From the Institute of Biophysics, Academy of Sciences
of the Czech Republic, CZ-61265 Brno, Czech Republic and the
¶ Masaryk Memorial Cancer Institute, CZ-65653 Brno, Czech
Republic
Received for publication, February 8, 2001
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ABSTRACT |
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The p53 gene encodes a nuclear phosphoprotein
that is biologically activated in response to genotoxic stresses
including treatment with anticancer platinum drugs. The DNA binding
activity of p53 protein is crucial for its tumor suppressor function.
DNA interactions of active wild-type human p53 protein with DNA
fragments and oligodeoxyribonucleotide duplexes modified by antitumor
cisplatin and its clinically ineffective trans isomer
(transplatin) were investigated by using a gel mobility shift assay. It
was found that DNA adducts of cisplatin reduced binding affinity of the
consensus DNA sequence to p53, whereas transplatin adducts did not.
This result was interpreted to mean that the precise steric fit
required for the formation and stability of the tetrameric complex of
p53 with the consensus sequence cannot be attained, as a consequence of
severe conformational perturbations induced in DNA by cisplatin
adducts. The results also demonstrate an increase of the binding
affinity of p53 to DNA lacking the consensus sequence and modified by
cisplatin but not by transplatin. In addition, only major 1,2-GG
intrastrand cross-links of cisplatin are responsible for this
enhanced binding affinity of p53. The data base on structures of
various DNA adducts of cisplatin and transplatin reveals distinctive
structural features of 1,2-intrastrand cross-links of cisplatin,
suggesting a unique role for this adduct in the binding of p53 to DNA
lacking the consensus sequence. The results support the hypothesis that
the mechanism of antitumor activity of cisplatin may also be associated
with its efficiency to affect the binding affinity of platinated DNA to
active p53 protein.
It is well established that platinum coordination complexes
exhibit antitumor effects (1, 2). The success of platinum complexes in
killing tumor cells results from their ability to form on DNA various
types of covalent adducts (3). The first platinum complex introduced in
the clinic is cis-diamminedichloroplatinum(II) (cisplatin)1 (1, 4). Although
the antitumor effects of cisplatin were discovered more than 30 years
ago, the mechanism of its antitumor activity has not yet been fully
understood. It has been shown (3) that this bifunctional platinum
complex forms on DNA mainly intrastrand cross-links (CLs) between
neighboring purine residues (~90%). Other minor adducts are
intrastrand CLs between two purine nucleotides separated by one or more
nucleotides and interstrand CLs; a few adducts remain
monofunctional. Transplatin (trans isomer of cisplatin) is
clinically ineffective; thus both isomers have been widely used in
studies of the structure-pharmacological activity relationship of
platinum complexes (5). In these studies, one searches for differences
between active and inactive compounds that may be responsible for the
differences in the pharmacological effect. Transplatin-DNA adducts are
interstrand CLs, and a relatively large portion of adducts remains
monofunctional (6, 7).
The adducts formed on DNA by bifunctional platinum compounds are
capable of terminating DNA synthesis (1) and triggering several
cellular processes such as apoptosis (8) or repair of lesions (9, 10).
It has become clear that under some circumstances p53 can play a major
role in the activation of apoptosis (11, 12) and actively participates
in various processes of DNA repair via its ability to interact with
components of the repair machinery and by its various biochemical
activities (13).
The tumor suppressor protein p53 is a nuclear phosphoprotein consisting
of 393 amino acids and containing four major functional domains (14).
The transcriptional activation domain is located at the N terminus,
whereas the sequence-specific DNA-binding domain is within the central
part of p53. The C-terminal portion, which interacts with DNA in a
nonspecific manner, contains an oligomerization and regulatory domains.
p53 is a potent mediator of cellular responses against genotoxic
insults (13) that exerts its effect through transcriptional regulation.
Upon exposure to genotoxic compounds, p53 protein levels increase due
to several post-transcriptional mechanisms. Cisplatin induces apoptosis
in cells expressing either wild-type (wt) or mutant p53 so that a
consensus on the significance of p53 for response to cisplatin has not
been attainable (15). Nevertheless, on average, cells with mutant p53
are more resistant to the effect of cisplatin (16). Hence, it seems
reasonable to conclude that p53 can control the processing of DNA
adducts of platinum, depending on the cell type.
The tumor suppressor function of p53 protein is crucially related to
its DNA binding activity. Active wt p53 binds as a tetramer to over 100 different response elements naturally occurring in the human genome.
These response elements, which show functionality, differ in the
details of their specific base sequence, but all contain two tandem
consensus decamers, each a pentameric inverted repeat. Most consensus
decamers, separated in the binding unit by 0-21 base pairs (bp),
follow the consensus sequence pattern (17) PuPuPuC(A/t)|(T/a)GPyPyPy,
where Pu and Py are purines and pyrimidines, respectively, and the
vertical bar denotes the center of pseudodyad symmetry. Four molecules
of the DNA-binding domain of p53 bind the response elements with high
cooperativity. They also bend DNA. It has been suggested (18) that this
bending is localized mostly at the two pentamer CA|TG junctions in
the consensus DNA response element (CDRE) (by 25-28° at each
junction) toward the major groove. Active wt p53 also over-twists the
DNA response element by ~70°. This DNA twisting is uniformly
distributed among the pentamers. It has also been suggested that due to
many functions of p53 protein the demands for binding specificity and selectivity are necessarily extraordinary, which is accomplished through its tetrameric association with a repetitive binding site. Precise steric fit accommodated through both DNA bending and twisting appears extremely important in this binding site. Because DNA bending
and twisting are coupled in the p53-DNA complex, the binding specificity of the p53 system as well as complex stability could be
fine-tuned by agents that affect DNA bending and twisting.
It has been shown (3) that DNA adducts of cisplatin and other platinum
bifunctional compounds distort the conformation of DNA, including
bending and changes in the twist angle. Thus, formation of the adducts
by platinum compounds in the CDRE could affect its binding affinity to
p53. No direct interaction between active wt p53 and
platinum-modified DNA in cell-free media has yet been
reported. Only binding of latent p53 protein, lacking sequence-specific DNA binding, to DNA modified by cisplatin has been
reported (19). In the present work, we have used gel mobility shift
assay methodology to investigate the binding affinity of the active
human wt p53 system to the CDRE modified by either antitumor cisplatin
or its clinically inefficient trans isomer in a cell-free
medium. We have also examined binding of DNA lacking CDRE and modified
by cisplatin or transplatin to reveal affinity of active wt p53 to
platinated DNA containing no consensus nucleotide sequence. Thus, these
studies could provide insight into the relative cytotoxicities of these
two isomers, thereby potentially aiding in the rational design of new
platinum drugs as well as illuminating aspects of the role of p53 in
chemotherapy by platinum compounds.
DNA--
For CDRE binding of the p53 protein we used a small
PvuII (474 bp) fragment of pPGM1 plasmid derived from
pBluescript SK II+ DNA (2961 bp, Stratagene) by cloning the
p53 20-bp CDRE 5'-AGACATGCCTAGACATGCCT-3'/5'-AGGCATGTCTAGGCATGTCT-3' into the HindIII site. pPGM1 and pBluescript SK
II+ DNAs were purified using Qiagen kits.
Ethanol-precipitated plasmids were resuspended in TE buffer (10 mM Tris-HCl, 0.1 mM Na3EDTA (pH
7.5)) and stored at 4 °C. The synthetic oligodeoxyribonucleotides (Fig. 1) were purchased from IDT, Inc.
(Coralville, IA) and purified as described previously (20, 21); in the
present work their molar concentrations are related to the whole
duplexes.
Purification of the Active Wild-type Human p53 Protein--
The
human wt p53 protein was expressed in baculovirus-infected recombinant
Sf9 insect cells. The details of the purification were described
previously (22). The protein concentration was determined by the
Bradford method. In the present paper the concentration of the p53
protein is related to tetrameric protein units.
Platination Reactions--
Cisplatin and transplatin were
purchased from Sigma. Monodentate
diethylenetriaminechloroplatinum(II) chloride ([Cl(dien)Pt]Cl) was
kindly provided by Dr. G. Natile (University of Bari, Italy). Short
PvuII fragments of pPGM1 and pBluescript II SK+
plasmid DNAs (474 and 448 bp, respectively) and an
oligonucleotide duplex (oligo-CDRE) were incubated with
cisplatin or transplatin in 10 mM NaClO4 at
37 °C for 48 h in the dark. The number of platinum atoms bound
per nucleotide (rb value) was determined by
flameless atomic absorption spectrophotometry or differential pulse
polarography (23). The number of interstrand CLs formed by cisplatin in
the oligo-CDRE duplex was determined using polyacrylamide gel
electrophoresis (PAGE) under denaturing conditions in the same way as
described in previous reports (24, 25). The oligonucleotide duplexes
containing single site-specific adducts of cisplatin were prepared and
characterized as described previously (20, 26-28).
Preparation of DNA-Protein Complexes--
Formation of the
complexes of the p53 protein with the 474- or 448-bp-long
PvuII fragments of pPGM1 or pBluescript SK II+,
respectively, unplatinated or modified by cisplatin or transplatin was
examined in a buffer containing 5 mM Tris-HCl, pH 7.6, 0.5 mM Na3EDTA, 50 mM KCl, 0.01%
Triton X-100 in a total volume of 15 µl. The nonmodified or
platinated 474- or 448-bp fragment was mixed with a nonmodified
2513-bp-long fragment of pPGM1. The final amounts of the short and long
fragments in the reactions were 150 and 850 ng, respectively (the molar
ratio of these fragments was ~1). The molar ratio of p53 to the 474- or 448-bp fragment was 0-6. Samples with p53 protein were incubated in
ice for 30 min. After the incubation was completed, 3 µl of the
loading buffer (50% glycerol, 50 mM Na3EDTA,
2% bromphenol blue) was added, and the samples were loaded on the 1%
agarose gel precooled to 4 °C and electrophoresed in 0.5× TBE
buffer (0.09 M Tris borate, 2 mM
Na3EDTA (pH 8.0)). The gel was finally stained by ethidium bromide.
Formation of the complexes of p53 with the oligonucleotide duplexes was
examined in the same buffer as that used for analysis of the complexes
of p53 with the plasmid fragments (see above) in a total volume of 12 µl. The nonmodified or platinated duplexes were mixed with the
nonmodified 2513-bp-long fragment of pPGM1. The final amounts of the
duplexes and long fragment in the reactions were 20 and 120 ng,
respectively. The molar ratio of p53 to duplex was 0-3. Samples with
p53 were incubated in ice for 30 min. After the incubation was
completed, 3 µl of the loading buffer (50% glycerol, 50 mM Na3EDTA, 2% bromphenol blue) was added, and
the samples were loaded on the native 5% polyacrylamide gel
(mono:bis(acrylamide) ratio, 29:1) precooled to 4 °C in 0.5× TBE
buffer. The radioactivity associated with the bands was quantified by
means of a Molecular Dynamics PhosphorImager (Storm 860 system with
ImageQuant software).
The primary p53 monoclonal antibody (mAb) DO-1 (purified and
characterized as described in Ref. 29) was also added to the p53-DNA
complex (the molar ratio of mAb to p53 tetramer was 3), the mixture was
incubated for an additional 30 min at 20 °C, and the resulting
p53-DNA-mAb complexes were loaded on the gels.
Other Chemicals--
T4 polynucleotide kinase and
[ Binding of p53 Protein to Platinated DNA Containing the Consensus
Response Element--
pPGM1 plasmid was cleaved by PvuII
(blunt end-forming enzyme, which cuts twice within the pPGM1). This
cleavage produced 474- and 2513-bp fragments containing and lacking
CDRE, respectively. The two fragments were separated on the agarose
gel, extracted, and purified. The 474-bp fragment was further globally
modified by cisplatin or transplatin at rb = 0.02-0.1, and the unplatinated 2513-bp fragment was added as the
nonspecific competitor. This mixture was incubated with various amounts
of wt p53 (at molar ratios of p53 to 474-bp fragment in the range of
0-6) and analyzed using agarose gel electrophoresis. The incubation of the unplatinated 474-bp fragment with increasing amounts of p53 resulted in the appearance of a new, more slowly migrating species, with a concomitant decrease of the intensity of the band corresponding to the 474-bp fragment incubated in the absence of p53 (shown for
p53:474-bp fragment ratio of 0.95 in Fig.
2, lane 6). This result was in
agreement with the previously published reports and demonstrated
formation of a sequence-specific complex between DNA and p53 protein
(30). Importantly, addition of DO-1 mAb (which maps to the N-terminal
domain of p53) produced supershifted complexes that migrated still more
slowly than the p53-474-bp complex (Fig. 2, lane 11),
confirming the presence of p53 in the more slowly migrating species. In
contrast, incubation of the 474-bp fragment modified by cisplatin at
rb = 0.02-0.06 with p53 (in the presence of the
unplatinated 2513-bp fragment) considerably reduced the yield of the
species migrating more slowly in the agarose gel (shown for a p53 to
474-bp fragment ratio of 0.95 in Fig. 2, lanes 7-9). This
result is consistent with the idea that cisplatin adducts efficiently
reduce binding affinity of the CDRE to active p53. The same experiments
were performed with the 474-bp fragment globally modified by
transplatin. No reduction in the intensity of the band corresponding to
the sequence-specific p53-DNA complex as a consequence of the
modification by transplatin was noticed even at rb
as high as 0.1 (shown for rb = 0.06 and a p53 to
474-bp fragment ratio of 0.95 in Fig. 2, lane 10). This
result demonstrates the inefficiency of transplatin adducts to reduce
the binding affinity of the CDRE to the active p53 protein.
Further studies were performed using a short (20 bp)
oligodeoxyribonucleotide duplex, oligo-CDRE (Fig. 1) whose sequence
follows the consensus sequence pattern (17). The duplex was globally modified by cisplatin or transplatin to rb in the
range of 0.02-0.06, and the unplatinated PvuII fragment of
pPGM1, which was 2513 bp long and contained no CDRE, was added
as the nonspecific competitor. These mixtures were incubated with p53
at various p53 to duplex molar ratios (0.1-3) and analyzed using
native PAGE (Fig. 3). Incubation of the
unplatinated oligo-CDRE with increasing amounts of p53 resulted in the
appearance of the new, more slowly migrating species, with a
concomitant decrease of the intensity of the band corresponding to the
20-bp duplex incubated in the absence of p53 (shown for a p53 to duplex
ratio of 0.3 in Fig. 3, lane 2). This result confirmed the
formation of the complex between oligo-CDRE and p53. In contrast, the
incubation of oligo-CDRE modified by cisplatin at rb = 0.02-0.06 with p53 reduced the yield of the species migrating more
slowly in the gel. For instance, the modification of oligo-CDRE by
cisplatin at rb = 0.05 already completely inhibited
formation of the complex between this duplex and p53 (shown for a p53
to duplex molar ratio of 0.3 in Fig. 3, lane 4). It was also
verified using PAGE under denaturing conditions that the modification
of oligo-CDRE by cisplatin at rb = 0.05 also
afforded duplexes interstrand-cross-linked by this drug (data not
shown). The quantitative evaluation (24, 25) of the radioactivities
associated with the bands corresponding to interstrand cross-linked
duplexes and duplexes containing no interstrand CL (24, 25) revealed
~20% of duplexes containing at least one interstrand CL of
cisplatin.
Oligo-CDRE was also globally modified by transplatin and incubated with
p53. No reduction of the intensity of the band corresponding to the
p53-oligo-CDRE complex was noticed even at so high an
rb as 0.05 (Fig. 3, lane 6),
i.e. under conditions when cisplatin adducts inhibited
formation of the complex between p53 and the duplex completely (Fig. 3,
lane 4). Thus, these experiments confirmed that transplatin
adducts do not affect the binding affinity of the CDRE to p53 protein.
Binding of Active p53 Protein to Platinated DNA Lacking the
Consensus Response Element--
We also investigated binding of p53 to
the 448-bp fragment of the pBluescript II SK+ plasmid
lacking CDRE but modified by cisplatin or transplatin. The plasmid was
cleaved by PvuII, which yielded the 448- and 2513-bp fragments. The longer fragment was identical to that produced by the
PvuII cleavage of pPGM1, whereas the shorter fragment only differed from the shorter fragment produced by PvuII
cleavage of pPGM1 by lacking CDRE. The 448-bp fragment was globally
modified by cisplatin or transplatin at rb = 0.01-0.08. After the 448-bp fragment was platinated, the unplatinated
2513-bp fragment was added as the nonspecific competitor. These
mixtures were incubated with p53 at various p53 to 448-bp fragment
molar ratios (in the range of 0.5-6) and analyzed using agarose gel
electrophoresis. Incubation of the unplatinated PvuII
fragments with increasing amounts of p53 resulted in no changes in the
migration of these fragments, demonstrating no effect on formation of
the complex between p53 and DNA lacking CDRE (shown for a p53 to 448-bp
fragment ratio of 6 in Fig. 4, lane
6). On the other hand, a new species migrating in the gel
considerably more slowly was observed if the 448-bp fragment modified
by cisplatin at rb = 0.01-0.08 was analyzed (shown
for a p53 to 448-bp fragment ratio of 6 in Fig. 4, lanes
7-9). This result demonstrated formation of the complex between
p53 and DNA lacking CDRE and modified by cisplatin (30). Importantly,
supershifted complexes were noticed as a consequence of addition of mAb
DO-1 to the complex of p53 with the 448-bp fragment modified by
cisplatin (Fig. 4, lane 11). The same experiments were
performed with the 448-bp fragment globally modified by clinically
ineffective transplatin. No more slowly migrating species as a
consequence of the modification by transplatin was noticed at
rb = 0.08 and a p53 to DNA ratio of 6 (shown for
rb = 0.08 and a p53 to 448-bp fragment ratio of 6 in
Fig. 4, lane 10). Thus, these results indicate that the binding affinity of p53 to DNA lacking CDRE is enhanced selectively by
the modification by cisplatin and not by transplatin.
Cisplatin forms several types of adducts, which occur in DNA with a
different frequency and differently distort the conformation of DNA
(3). To determine which specific adduct of cisplatin represents a
structural motif responsible for recognition of cisplatin-modified DNA
lacking CDRE by p53, we prepared a series of 20-bp duplexes with blunt
ends. The nucleotide sequences of these oligonucleotides were designed
(Fig. 1) so that they did not follow the consensus sequence pattern
(17), but they allowed us to prepare the duplexes containing a single
site-specific adduct of cisplatin, such as 1,2-GG or 1,3-GTG
intrastrand CL in d(TGGT)/d(ACCA) or d(TGTGT)/d(ACACA), respectively,
and 1,2-GG interstrand CL or a site-specific monofunctional adduct of
the model platinum compound [Cl(dien)Pt]Cl (20) in the duplex
d(TGCT)/d(AGCA). The duplexes containing the single adduct were
prepared and purified, and the unplatinated PvuII fragment
of pPGM1, which was 2513 bp long and lacked CDRE, was added as
the nonspecific competitor. These mixtures were incubated with p53 at
various p53 to duplex molar ratios (0.1-3) and analyzed using native
PAGE. Incubation of the mixtures containing unplatinated oligonucleotide duplexes with p53 did not result in the appearance of
any more slowly migrating species (shown for a p53 to duplex ratio of 3 in Fig. 5, lane 2). Similarly,
no more slowly migrating species that would demonstrate formation of
the complex between p53 and the platinated duplex was observed if p53
was incubated with the duplexes containing the single site-specific
1,3-GTG intrastrand, 1,2-GG interstrand CL of cisplatin, or
monofunctional adduct of [Cl(dien)Pt]Cl (shown for a p53 to duplex
ratio of 3 in Fig. 5, lanes 8, 10,
12). In contrast, incubation of the d(TGGT)d(ACCA) duplex
containing 1,2-GG intrastrand CL of cisplatin with p53 did result in
the occurrence of the species migrating more slowly in the gel (shown
for a p53 to duplex ratio of 0.93-3 in Fig. 5, lanes 4-6).
These results demonstrate that among DNA adducts of cisplatin major
intrastrand adducts between neighboring purine residues only produce a
structural motif responsible for enhancement of the binding affinity of
active p53 to cisplatin-modified DNA lacking CDRE.
Interestingly, the protein concentration at which half of the free
d(TGGT)/d(ACCA) duplex containing the single 1,2-intrastrand CL was
bound, was ~1.5 × 10 DNA Adducts of Antitumor Cisplatin Reduce the Binding Affinity of
the Consensus Response Sequence to Active p53 Protein, whereas the
Adducts of Clinically Ineffective Transplatin Do Not--
It has been
shown that sequence-dependent conformational variability of
response elements plays a critical role in the sequence-specific binding of p53 to DNA and the stability of the resulting complex. Extraordinary demands for this binding specificity and selectivity of
p53 are closely related to its tetrameric association with CDRE, in
which a precise steric fit is extremely important. It has been
suggested (18) that steric fit is accommodated through strongly
correlated DNA bending localized at the two highly bendable CA|TG
junctions and twisting uniformly distributed between the pentamers of
the CDRE. The consensus sequences investigated in the present work
contained several sites at which bifunctional adducts of cisplatin
strongly distorting DNA conformation are formed. In particular, they
contained the sites at which major intrastrand CLs between adjacent
purines (at d(GG) or 5'-d(AG)) are formed. For instance, the structure
of these adducts determined by NMR methods has revealed (31, 32) that
these adducts induce the overall helix bend of 40-78° toward the
major groove, DNA unwinding of 25-27°, severe perturbation of
hydrogen bonding within the 5'-coordinated GC bp, and distortion
extended over at least 4-5 bp at the site of the CL. Interestingly,
the CDREs tested in the present work also contain the sites at which
cisplatin forms less frequent interstrand CLs. Formation of these
lesions in the CDREs investigated in the present work was confirmed by PAGE under denaturing conditions (see above). The interstrand CL, which
is preferentially formed by cisplatin between opposite G
residues in the 5'-GC/5'-GC sequence (24), induces several irregularities in the cross-linked base pairs and their immediately adjacent pairs (33). The cross-linked G residues are not paired with
hydrogen bonds to the complementary cytosines, which are located
outside the duplex and not stacked with other aromatic rings. All other
base residues are paired, but distortion extends over at least 4 bp at
the site of the CL. In addition, the
cis-diammineplatinum(II) bridge resides in the minor groove,
and the double helix is locally reversed to a left-handed, Z-DNA-like
form. This adduct induces the helix unwinding by ~80° relative to
B-DNA and also the bending of ~40° of the helix axis at the
cross-linked site toward the minor groove. Thus, cisplatin formed in
the CDREs investigated in the present work bifunctional adducts, which
strongly disturb its secondary structure. The result of these
perturbations is that the precise steric fit required for the formation
and stability of the tetrameric complex of p53 with the consensus
nucleotide sequence cannot be attained, so that p53 does not bind to
its CDRE.
We demonstrated in the present work that 1,2-intrastrand CLs of
cisplatin formed in the CDRE reduce its binding affinity to p53,
whereas the same adducts formed in the sequences that do not follow the
CDRE pattern afford DNA enhanced binding affinity to p53. Hence, the
CDRE should have some intrinsic specific feature absent in the usual
B-DNA that precludes p53 from binding to CDRE after its modification by
cisplatin. It has been shown (18) that CDREs are already intrinsically
curved in the flexible CATG tetramers, with the directionality close to
that in the p53-DNA complexes. We suggest that unique alterations
induced in the consensus sequence simultaneously by 1,2-intrastrand CLs
of cisplatin and the intrinsic curvature of the CDRE are structural
factors responsible for the reduced affinity of p53 to its consensus
sequence modified by cisplatin.
Clinically ineffective transplatin also forms various types of adducts
in DNA. It forms mainly monofunctional adducts at G residues and some
amount of interstrand CLs between complementary G and C residues (6, 7,
25). Monofunctional adducts of transplatin (and cisplatin) affect DNA
conformation only slightly, without bending (20), and DNA unwinding is
very small (~6°) (34). In addition, the conformational alterations
induced by the interstrand CL of transplatin are much less severe than
those induced by the CLs of cisplatin (33, 35). The duplex is slightly distorted on both sides of the CL, but all bases are still paired and
hydrogen-bonded. The CL of transplatin only unwinds the double helix by
~12° and induces a slight, flexible bending of ~20° of its axis toward the minor groove. We conclude that these relatively subtle structural perturbations induced by transplatin in the CDRE have
no substantial effect on the formation of the tetrameric complex of p53
with the CDRE.
1,2-Intrastrand Cross-links of Cisplatin Formed in DNA Lacking the
Consensus Nucleotide Sequence Increase DNA Binding Affinity to Active
wt p53 Protein--
The results of the present work also demonstrate
enhancement of the binding affinity of p53 to DNA lacking CDRE due to
its modification by cisplatin. However, this binding affinity of p53 is
considerably lower (roughly by one order of magnitude) than that to the
unplatinated CDRE. Importantly, no change of the binding affinity to
p53 of DNA lacking CDRE is observed due to its modification by
clinically ineffective transplatin; thus this enhancement is specific
for cisplatin-modified DNA. Also importantly, among DNA adducts of
cisplatin only the 1,2-intrastrand CLs are responsible for this
increase in DNA binding to p53. Hence, 1,2-intrastrand CLs distort DNA
lacking the CDRE in a specific way, producing a structural motif
recognized by p53. We propose that directional and stable bending of
DNA lacking the CDRE toward its major groove due to formation of the
1,2-intrastrand CL of cisplatin affords a structural element exhibiting
this specific affinity to p53. In this way a stable flexure of DNA by
1,2-intrastrand CL provides an opportunity for more stable contacts
between p53 and DNA lacking CDRE. In other words, the lesions such as
1,2-intrastrand CLs that efficiently induce the directional and fixed
bend in DNA toward the major groove, thus providing a stable pre-bent
site on DNA to p53, serve as a structural motif for recognition of DNA
lacking CDRE by p53.
The observation that other minor bifunctional DNA adducts formed by
cisplatin in usual B-DNA lacking CDRE, which also bend and unwind DNA,
are not recognized by p53 deserves further discussion. The intrastrand
CL formed by cisplatin between two G residues separated by a third base
also bends the helix axis toward the major groove by ~30° (36) and
locally unwinds DNA by ~19° (26). However, another important
feature of the conformational alteration induced by this lesion is that
DNA is locally denatured and flexible at the site of the adduct (36),
the structural feature much less pronounced in the 1,2-intrastrand
adduct. Furthermore, interstrand CLs of cisplatin also distort DNA
distinctly differently and more severely than intrastrand adducts (see
above). These significant structural differences between the latter
minor adducts and the major 1,2-intrastrand CLs suggest that their
recognition and binding by active p53 will be different and restricted
to the structural features identical or similar to those induced in DNA
by the 1,2-intrastrand adduct of cisplatin. Consistent with this idea
is the observation that there are also significant structural
differences between 1,2-intrastrand CLs of cisplatin and the adducts
formed on DNA by transplatin (see above), which do not form the lesions
recognized by p53 on DNA lacking CDREs. Thus, the data base on
the structures of cisplatin and transplatin adducts reveals their
propensity to distort DNA in very different ways. The distinctive
structural features of 1,2-intrastrand CLs of cisplatin suggest a
unique role for this adduct in the enhancement of the binding of p53 to
platinated DNA segments lacking CDRE, which is, however, weaker than
the binding of p53 to unplatinated CDRE.
Biological Implications--
There is substantial evidence
suggesting that p53 plays a central role in the cellular response to
DNA damage. It is also clear that p53 function may only be one of many
factors that modulate cisplatin sensitivity, and the effects may be
different for various cell types. In addition, sequence-specific DNA
binding is one of the key biochemical activities responsible for much
of the biological function of p53. Hence, the observation described in the present work demonstrating that cisplatin adducts formed in the
CDRE reduce its binding affinity to active p53 may affect these key
biochemical activities. Because the affinity of p53 to the CDRE is not
affected by the adducts of clinically ineffective transplatin, we
suggest that the reduced affinity of the active p53 protein to the CDRE
due to its modification by cisplatin is relevant to the biological
activity of this drug.
A possible intriguing scenario for response to cellular exposure to
cisplatin is also that associated with another important result of the
present work demonstrating a relatively weak but significant increase
in the binding affinity of p53 to major adducts of
cisplatin-1,2-intrastrand CLs formed in the parts of DNA lacking CDRE.
This increase in the binding affinity of p53 is specific for DNA
modified by cisplatin and does not occur if DNA is modified by
clinically ineffective transplatin. This result is consistent with the
view that the mechanism of antitumor activity of cisplatin may also be
associated with its efficiency to promote binding activity of p53 in
the segments of DNA lacking CDRE. We suggest that 1,2-intrastrand CLs
formed by cisplatin in DNA segments lacking CDRE may be sufficient to
hijack p53 protein, keeping it away from its natural targets. Thus, the
resultant complexes may divert p53 from its natural functions and/or
may protect the cisplatin damage from its recognition by other cellular
components. Alternatively, it has been shown (37) that p53 enhances
binding of DNA modified by cisplatin to chromosomal high mobility group
(HMG) 1 protein. HMG domain proteins are known to specifically bind
1,2-intrastrand CLs of cisplatin and thus mediate antitumor effects of
this drug (3). The details of how the binding of HMG domain proteins to
cisplatin-modified DNA sensitizes tumor cells to cisplatin are still
not completely resolved, but possibilities such as shielding cisplatin-DNA adducts from excision repair or titrating away these proteins from their transcriptional regulatory function have
been suggested as clues to how these proteins are involved in the
antitumor activity (9). It is therefore possible that p53 and HMG1
proteins encounter each other at the sites of 1,2-intrastrand adducts
of cisplatin (formed outside the CDRE). This interaction could further promote keeping p53 away from its natural target and/or promote the binding affinity of HMG1 protein to cisplatin-modified DNA, with
the consequences for the antitumor effect of cisplatin mentioned above.
More detailed information about cellular consequences of interactions of platinated DNA and p53 protein is required before any
definite conclusions can be drawn about how these interactions contribute to the mechanism of the biological activity of
cisplatin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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[in a new window]
Fig. 1.
Sequences of the 20-bp-long synthetic
oligodeoxyribonucleotide duplexes used in the present study, with their
abbreviations. The top and bottom strands of each pair are
designated top and bottom, respectively, in the text. The bold
letters in the top strands of d(TGGT)/d(ACCA) and
d(TGTGT)/d(ACACA) duplexes indicate the location of the intrastrand CL
after modification of the oligonucleotides by cisplatin. The bold
letters in both strands of d(TGCT)/d(AGCA) duplex indicate
the sites involved in the interstrand CL of cisplatin. The bold
letter in the top strand of the d(TGCT)/d(AGCG) duplex also
indicates the location of the monofunctional adduct of [Cl(dien)Pt]Cl
in this duplex.
-32P]ATP used for 5'-end radioactive labeling of the
top strands of the oligonucleotide duplexes were purchased from New
England Biolabs (Beverly, MA) and Amersham Pharmacia Biotech,
respectively. Acrylamide, bis(acrylamide), agarose, and urea were from
Merck KgaA (Darmstadt, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Binding of p53 to the PvuII
fragment of pPMG1 DNA, which was 474-bp-long and contained
CDRE. The fragment was unplatinated (lanes 1 and
6) or globally modified by cisplatin (lanes 2-4
and 7-9) or transplatin (lanes 5 and
10). A gel mobility retardation assay was performed in the
presence of the unplatinated 2513-bp nonspecific competitor
(PvuII fragment of pPMG1 lacking CDRE) in 1% agarose gel;
concentrations of the 474- and 2513-bp fragments were 10 and 57 µg/ml
(3.3 and 3.5 × 10 8 M),
respectively, and the concentration of the p53 protein was 0 (lanes 1-5) or 3.14 × 10
8
M (lanes 6-11). rb values
were as follows: 0 (lanes 1, 6, and
11); 0.02 (lanes 2 and 7); 0.04 (lanes 3 and 8); 0.06 (lanes 4, 5, 9, 10). Lane 11 was the same as lane 6,
but mAb DO-1 was added at a molar ratio of mAb to p53 of 3. For
other details, see "Experimental Procedures."
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Fig. 3.
Binding of p53 to the 20-bp duplex containing
CDRE (oligo-CDRE). The duplex was unplatinated (lanes 1 and 2) or globally modified to rb = 0.05 by cisplatin (lanes 3 and 4) or transplatin
(lanes 5 and 6). A gel mobility retardation assay
was performed in the presence of the unplatinated 2513-bp nonspecific
competitor (PvuII fragment of pPMG1 lacking CDRE) in 5%
native polyacrylamide gel; concentrations of the oligo-CDRE and 2513-bp
fragment were 1.6 and 10 µg/ml (1.26 × 10 7 and 6 × 10
9 M), respectively, and the
concentration of p53 was 0 (lanes 1, 3, and
5) or 3.9 × 10
8
M (lanes 2, 4, and 6). The
oligo-CDRE was radioactively labeled at the 5'-end of the top
strand. For other details, see "Experimental
Procedures."
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Fig. 4.
Binding of p53 to the PvuII
fragment of pBluescript SK II+ DNA,
which was 448-bp-long and lacked CDRE. The fragment was
unplatinated (lanes 1 and 6) or globally modified
by cisplatin (lanes 2-4, 7-9, and
11) or transplatin (lanes 5 and 10). A
gel mobility retardation assay was performed in the presence of the
unplatinated 2513-bp nonspecific competitor (PvuII fragment
of pPMG1 lacking CDRE) in 1% agarose gel; concentrations of the 448- and 2513-bp fragments were 10 and 57 µg/ml (3.1 and 3.5 × 10 8 M), respectively, and the
concentration of p53 was 0 (lanes 1-5) or 1.88 × 10
7 M (lanes 6-11).
rb values were as follows: 0 (lanes 1 and
6); 0.01 (lanes 2 and 7); 0.05 (lanes 3 and 8); 0.08 (lanes 4,
5, and 9-11). Lane 11 was the
same as lane 9, but mAb DO-1 was added at a molar
ratio of mAb to p53 of 3. For other details, see
"Experimental Procedures."
View larger version (21K):
[in a new window]
Fig. 5.
Binding of p53 protein to the 20-bp
oligonucleotide duplexes lacking CDRE and containing no, or a single,
site-specific adduct of cisplatin. A gel mobility retardation
assay was performed in the presence of the unplatinated 2513-bp
nonspecific competitor (PvuII fragment of pPMG1 lacking
CDRE) in 5% native polyacrylamide gel; concentrations of the
oligonucleotide duplexes and 2513-bp fragments were 1.6 and 10 µg/ml
(1.26 × 10 7 and 6 × 10
9 M), respectively, and the
concentration of p53 was 0 M (lanes 1,
3, 7, 9, and 11), 1.17 × 10
7 M (lane 4),
1.53 × 10
7 M (lane
5), or 1.88 × 10
7 M
(lanes 2, 6, 8, 10, and
12). Unplatinated duplex d(TGGT)/d(ACCA), lanes 1 and 2; d(TGGT)/d(ACCA) containing 1,2-GG intrastrand CL,
lanes 3-6; d(TGTGT)/d(ACACA) containing 1,3-GTG
intrastrand CL, lanes 7 and 8; d(TGCT)/d(AGCA)
containing 1,2-GG interstrand CL, lanes 9 and 10;
d(TGCT)/d(AGCA) containing a monofunctional adduct of [Cl(dien)Pt]Cl
at the central G residue in the top strand (lanes 11 and
12). The oligonucleotide duplexes were radioactively labeled
at the 5'-end of the top strand. For other details, see
"Experimental Procedures." IEC, interstrand
CL.
7 M
(Fig. 5, lane 5), whereas that at which half of the
unplatinated oligo-CDRE was bound was roughly 10 times lower. This
rough comparison shows that the binding affinity of p53 to
1,2-intrastrand cross-link of cisplatin formed in DNA lacking CDRE is
lower than the binding affinity of p53 to unplatinated CDRE.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. B. Vojtesek for critical reading of the manuscript and helpful discussions.
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FOOTNOTES |
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* This work was supported in part by the Grant Agency of the Czech Republic (Grants 305/99/0695, 305/01/0418, and 301/00/P094), the Grant Agency of the Academy of Sciences of the Czech Republic (Grant A5004101), and the Internal Grant Agency of the Ministry of Health of the Czech Republic (Grant NL6058-3/2000).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by an International Research Scholar's award from the Howard Hughes Medical Institute and the Wellcome Trust (United Kingdom).
To whom correspondence should be addressed: Institute of
Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-61265 Brno, Czech Republic. Tel.: 420-5-41517148; Fax: 420-5-41240499; E-mail: brabec@ibp.cz.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M101224200
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ABBREVIATIONS |
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The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); CL, cross-link; transplatin, trans-diamminedichloroplatinum(II); wt, wild-type; bp, base pair(s); CDRE, consensus DNA response element; [Cl(dien)Pt]Cl, diethylenetriaminechloroplatinum(II) chloride; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; HMG, high mobility group.
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