition elements. J Mol Biol 348, 589–596.
8 Muller P, Ceskova P & Vojtesek B (2005) Hsp90 is
essential for restoring cellular functions of temperaturesensitive p53 mutant protein but not for stabilization
and activation of wild-type p53: implications for cancer
therapy. J Biol Chem 280, 6682–6691.
9 Selivanova G (2004) p53: fighting cancer. Curr Cancer
Drug Targets 4, 385–402.
10 Nagaich AK, Appella E & Harrington RE (1997) DNA
bending is essential for the site-specific recognition of
DNA response elements by the DNA binding domain
of the tumor suppressor protein p53. J Biol Chem 272,
14842–14849.

4704

11 Kim E & Deppert W (2003) The complex interactions
of p53 with target DNA: we learn as we go. Biochem
Cell Biol 81, 141–150.
12 Palecek E, Brazda V, Jagelska E, Pecinka P, Karlovska
L & Brazdova M (2004) Enhancement of p53 sequencespecific binding by DNA supercoiling. Oncogene 23,
2119–2127.

13 Dornan D, Shimizu H, Perkins ND & Hupp TR
(2003) DNA-dependent acetylation of p53 by the
transcription coactivator p300. J Biol Chem 278,
13431–13441.
14 Pospisilova S, Brazda V, Kucharikova K, Luciani MG,
Hupp TR, Skladal P, Palecek E & Vojtesek B (2004)
Activation of the DNA-binding ability of latent p53
protein by protein kinase C is abolished by protein
kinase CK2. Biochem J 378, 939–947.
15 Selivanova G, Iotsova V, Kiseleva E, Strom M,
Bakalkin G, Grafstrom RC & Wiman KG (1996) The
single-stranded DNA end binding site of p53 coincides
with the C-terminal regulatory region. Nucleic Acids Res
24, 3560–3567.
16 Hupp TR, Meek DW, Midgley CA & Lane DP (1992)
Regulation of the specific DNA binding function of
p53. Cell 71, 875–886.
17 Fojta M, Pivonkova H, Brazdova M, Nemcova K,
Palecek J & Vojtesek B (2004) Investigations of the
supercoil-selective DNA binding of wild type p53
suggest a novel mechanism for controlling p53 function.
Eur J Biochem 271, 3865–3876.
18 Brazdova M, Palecek J, Cherny DI, Billova S, Fojta M,
Pecinka P, Vojtesek B, Jovin TM & Palecek E (2002)
Role of tumor suppressor p53 domains in selective binding to supercoiled DNA. Nucleic Acids Res 30, 4966–
4974.
19 Espinosa JM & Emerson BM (2001) Transcriptional
regulation by p53 through intrinsic DNA ⁄ chromatin
binding and site-directed cofactor recruitment. Mol Cell
8, 57–69.

20 Brazda V, Brazdova-Jagelska E, Fojta M & Palecek E
(2006) Searching for target sequences by p53 protein is
influenced by DNA length: a role for linear drifting of
the protein along DNA. Biochem Biophys Res Commun
341, 470–477.
21 Ceskova P, Valik D & Vojtesek B (2003) What we currently know about the structure and function of the p53
homologue ) p73 protein: facts, hypotheses and expectations. Folia Biol 49, 1–8.
22 Ichimiya S, Nakagawara A, Sakuma Y, Kimura S, Ikeda
T, Satoh M, Takahashi N, Sato N & Mori M (2000) p73:
structure and function. Pathol Int 50, 589–593.
23 Levrero M, De Laurenzi V, Costanzo A, Gong J, Wang
JY & Melino G (2000) The p53 ⁄ p63 ⁄ p73 family of transcription factors: overlapping and distinct functions.
J Cell Sci 113, 1661–1670.

FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS


ˇ
´
H. Pivonkova et al.

24 Oniscu A, Sphyris N, Morris RG, Bader S & Harrison
DJ (2004) p73alpha is a candidate effector in the p53
independent apoptosis pathway of cisplatin damaged
primary murine colonocytes. J Clin Pathol 57, 492–498.
25 Gong JG, Costanzo A, Yang HQ, Melino G, Kaelin
WG Jr, Levrero M & Wang JY (1999) The tyrosine
kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806–809.
26 Jamieson ER & Lippard SJ (1999) Structure, recognition, and processing of cisplatin-DNA adducts. Chem
Rev 99, 2467–2498.

27 Brabec V (2002) DNA modifications by antitumor platinum and ruthenium compounds: their recognition and
repair. In Progress in Nucleic Acid Research and
Molecular Biology (Moldave K, ed.), pp. 1–68. Academic Press Inc., San Diego.
28 Takahara PM, Rosenzweig AC, Frederick CA &
Lippard SJ (1995) Crystal structure of double-stranded
DNA containing the major adduct of the anticancer
drug cisplatin. Nature 377, 649–652.
29 Zlatanova J, Yaneva J & Leuba SH (1998) Proteins that
specifically recognize cisplatin-damaged DNA: a clue to
anticancer activity of cisplatin. FASEB J 12, 791–799.
30 Kasparkova J & Brabec V (1995) Recognition of DNA
interstrand cross-links of cis-diamminedichloroplatinum(Ii) and its trans isomer by DNA-binding proteins.
Biochemistry 34, 12379–12387.
31 Kasparkova J, Pospisilova S & Brabec V (2001) Different recognition of DNA modified by antitumor cisplatin
and its clinically ineffective trans isomer by tumor suppressor protein p53. J Biol Chem 276, 16064–16069.
32 Kasparkova J, Fojta M, Farrell N & Brabec V (2004)
Differential recognition by the tumor suppressor protein
p53 of DNA modified by the novel antitumor trinuclear
platinum drug BBR3464 and cisplatin. Nucleic Acids
Res 32, 5546–5552.
33 Fojta M, Pivonkova H, Brazdova M, Kovarova L,
Palecek E, Pospisilova S, Vojtesek B, Kasparkova J &
Brabec V (2003) Recognition of DNA modified by antitumor cisplatin by ‘latent’ and ‘active’ protein p53.
Biochem Pharmacol 65, 1305–1316.
34 Pivonkova H, Brazdova M, Kasparkova J, Brabec V &
Fojta M (2006) Recognition of cisplatin-damaged DNA
by p53 protein: critical role of the p53 C-terminal
domain. Biochem Biophys Res Commun 339, 477–484.
35 Wetzel CC & Berberich SJ (1998) DNA binding activities of p53 protein following cisplatin damage of ovarian cells. Oncol Res 10, 151–161.
36 Wetzel CC & Berberich SJ (2001) p53 binds to cisplatin-damaged DNA. Biochim Biophys Acta 1517, 392–

397.
37 Stehlikova K, Kasparkova J, Novakova O, Martinez A,
Moreno V & Brabec V (2006) Recognition of DNA
modified by trans-[PtCl2NH3(4-hydroxymethylpyridine)]

Cisplatin effects on p53 ⁄ p73 DNA recognition

38

39

40

41
42

43

44

45

46

47

48

49

50

51

by tumor suppressor protein p53 and character of DNA
adducts of this cytotoxic complex. FEBS J 273, 301–314.
Vojtesek B, Bartek J, Midgley CA & Lane DP (1992)
An immunochemical analysis of the human nuclear
phosphoprotein p53. New monoclonal antibodies and
epitope mapping using recombinant p53. J Immunol
Methods 151, 237–244.
Brazda V, Palecek J, Pospisilova S, Vojtesek B & Palecek
E (2000) Specific modulation of p53 binding to consensus
sequence within supercoiled DNA by monoclonal antibodies. Biochem Biophys Res Commun 267, 934–939.
Anderson ME, Woelker B, Reed M, Wang P &
Tegtmeyer P (1997) Reciprocal interference between the
sequence-specific core and nonspecific C-terminal DNA
binding domains of p53: implications for regulation.
Mol Cell Biol 17, 6255–6264.
Hupp TR & Lane DP (1994) Allosteric activation of
latent p53 tetramers. Curr Biol 4, 865–875.
Wolcke J, Reimann M, Klumpp M, Gohler T, Kim E
& Deppert W (2003) Analysis of p53 ‘latency’ and ‘activation’ by fluorescence correlation spectroscopy: evidence for different modes of high affinity DNA binding.
J Biol Chem 278, 32587–32595.
Ayed A, Mulder FA, Yi GS, Lu Y, Kay LE &
Arrowsmith CH (2001) Latent and active p53 are identical in conformation. Nat Struct Biol 8, 756–760.
Yakovleva T, Pramanik A, Terenius L, Ekstrom T &
Bakalkin G (2002) p53 latency ) out of the blind alley.
Trends Biochem Sci 27, 612–618.
Kim E, Albrechtsen N & Deppert W (1997) DNA-conformation is an important determinant of sequence-specific DNA binding by tumor suppressor p53. Oncogene

15, 857–869.
McKinney K, Mattia M, Gottifredi V & Prives C
(2004) p53 linear diffusion along DNA requires its C
terminus. Mol Cell 16, 413–424.
Brazda V, Brazdova-Jagelska E, Fojta M & Palecek E
(2006) Searching for targeted sequences by p53 protein
is influenced by DNA length. Biochem Biophys Res
Commun 341, 470–477.
Cho Y, Gorina S, Jeffrey PD & Pavletich NP (1994)
Crystal structure of a p53 tumor suppressor–DNA complex: understanding tumorigenic mutations [see comments]. Science 265, 346–355.
Cullinane C, Mazur SJ, Essigmann JM, Phillips DR &
Bohr VA (1999) Inhibition of RNA polymerase II transcription in human cell extracts by cisplatin DNA
damage. Biochemistry 38, 6204–6212.
Evans GL & Gralla JD (1992) Cisplatin-induced
imbalances in the pattern of chimeric marker gene
expression in HeLa cells. Biochem Biophys Res
Commun 184, 1–8.
Jiang M, Yi X, Hsu S, Wang CY & Dong Z (2004)
Role of p53 in cisplatin-induced tubular cell apoptosis:

FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS

4705


ˇ
´
H. Pivonkova et al.

Cisplatin effects on p53 ⁄ p73 DNA recognition

52

53

54

55

dependence on p53 transcriptional activity. Am J
Physiol Renal Physiol 287, F1140–F1147.
Houldsworth J, Xiao H, Murty VV, Chen W, Ray B,
Reuter VE, Bosl GJ & Chaganti RS (1998) Human
male germ cell tumor resistance to cisplatin is linked to
TP53 gene mutation. Oncogene 16, 2345–2349.
Ganjavi H, Gee M, Narendran A, Freedman MH &
Malkin D (2005) Adenovirus-mediated p53 gene therapy
in pediatric soft-tissue sarcoma cell lines: sensitization to
cisplatin and doxorubicin. Cancer Gene Ther 12, 397–
406.
Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk
J, Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA &
Sen S (2004) Phosphorylation by aurora kinase A
induces Mdm2-mediated destabilization and inhibition
of p53. Nat Genet 36, 55–62.
Dart DA, Picksley SM, Cooper PA, Double JA &
Bibby MC (2004) The role of p53 in the chemotherapeutic responses to cisplatin, doxorubicin and 5-fluorouracil treatment. Int J Oncol 24, 115–125.

4706

56 Datta K, Shah P, Srivastava T, Mathur SG,
Chattopadhyay P & Sinha S (2004) Sensitizing glioma
cells to cisplatin by abrogating the p53 response with
antisense oligonucleotides. Cancer Gene Ther 11, 525–
531.
57 Clarke PA, Pestell KE, Di Stefano F, Workman P &
Walton MI (2004) Characterisation of molecular events
following cisplatin treatment of two curable ovarian
cancer models: contrasting role for p53 induction and
apoptosis in vivo. Br J Cancer 91, 1614–1623.
58 Burger H, Nooter K, Boersma AW, Kortland CJ & Stoter G (1997) Lack of correlation between cisplatininduced apoptosis, p53 status and expression of Bcl-2
family proteins in testicular germ cell tumour cell lines.
Int J Cancer 73, 592–599.
´
59 Kim SD, Vrana O, Kleinwachter V, Niki K & Brabec V
ă
(1990) Polarographic determination of subnanogram
quantities of free platinum in reaction mixture with
DNA. Anal Lett 23, 1505–1518.

FEBS Journal 273 (2006) 4693–4706 ª 2006 The Authors Journal compilation ª 2006 FEBS