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 th
Trang 1DNA binding of p53 and p73 proteins in a target
site-dependent manner
Hana Pivonˇkova´1, Petr Pecˇinka1, Pavla Cˇ esˇkova´2
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
The tumor suppressor protein p53 is known as a
tran-scription factor involved in cell cycle control [1–3] It
plays a crucial role in preventing malignant
transfor-mation 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
ele-ments [p53 DNA-binding sites (p53DBSs)] in
promot-ers 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 p53-binding sites encompassing over 540 loci in the human genome [4] A typical p53DBS consists of two tandem copies of the motif RRRCWWGYYY (where R¼ A
or G, Y¼ 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
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,
Kra´lovopolska´ 135, CZ-612 65 Brno,
Czech Republic
Fax: +420 541211293
Tel: +420 541517197
E-mail: fojta@ibp.cz
(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, regu-lation 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 previ-ously 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 signifi-cantly, 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 bifunc-tional 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 cisplatin-treated cells
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.
Trang 2DNA 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
signifi-cantly to the stability of the p53–DNA complexes [10]
In addition, interactions of the p53 protein with
cer-tain 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-trans-lational modifications (phosphorylation, acetylation)
within the protein C-terminal domain [3,13,14]
Trun-cated forms of p53 lacking a negative-regulating
seg-ment 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
conformation-selective 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
homo-log 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
dam-age 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
cros-slinks (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
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 pro-posed that these interactions are important for the anticancer activity of the drug [26,29,30]
Interactions of the p53 protein with cisplatin-modi-fied 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 tri-nuclear 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 inhib-itory 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 pro-tein–DNA interactions to cisplatin DNA treatment correlated with the occurrence of sequence motifs forming the cisplatin IACs (namely GG and AG dou-blets) within the given p53DBS Binding of both pro-teins to mutated target sites not containing these motifs was not significantly affected by the DNA cis-platination 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, consti-tutively active p53(1–363) to eliminate the sequence-nonspecific p53 interactions with the cis-platinated DNA, which have been shown to be mediated pri-marily by the p53 C-terminal DNA-binding site (CTDBS) [34] In the presence of competitor nonspe-cific DNA, sequence-spenonspe-cific 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
Trang 3Fig 1 Scheme of DNA substrates used in this work All p53 DNA-binding sites (p53DBSs) were placed in the center of 50-mer oligonucleo-tides (oligos), being flanked with the sequences shown on the top (the same stretches flank the p53DBSs in the pPGM1 and pPGM4 plas-mids) 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.
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 32 P-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, R73dand 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 super-shifted bands are denoted as SR 53 , SR 73d and SR 73b ; 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.
Trang 4and p73d proteins to the DNA targets caused the
for-mation 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
DO-1 antibody [DO-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
dif-fered to some extent (which was manifested by
differ-ent 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
modi-fication with cisplatin causes dose-dependent inhibition
of the full-length (fl) p53 sequence-specific DNA
bind-ing 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
Inter-action of the protein with any of these targets was
sig-nificantly affected by the cisplatin treatment, but the
levels of inhibition observed with individual p53DBSs
at the same degree of global DNA cis-platination
dif-fered significantly The steepest decrease in p53–DNA
binding with degree of DNA modification was
exhib-ited by the mdm2 target The R53 band due to the
p53–mdm2 complex exhibited only 10% intensity for
rb¼ 0.02, compared to the R53 band due to protein
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 tar-gets retained 75% and 53% of the p53-binding capa-city at rb¼ 0.02, respectively (Fig 3A) Increasing the DNA modification degree to rb¼ 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 rb¼ 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 cisp-latin treatment, contains only one GGG triplet within the p53DBS The PGM1 site possesses two AGG tri-plets 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-react-ive motifs were eliminated Two p53DBSs were dercisplatin-react-ived 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)
Trang 5We 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 rb¼ 0.02
resulted in a decrease of p53 binding to the p21a
tar-get by about 15%, which represented weaker
inhibi-tion than observed with the p21 target (25% decrease;
Fig 3) More conspicuous differences between the
p21a and p21 targets appeared at rb¼ 0.04 (35% or
58% inhibition, respectively) At rb¼ 0.06, the p21a
target retained 45% of the p53 binding, thus
exhibit-ing at least four times higher bindexhibit-ing 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
treat-ment 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 rb¼ 0.02 or 0.04 and about 10% inhibi-tion for rb¼ 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
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 R 53 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 R 53 band resulting from p53 binding to unmodified DNA was taken as 1.0, and the intensities of bands correspond-ing to p53 bindcorrespond-ing to the same but cisplatin-treated substrate were normalized to this).
Trang 6We also performed parallel experiments with fl p53
(expressed in insect cells) Like p53(1–363), the fl
pro-tein 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
sequence-specific 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 R73dband decreased almost linearly with
increase in the cis-platination level; for rb¼ 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 rb¼ 0.02 or 0.04 had no significant effect on
its interaction with either of the p73 isoforms; at rb¼
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
modifica-tion on the competimodifica-tion between two p53 target sites
for the protein (Fig 6) The 474 base pair fragments
of plasmids pPGM1 or pPGM4 were used as the sequence-specific competitors, and changes in p53(1– 363) binding to the32P-labeled 50-mer targets were fol-lowed We first tested the effect of the presence of the competitor fragments (unmodified or treated with cisp-latin) on p53 binding to the unmodified PGM1 probe (Fig 6A) Addition of either of the unmodified frag-ments (70 ng per sample) resulted in a partial decrease (by 35–45%) of the R53band 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 frag-ment was cis-platinated to rb¼ 0.04 or 0.06, its pres-ence had practically no effect on the R53 band intensity, suggesting that the modified pPGM1 frag-ment 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 nonspe-cific competitor DNA), and performed a p53-binding assay (Fig 6B) In the unmodified DNA, the compet-itor pPGM1 fragment caused about 70% inhibition of p53(1–363) binding to either of the two targets Modi-fication of the p21⁄ pPGM1 mixture resulted in
rb-dependent inhibition of p53 binding to the p21 tar-get, 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 inhibi-tion effect was detectable only at rb¼ 0.04 and 0.06 The apparent lack of the cisplatin effect at rb¼ 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 (rel-atively 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 inten-sity of the R53 band on the autoradiogram increased with the degree of DNA modification The increase was already significant at rb¼ 0.02 At rb¼ 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
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.
Trang 7under the same conditions The results of these model
competition experiments suggest that global
modifica-tion of DNA with cisplatin may shift the distribumodifica-tion
of the p53 protein among different target sites,
depend-ing 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-non-specific 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
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) cisPt-DNA recognition [34] On the other hand, sequence-specific 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 bond-ing within the base pairs [26–28] Cisplatin adducts occurring within p53DBS can therefore be expected to cause severe deformations of the binding site with con-comitant destabilization of the p53–DNA interaction (or even prevention of target recognition by the pro-tein)
DNA binding of the C-terminally truncated p53(1–363) protein
In this work, we studied the effects of cisplatin treat-ment 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 constitu-tively active for sequence-specific DNA binding [16] Models of p53 latency considering the (post-transla-tionally unmodified) p53 C-terminus solely as a negat-ive 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 bind-ing to target sites adoptbind-ing 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 medi-ated 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 evalua-tion of the protein-binding affinities for 20 natural p53 recognition elements [7] A comparative study invol-ving four of them showed practically the same cooper-ative binding of the fl p53 as exhibited by p53CT [7]
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.
Trang 8Likewise, 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
comparat-ive 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
random-sequence natural genetic material that can
accommo-date the cisplatin adducts regardless of the reactivity
of the particular p53DBS The frequency of DNA modification within the p53DBSs could thus be expec-ted 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 pri-marily 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 corre-lation 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 suit-able for formation of the IACs inside the p53DBSs (Fig 1)
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 frag-ments of plasmids pPGM1 or pPGM4 (and with unmodified calf thymus DNA) prior to addition of the p53 protein When the com-petitor 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 cisplatin-resistant p53DBS, and its cis-platination did not change its competitiveness for p53(1– 363) In (B), the32P-labeled targets p21 (i) and p21b (ii) were treated with cisplatin together with the competitor pPGM1 frag-ment, and this was followed by the p53-binding 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 con-ditions [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 R 53 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.
Trang 9In 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
cisplat-in IAC, wherever it is withcisplat-in the p53DBS, can fully
abrogate p53 sequence-specific DNA binding, or
whe-ther 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
appar-ent sensitivity of p53 (or p73) DNA binding to
cis-pla-tination was influenced primarily by the probability of
adduct formation within the target sites, regardless of
the positions of the cisplatin adducts The adduct
posi-tioning may be nevertheless be important with respect
to the stereochemistry of the protein–DNA recognition
(cis-platination induces significant bending and
tor-sional deformations of the DNA double helix [28]) and
the availability of functional groups ensuring the
essen-tial protein–DNA contacts For example, formation of
the cisplatin crosslinks within the CWWG box (which
represents an area where the p53 Arg248 residue
inter-acts 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
pre-liminary results (M Fojta et al., unpublished data)
suggest that the behavior of cisplatin-treated target
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 unmodi-fied PGM1, PGM4, p21 and p21b targets in the pres-ence of unmodified or cis-platinated (rb¼ 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 50-mer 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 seg-ments 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 cisplatin-reactive 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 recogni-tion However, it should be emphasized that such conclusions need not be applicable to the post-translationally unmodified form of fl p53, which exhibits apparently weaker binding to p53DBS but sig-nificant 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
Trang 10DNA-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
there-fore more easily modified by the drug [50] However,
to our knowledge, no systematic study of the
sensitiv-ity of various promoters (and particularly those
con-trolled 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
involves cell cycle arrest via p21WAF1⁄ CIP1 induction and activation of DNA repair processes that, in gen-eral, confer chemoresistance to cancer cells The other pathway leads to programmed cell death through acti-vation 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 homo-logs, it has not yet been clarified how the checkpoint proteins decide which pathway to activate Partic-ularly, no unambiguous correlation between wild-type p53 expression and cancer cell susceptibility to cisplat-in-induced apoptosis has been established Although some authors reported a clear p53-dependent
apoptot-ic 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 apop-tosis 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 cisp-latin 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 sug-gest that the expression of various p53 downstream genes might be differentially affected in the cisplatin-treated 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)
Fig 7 The effect of DNA modification with cisplatin on p53(1–363) binding to p53 DNA-binding sites (p53DBSs) flanked by stret-ches 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.