DNA Interstrand Cross-links of the Novel Antitumor Trinuclear
Platinum Complex BBR3464
CONFORMATION, RECOGNITION BY HIGH MOBILITY GROUP DOMAIN
PROTEINS, AND NUCLEOTIDE EXCISION REPAIR*
Jana
Kasparkova
§,
Jana
Zehnulova,
Nicholas
Farrell¶
, and
Viktor
Brabec**
From the Institute of Biophysics, Academy of Sciences
of the Czech Republic, Kralovopolska 135, CZ-61265 Brno, Czech
Republic and the ¶ Department of Chemistry, Virginia Commonwealth
University, Richmond, Virginia 23284-2006
Received for publication, August 6, 2002, and in revised form, August 25, 2002
 |
ABSTRACT |
The novel phase II antitumor polynuclear platinum
drug BBR3464
([(trans-PtCl(NH3)2)2(µ-trans-Pt(NH3)2(NH2(CH2)6NH2)2)](NO3)4) forms intra- and interstrand cross-links (CLs) on DNA (which is the
pharmacological target of platinum drugs). We examined first in
our recent work how various intrastrand CLs of BBR3464 affect the
conformation of DNA and its recognition by cellular
components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the
present work, we have extended the studies on the DNA interstrand CLs
of this drug. The results have revealed that the interstrand CLs are
preferentially formed between guanine residues separated by 2 base
pairs in both the 3' 
3' and 5' 5' directions. The major
1,4-interstrand CLs distort DNA, inducing a directional bending of the
helix axis and local unwinding of the duplex. Although such distortions
represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of
BBR3464. On the other hand, in contrast to intrastrand adducts of
BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide
excision repair. It has been suggested that interstrand CLs of BBR3464
could persist considerably longer in cells compared with intrastrand
adducts, which would potentiate the toxicity of the interstrand lesions
to tumors sensitive to this polynuclear drug.
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INTRODUCTION |
It is now well established that polynuclear platinum compounds in
which two or three platinum coordination units are linked in a linear
fashion compose an important new class of anticancer drugs [1,2]. The
first clinical compound, currently denoted BBR3464 (see Fig.
1A), is a bifunctional trinuclear DNA-binding agent with an
overall 4+ charge (3). The phase I trials (4, 5) demonstrated a clear
pattern of responses in cancers not normally treatable with cisplatin
(cis-diamminedichloroplatinum(II)) (see Fig. 1A),
including responses in melanoma and pancreatic and lung cancer.
Objective responses in phase II were verified in relapsed ovarian
cancer and non-small cell lung cancer (6, 7). Pre-clinical studies
indicated activity in p53 mutant tumors and a minimum induction of p53
following BBR3464 treatment. The interactions of antitumor polynuclear
platinum compounds with target DNA (for reviews, see Refs. 1, 2, and 8)
are distinct from those of the mononuclear based cisplatin family and,
indeed, unlike those of any DNA-damaging agent in clinical use. Hence,
it is important to understand these novel interactions to the greatest extent possible and how they affect DNA structure and function to
exploit the full clinical potential of these new agents.
Bifunctional binding of BBR3464 to double-stranded DNA preferentially
involves guanine residues and is characterized by the rapid formation
of long-range intra- and interstrand cross-links (CLs)1 in which the
platinated sites are separated by 1 or more bp (9). Quantitation
of DNA interstrand cross-linking in natural and linear DNAs indicated
~20% of the DNA to be interstrand cross-linked. This value is
significantly higher than that for cisplatin (10, 11); on the other
hand, an intriguing aspect of BBR3464 is that long-range delocalized
intrastrand adducts are equally or even more probable than interstrand
CLs (9). As the [Pt,Pt] intrastrand CLs of BBR3464 are analogs of the
major adducts of cisplatin, which forms ~90% bifunctional
intrastrand adducts between neighboring purine residues on DNA, we
examined first in our recent work (12) how the structures of the
various types of the intrastrand CLs of BBR3464 affect conformational
properties of DNA and how these adducts are recognized by the HMGB1
protein and removed from DNA during in vitro nucleotide
excision repair (NER) reactions. These studies were performed because
some structures altered by platinum adducts, such as stable directional
bending and unwinding, attract various damaged DNA-binding proteins
such as those containing HMG domains (13-15). The binding of these
proteins has been postulated to mediate the antitumor properties of the
platinum drugs (14, 15). In addition, several reports have demonstrated
that intrastrand CLs of cisplatin are removed from DNA during NER
reactions and that NER is also a major mechanism contributing to
cisplatin resistance (16-18). Recent work has revealed that
intrastrand CLs of BBR3464 create a local conformational distortion,
but that none of these intrastrand adducts results in a stable
curvature such as that observed for the major 1,2-intrastrand CL of
cisplatin (12). In addition, we have observed also, in contrast to the
1,2-GG intrastrand CL of cisplatin, no recognition of the intrastrand CLs of BBR3464 by HMGB1 proteins, but we have observed effective removal of these adducts from DNA by NER. We have suggested that the
processing of the intrastrand CLs of BBR3464 in tumor cells sensitive
to this drug may not be relevant to its antitumor effects because these
CLs do not persist to potentiate the anticancer effect of this drug.
This also implies that polynuclear platinum compounds apparently
represent a novel class of platinum anticancer drugs acting by a
different mechanism than cisplatin and its analogs.
On the other hand, BBR3464 also forms interstrand CLs on DNA with a
considerably higher frequency compared with cisplatin (9). In general,
DNA interstrand CLs could be even more effective lesions than
intrastrand adducts in terminating DNA or RNA synthesis in tumor cells
and thus could be even more likely candidates for the genotoxic lesion
relevant to the antitumor effects of BBR3464. In addition, the
interstrand CLs pose a special challenge to repair enzymes because they
involve both strands of DNA and would not be repaired so readily as
intrastrand lesions because these repair enzymes would require the
information in the complementary strand for resynthesis. In this way,
the interstrand CLs might persist considerably longer than intrastrand
adducts, which would result in a higher cytotoxicity of the interstrand
lesions. Interestingly, the interstrand CLs formed by cisplatin, which
still remain important candidates for a genotoxic lesion relevant to
the antitumor effects of this platinum drug, preferentially form
between guanine residues in the 5'-GC/5'-GC sequence. This adduct
induces several irregularities in DNA (19-21). The guanine residues
interstrand cross-linked by cisplatin 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 76-80° relative to B-DNA and also the bending of
20-40° of the helix axis at the cross-linked site toward the minor
groove. In addition, the interstrand CL of cisplatin is specifically
recognized by the full-length HMGB1 protein (22) and its domain
B2 and is not removed by the
NER system under conditions in which the 1,2-GG intrastrand CL is
readily excised (16).
Cellular pharmacology studies in L1210 and osteosarcoma cells using
alkaline elution show the persistence of interstrand CLs with time,
consistent with a slower rate of repair (23, 24). Hence, data on
conformation, recognition by HMGB1 domain proteins, and NER of DNA
interstrand CLs of BBR3464 are needed to obtain greater insight into
which DNA adduct of BBR3464 is the more likely lesion responsible for
the antitumor effects of this polynuclear platinum drug.
In this work, we continue our investigations of the DNA interactions of
BBR3464 in cell-free medium to address further fundamental questions
about the mechanism of antitumor activity of this novel platinum drug.
In a previous report (9) using an indirect assay employing
transcription mapping of the DNA fragment globally modified by BBR3464,
some sites in DNA involved in the interstrand CLs have been already
suggested. However, no systematic study on the sequence specificity of
these lesions has been performed. Therefore, in this work, we examined
in detail which sites are preferentially involved in the interstrand
CLs of BBR3464. In addition, we also studied how interstrand CLs of
BBR3464 affect the local conformation of DNA (in particular, bending
and unwinding) and how these adducts are recognized by the HMGB1 domain
proteins and removed from DNA during in vitro NER reactions.
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EXPERIMENTAL PROCEDURES |
Chemicals--
BBR3464 (see Fig. 1A) was prepared by
standard methods. Cisplatin (see Fig. 1A), dimethyl sulfate
(DMS), KMnO4, diethyl pyrocarbonate (DEPC), KBr, and
KHSO5 were obtained from Sigma (Prague, Czech Republic).
The stock solutions of platinum compounds were prepared at a
concentration of 5 × 10
4 M in 10 mM NaClO4 and stored at 4 °C in the dark.
The synthetic oligodeoxyribonucleotides (see Fig. 1B) were
synthesized and purified as described previously (25). Expression and
purification of rat recombinant full-length HMGB1 protein and its
domains A (residues 1-84) and B (residues 85-180) (HMGB1a and HMGB1b,
respectively) were carried out as described (26, 27). Human recombinant replication protein A (RPA) was purified from Escherichia
coli (28) and was a kind gift of John J. Turchi. T4 DNA ligase and T4 polynucleotide kinase were purchased from New England Biolabs Inc.
(Beverly, MA). Acrylamide, bisacrylamide, urea and NaCN were from Merck
(Darmstadt, Germany). [
-32P]ATP was from
Amersham Biosciences. ATP was from Roche Molecular Biochemicals
(Mannheim, Germany).
Platination of Oligonucleotides--
The single-stranded
oligonucleotides (the top strands of the duplexes in Fig.
1B, except [TGGT] and [GTG(NER)]) were reacted in
stoichiometric amounts with the monoaqua derivative of BBR3464. The
platinated oligonucleotides were re-purified by ion-exchange fast
protein liquid chromatography (FPLC). We verified that the modified
oligonucleotides contained three platinum atoms by platinum flameless
atomic absorption spectrophotometry and by measurement of the optical
density. Using DMS footprinting of platinum on DNA (10, 29, 30), we
also verified that in the platinated top strands of all duplexes, N-7
of a single guanine (G) residue was not accessible for reaction with
DMS. The oligonucleotides were then treated with hot piperidine and
analyzed by denaturing 24% PAGE. For the unmodified
oligonucleotides, shortened fragments due to cleavage of the strand at
one methylated G residue were observed on the gel. However, no such
bands were detected for the oligonucleotides modified by BBR3464. These
results indicate that one BBR3464 molecule was coordinated to a single
G residue in the top strands of all duplexes. The platinated top
strands were allowed to anneal with unplatinated complementary strands (the bottom strands in Fig. 1B) in 0.1 M
NaClO4. The resulting products were analyzed by FPLC in an
alkaline gradient. Using this denaturing gradient, non-interstrand
cross-linked strands were eluted as a 20-23-nucleotide single strand,
whereas the interstrand cross-linked strands were eluted later in a
single peak as a higher molecular mass species. Only this single peak
was collected so that the samples of the interstrand cross-linked
duplexes contained no single-stranded molecules. Alternatively, the
products were separated on denaturing 8 M urea and 12%
polyacrylamide gel; the bands corresponding to interstrand cross-linked
duplexes were analyzed by densitometry or were cut off from the gel,
eluted, precipitated by ethanol, and dissolved in 50 mM
NaCl. Both procedures for the purification of interstrand cross-linked
duplexes provided products whose subsequent analysis (see below) gave
identical results. FPLC purification and flameless atomic absorption
spectrophotometry measurements were carried out in an Amersham
Biosciences FPLC system with a Mono Q HR 5/5 column and with a Unicam
939 AA spectrometer equipped with a graphite furnace, respectively.
Duplexes [TGGT] and [GTG(NER)] (see Fig. 1B) containing
a single 1,2-GG or 1,3-GTG intrastrand CL of BBR3464 or cisplatin in
the top strand were prepared as described (10, 12, 29-32). The
unmodified or intrastrand CLs containing duplexes used in the studies
of recognition by HMGB1 domain proteins and RPA were purified by
electrophoresis on native 15% polyacrylamide gel
(monoacrylamide/bisacrylamide ratio of 29:1). Other details have been
described previously (10, 25, 33).
Chemical Modifications--
The modifications by
KMnO4, DEPC, and KBr/KHSO5 were performed as
described previously (33-36). The strands of the duplexes were
5'-end-labeled with [-32P]ATP. In the case of the
platinated oligonucleotides, the platinum complex was removed after
reaction of the DNA with the probe by incubation with 0.2 M
NaCN (pH 11) at 45 °C for 10 h in the dark.
Ligation and Electrophoresis of
Oligonucleotides--
Unplatinated single strands (top strands in Fig.
1B) and the duplexes containing a unique interstrand CL were
5'-end-labeled with [-32P]ATP using T4 polynucleotide
kinase. The single-stranded, unplatinated oligonucleotides were
subsequently annealed with their phosphorylated complementary strands.
Unplatinated or interstrand CL-containing duplexes were allowed to
react with T4 DNA ligase. The resulting samples along with
ligated unplatinated duplexes were subsequently examined on native
8% polyacrylamide gel (monoacrylamide/bisacrylamide ratio of 29:1).
Other details of these experiments were as described in previously
(37-39).
Gel Mobility Shift Assay--
The 21-mer oligonucleotides
(bottom strands of duplexes [1,4;3'-3'(21)] and [1,4;5'-5'(21)]
shown in Fig. 1B to which one adenine residue was added to
their 3'-ends and one adenine residue was removed from their 5'-ends)
were 5'-end-labeled and annealed (see above) to their complementary
strands (top strands of duplexes [1,4;3'-3'(21)] and
[1,4;5'-5'(21)] shown in Fig. 1B), which were either
unplatinated (controls) or contained monofunctional adducts of BBR3464
on the central G residue. The platinated duplexes, which had blunt
ends, were further incubated for 24 h in 0.1 M NaClO4 at 37 °C to form interstrand CLs. The
oligonucleotides containing interstrand CLs were separated by
electrophoresis on denaturing 12% polyacrylamide gel, isolated from
the gel by elution, extracted by phenol/chloroform and chloroform,
precipitated by ethanol, and used as DNA probes in the experiments with
HMGB1a and HMGB1b proteins and RPA. The 149-bp probes with blunt ends used in the experiments with the full-length HMGB1 protein were prepared in the same way as those used in the NER assays (see below).
Reactions with the Full-length HMGB1 Protein and Its
Domains--
The unplatinated and platinated duplexes (0.4 nM) were incubated with increasing concentrations of HMGB1a
or HMGB1b protein in 20-µl sample volumes containing 10 mM HEPES (pH 7.5), 10 mM MgCl2, 50 mM LiCl, 100 mM NaCl, 1 mM
spermidine, 0.2 mg/ml bovine serum albumin, and 0.05% Nonidet P40.
Samples were incubated on ice for 30 min and then brought to 7% in
sucrose and 0.017% in xylene cyanol prior to loading on prerun and
precooled (4 °C) native 6% polyacrylamide gels
(monoacrylamide/bisacrylamide ratio of 29:1). The gels were
electrophoresed for 3 h and visualized using an Amersham
Biosciences PhosphorImager (Storm 860 system), and the bands were
quantitated with ImageQuant software. The experiments with the
full-length HMGB1 protein were performed in the same way, but with
minor modifications. The labeled 149-bp DNA probes were titrated with
the protein in the presence of 0.1 mg/ml sonicated calf thymus DNA in
buffer composed of 10 mM HEPES (pH 7.9), 10 mM
MgCl2, 150 mM NaCl, 0.2 mg/ml bovine serum
albumin, 20% (v/v) glycerol, and 1 mM dithiothreitol.
Reactions with RPA--
32P-Labeled DNA substrates
(0.5 nM) and the amounts of RPA indicated were incubated at
20 °C in 20-µl reactions containing 25 mM HEPES-KOH
(pH 8.3), 30 mM KCl, 4 mM MgCl2, 1 mM EDTA, 0.9 mM dithiothreitol, 45 µg/ml
bovine serum albumin, and 10% glycerol. To assess binding at
equilibrium, reactions were stopped after 30 min by cooling the samples
to 0 °C. Following addition of gel loading buffer (4 µl)
containing 100 mM Tris-HCl (pH 8.3), 10% glycerol, and
0.05% orange G, the extent of binding was determined on native 6%
polyacrylamide gels. Electrophoresis was performed for 50 min at
4 °C; gels were dried and visualized using the Amersham Biosciences
PhosphorImager (Storm 860 system); and the radioactivities associated
with bands were quantitated with ImageQuant software.
Nucleotide Excision Assay--
The 149-bp substrates containing
a single central 1,4-interstrand CL of BBR3464 in the 5' 5' or 3'
3' direction were assembled from three oligonucleotide duplexes.
The central duplex was duplex [1,4;3'-3'(NER)] or
[1,4;5'-5'(NER)] (shown in Fig. 1B) to which two duplexes
(arms) with random base pair sequences with overhangs partially
overlapping those of the interstrand cross-linked duplex were ligated
(one to each side) by T4 DNA ligase. Both strands of the interstrand
cross-linked central duplexes were 5'-end-labeled with 32P
before ligation.
The intrastrand cross-linked substrates were prepared in the similar
following way. The 21-mer oligonucleotide (the top strand of duplex
[GTG(NER)] shown in Fig. 1B) was used for preparation of
linear 149-bp duplexes with the centrally located 1,3-GTG intrastrand CL of BBR3464 or cisplatin. Uniquely modified 21-mers were end-labeled to introduce a radiolabel at the 11th phosphodiester bond 5' to the CL
and annealed with the bottom strand of duplex [GTG(NER)]. Two
duplexes (arms) with random base pair sequences with overhangs partially overlapping those of the central platinated duplex were ligated to this intrastrand cross-linked duplex (one to each side) by
T4 DNA ligase.
Full-length substrates (unplatinated, containing the 1,4-interstrand CL
of BBR3464 or the 1,3-intrastrand CL of BBR3464 or cisplatin) were
separated from unligated products on denaturing 6% polyacrylamide gel,
purified by electroelution, re-annealed, and stored in annealing buffer
(50 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM MgCl2, and 1 mM
dithiothreitol) at 
20 °C. Other details of the purification
of DNA substrates for NER were the same as described previously (40,
41).
Oligonucleotide excision reactions were performed in cell-free extracts
(CFEs) prepared from the HeLa S3 and Chinese hamster ovary AA8 cell
lines as described (17, 42). These extracts were kindly provided by
J. T. Reardon and A. Sancar (University of North Carolina, Chapel
Hill, NC). In vitro repair of interstrand CLs of BBR3464 was
measured in an excision assay using these CFEs and 149-bp linear DNA
substrates (see above) as described previously (17) with minor
modifications. The reaction mixtures (25 µl) contained 10 fmol of
radiolabeled DNA, 50 µg of CFE, 20 µM dATP, 20 µM dCTP, 20 µM dGTP, and 20 µM TTP in reaction buffer (23 mM HEPES (pH
7.9), 44 mM KCl, 4.8 mM MgCl2, 0.16 mM EDTA, 0.52 mM dithiothreitol, 1.5 mM ATP, 5 µg of bovine serum albumin, and 2.5% glycerol)
and were incubated at 30 °C for 40 min. DNA was deproteinized and
precipitated by ethanol. Reaction products were treated
overnight with 0.4 M NaCN (pH 10-11) at 45 °C and
precipitated by ethanol prior to resolution on the gels. The excision
products were separated on denaturing 10% polyacrylamide gels and
visualized using the Amersham Biosciences PhosphorImager (Storm 860 system).
| |
RESULTS |
Sequence Specificity of Interstrand Cross-linking--
We
demonstrated in our previous work (9) that the preferential DNA-binding
sites of BBR3464 are G residues and that along with the delocalized
intrastrand CLs, the long-range interstrand CLs are the most frequent
lesions produced by this drug on DNA. In these CLs, the platinated
sites are separated by 1 or more base pairs. Bifunctional BBR3464 must
bind to DNA first through one end of the trinuclear unit. In this first
step of binding, the kinetic preferences are similar to those of
mononuclear species, i.e. they coordinate preferentially to
N-7 atoms of G residues. The array of adducts becomes, however,
different upon the coordination of the second platinum unit (9).
Considering these facts and our intention to investigate, in the
present work, interstrand CLs of BBR3464, we have designed a series of
synthetic oligodeoxyribonucleotide duplexes, [1,2], [1,4], and
[1,6], whose sequences are shown in Fig.
1B. The pyrimidine-rich top
strands of these duplexes contained only one G residue in the center
(shown in boldface in Fig. 1B). These top strands
were modified by BBR3464 so that they contained a single monofunctional
adduct of this platinum complex at the central G site. The duplexes
were also designed in such a way that their bottom (complementary)
strands contained a G residue in different positions symmetrical to the
single central cytosine (C) residue (complementary to the platinated G
residue in the top strand). In this way, the G residue in the top
strand with the monofunctionally attached BBR3464 may close to
the 1,2-, 1,4-, or 1,6-GG interstrand CL in duplex [1,2], [1,4], or
[1,6], respectively. The 1,2-interstrand CL is formed between G sites
in neighboring base pairs, whereas in 1,4- and 1,6-interstrand CLs, the
platinated G sites are separated by 2 and 4 base pairs, respectively. G
sites in the bottom strands involved in these interstrand CLs are also shown in boldface in Fig. 1B. The nucleotide
sequences of the duplexes were also designed in such a way that these
interstrand CLs may close to the G residues located on both
sides of the central C residue in the bottom strands, i.e.
in the 5' 5' or 3' 3' direction. The orientation of the
interstrand CL in the 5' 5' or 3' 3' direction can be explained
with the aid of the sequence of duplex [1,4] (Fig. 1B).
For instance, the 1,4-GG interstrand CL oriented in the 5' 5'
direction is that formed in duplex [1,4] between the central G
residue in the top strand and G8 in the bottom strand,
whereas the same CL oriented in the 3' 3' direction is that between
the central G residue in the top strand and G14 in the
bottom strand.
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Fig. 1.
Structures of platinum complexes
(A) and sequences of the synthetic
oligodeoxyribonucleotides used in this study and their abbreviations
(B). The boldface G residues in the
top strands of all duplexes except duplexes [TGGT] and [GTG(NER)]
indicate the location of the monofunctional adduct of BBR3464 formed
before the interstrand cross-linking reaction as described under
"Experimental Procedures." The boldface letters in the
top strands of duplexes [TGGT] and [GTG(NER)] indicate the location
of the intrastrand CL after modification of the oligonucleotides by
BBR3464 or cisplatin as described under "Experimental Procedures."
The boldface letters in the bottom strands indicate the
locations of the sites that could be involved in the interstrand CLs.
For duplexes [1,2], [1,4], and [1,6], the numbering of the
nucleotide residues in the bottom strand is also shown.
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The monoadducted top strands of duplexes [1,2], [1,4], and [1,6]
were hybridized with their complementary strands, and the hybrids were
incubated in 0.1 M NaClO4 at 37 °C. The
aliquots were withdrawn at various time intervals and analyzed by gel
electrophoresis under denaturing conditions. As shown in Fig.
2A for duplex [1,4] modified
by BBR3464, only one band was observed for the non-cross-linked duplex.
The subsequent incubation resulted in new bands migrating markedly more
slowly. Their intensity increased with the incubation time, with a
concomitant decrease in the intensity of the band corresponding to the
non-cross-linked duplex. This observation can be interpreted to mean
that interstrand CLs were formed. From the ratio of the sum of
intensities of all bands corresponding to cross-linked duplexes to the
sum of intensities of all bands, the percentage of interstrand CLs was
calculated (Fig. 2B). The highest rate of this interstrand
cross-linking reaction was observed in duplex [1,4]. The CLs in
duplex [1,2] were formed with a slightly lower rate, whereas the rate
of the cross-linking reaction in duplex [1,6] was markedly lower.
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Fig. 2.
Kinetics of DNA interstrand CL
formation by BBR3464. Duplexes [1,2], [1,4], and [1,6] were
formed by mixing their bottom strands with the complementary top strand
uniquely monoadducted by BBR3464 at the central G residue at 37 °C.
A, autoradiogram of a denaturing 8 M urea and
12% polyacrylamide gel of duplex [1,4] with a
32P-end-labeled bottom strand. The cross-linking reaction
was stopped by adjusting the NaOH concentration to 10 mM
and cooling the samples to 70 °C. inter designates
bands corresponding to the interstrand cross-linked fraction (see also
"Results"); SS indicates the unplatinated
strand. Lane 1, 0 h; lane 2, 1 h; lane 3, 3 h; lane 4, 8 h; lane
5, 24 h; lane 6, 48 h. B, the
percentage of interstrand cross-linking in duplexes [1,2] ( ),
[1,4] ( ), and [1,6] ( ) by BBR3464 calculated from the ratio
of the sum of the intensities of the bands corresponding to the
fragments containing an interstrand CL to the sum of the intensities of
all bands (corresponding to the non-cross-linked and cross-linked
oligonucleotides).
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The cross-linking reactions in duplexes [1,2], [1,4], and [1,6]
resulted in only one band or single peak in the FPLC profile designated
as interstrand (shown for duplex [1,4] in Fig. 2A). After
a 24-h reaction period, the bands or FPLC peaks corresponding to the
interstrand cross-linked duplexes were cut off from the gel or
collected after FPLC separation, respectively; the duplexes were
isolated (eluted from the bands and precipitated or only precipitated
from the FPLC fractions) and further characterized by Maxam-Gilbert
footprinting (10, 29, 43).
The samples of duplexes [1,2], [1,4], and [1,6] interstrand
cross-linked by BBR3464 in which the top strand was only 5'-end-labeled with 32P were reacted with DMS, which does not react with
platinated G residues because N-7 is no longer accessible (10, 29, 43). The adducts were removed by NaCN (29, 30), and then the sample was
treated with piperidine. In the unplatinated duplexes, the central G residue in the top strands was reactive with DMS (data not
shown). It was no longer reactive in all three cross-linked duplexes.
This observation confirms that the unique G residue in the top strands
remained platinated and was involved in the interstrand CL
contained in the single fraction of interstrand cross-linked
duplexes (10, 29, 43).
In additional studies, the 1,2-, 1,4-, and 1,6-interstrand cross-linked
duplexes in which the bottom strand was 5'-end-labeled with
32P were examined (Fig. 3).
The interstrand cross-linked duplexes were reacted with DMS. These
samples were then further treated with NaCN to remove the adducts and
finally also with piperidine. The treatment with piperidine of the
control unplatinated duplexes resulted in cleavage at all G sites in
the bottom strand (Fig. 3, NoPt lanes). If the cross-linked
duplexes treated with DMS and subsequently NaCN were cleaved (Fig. 3,
Pt/CN lanes), bands corresponding
to all G residues were observed that had the same intensity as the
corresponding bands seen for unplatinated duplexes, except for the G
residues marked by arrows in Fig. 3. This result proves that
these G residues were platinated and involved in the interstrand CL of
BBR3464. Interestingly, the band corresponding to G12 in
the bottom strand of interstrand cross-linked duplex [1,2] entirely
disappeared, whereas the bands corresponding to other G residues in the
bottom strand had the same intensity as the corresponding bands seen
for the unplatinated duplex. This result implies that the interstrand
CLs were formed by BBR3464 exclusively in the 3' 3' direction.
Somewhat different results were obtained if interstrand cross-linked
duplexes [1,4] and [1,6] were analyzed. As shown in Fig.
3B, the intensities of the bands corresponding to the two G
residues at positions 8 and 14 of the bottom strand of duplex [1,4]
were affected only by formation of the interstrand CL of BBR3464. The
intensities of these bands were approximately one-half of the
intensities observed for the corresponding bands seen for the
unplatinated duplex. This result can be interpreted to mean that the
1,4-interstrand CLs of BBR3464 are formed with approximately the same
preference in both the 3' 3' and 5' 5' directions. Similarly,
in the case of duplex [1,6], the intensities of the bands
corresponding to the two G residues at positions 7 and 17 of the bottom
strand of duplex [1,6] were affected only by formation of the
interstrand CL of BBR3464, but to a different extent (Fig.
3C). Whereas the intensity of the band corresponding to
G17 was reduced by only ~30%, the intensity of the band
corresponding to G7 was reduced more pronouncedly by
~70%. This result indicates that the 1,6-interstrand CLs are also
formed by BBR3464 in both directions, but with a clear preference for
formation of the CL in the 5' 5' direction. Thus, the results shown
in Fig. 3 demonstrate that although a short 1,2-interstrand CL of
BBR3464 is formed preferentially in the 3' 3' direction, the
growing length of these CLs reverses this preference to the opposite 5'
5' direction. Importantly, the sequences of the bottom strands in
duplexes [1,2] and [1,4] were chosen so that the 1,7- and
1,8-interstrand CLs could be also formed in both directions, but DMS
footprinting of the platinated sites in these duplexes interstrand
cross-linked by BBR3464 revealed no 1,7- or 1,8-interstrand CLs (Fig.
3, A and B). Similarly, no 1,8-interstrand CL was
formed in interstrand cross-linked duplex [1,6], in which this type
of CL was also possible (Fig. 3C). Thus, the maximum
possible length of the long-range interstrand CL of BBR3464 corresponds
to the CL in which the platinated sites are separated by 4 base
pairs.
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Fig. 3.
Maxam-Gilbert footprinting experiments.
Shown are autoradiograms of denaturing 8 M urea and 24%
polyacrylamide gels of the products of the reaction between DMS
(G-specific reactions) and duplexes [1,2] (A), [1,4]
(B), and [1,6] (C) either unmodified or
containing an interstrand CL of BBR3464. The bottom strands of the
duplexes were 5'-end-labeled. NoPt lanes, unplatinated
duplex; Pt/CN lanes, the duplex
containing an interstrand CL with platinum removed by NaCN after
modification by DMS. For other details, see "Results."
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Chemical Probes of DNA Conformation--
Among the interstrand
adducts, 1,4-interstrand CLs represent the DNA lesion formed most
readily by BBR3464. Therefore, additional experiments in this work were
focused mainly on this type of CL. The oligonucleotide duplexes
containing a single 1,4-interstrand CL between G residues oriented in
both the 3' 3' and 5' 5' directions (duplexes
[1,4;3'-3'(21)] and [1,4;5'-5'(21)] in Fig. 1B,
respectively) were further analyzed with chemical probes of DNA
conformation. The interstrand cross-linked duplexes were treated with
several chemical agents that are used as tools for monitoring the
existence of conformations other than canonical B-DNA. These agents
include KMnO4, DEPC, and bromine. They react preferentially with single-stranded DNA and distorted double-stranded DNA (33-36, 44). As we used for this analysis exactly the same methodology described in detail in our recent work aimed at DNA intrastrand CLs of
BBR3464 (12), the results are discussed briefly.
KMnO4 is hyperreactive with thymine (T) residues in
single-stranded nucleic acids and in distorted DNA compared with B-DNA (34, 36, 45, 46). The interstrand cross-linked duplexes showed strong
reactivity for the two 5'-T residues adjacent to the adduct (shown for
duplex [1,4;3'-3'] in Fig.
4A, ICL lane). A
somewhat weaker reactivity was also observed for the 3'-T residue adjacent to the platinated G residue involved in the CL.
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Fig. 4.
Chemical probes of DNA conformation.
Shown are the results from piperidine-induced specific strand cleavage
at KMnO4-modified (A),
KBr/KHSO5-modified (B and C), and
DEPC-modified (D) bases in duplex [1,4;3'-3'(21)]
unplatinated or containing a single 1,4-GG interstrand CL of BBR3464 in
the 3' 3' direction. The top (A and B) or
bottom (C and D) strands of the oligomers were
5'-end-labeled. ICL lanes, the duplex interstrand
cross-linked by BBR3464; DS lanes, unplatinated duplex;
SS lanes, unplatinated strand; G lanes, a
Maxam-Gilbert-specific reaction for the unplatinated duplex. A summary
of the reactivity of the chemical probes is given in E.  and  , strong and weak reactivity, respectively.
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DEPC carbetoxylates purines at N-7. It is hyperreactive with
unpaired and distorted adenine (A) residues in DNA and with left-handed Z-DNA (34, 36, 47, 48). Within the double-stranded oligonucleotides containing the interstrand CL, four base residues in the bottom strand
of duplex [1,4;3'-3'] became reactive (Fig. 4C, ICL
lane). The strongly reactive residues were readily identified as
the two A residues complementary to the strongly reactive T residues of
the top strand. The other two more weakly reactive residues were the
platinated G residue and adjacent 3'-A residue. A different reactivity
of DEPC was observed with duplex [1,4;5'-5'] containing the
interstrand CL formed by BBR3464 in the 5' 5' direction. The 3'-A
residue adjacent to the platinated G residue in the bottom strand was
only reactive (data not shown).
Bromination of C residues and formation of piperidine-labile sites are
observed when two simple salts, KBr and KHSO5, are allowed
to react with single-stranded or distorted double-stranded oligonucleotides (35). No reactivity of these residues was observed within the unplatinated duplexes (shown for the top and bottom strands
of duplex [1,4;3'-3'] in Fig. 4, B and D,
respectively, lanes DS). Within the double-stranded duplexes
containing the interstrand CL of BBR3464, no C residue in both strands,
including those complementary to the G residues involved in the CL, was reactive (shown for the top and bottom strands of duplex [1,4;3'-3'] in Fig. 4, B and D, respectively, ICL
lane). The results of the analysis of duplexes
[1,4;3'-3'] and [1,4;5'-5'] containing 1,4-GG interstrand CLs
formed by BBR3464 in the 3' 3' and 5' 5' direction, respectively, by chemical probes are summarized in Fig.
4E.
DNA Unwinding and Bending--
Among the alterations of secondary
and tertiary structures of DNA to which it may be subject, the role of
intrinsic bending and unwinding of DNA is increasingly recognized as of
potential importance in regulating replication and transcription
functions through specific DNA-protein interactions. For DNA adducts of cisplatin, the structural details responsible for bending and subsequent protein recognition have recently been elucidated (14, 15).
Given the recent advances in our understanding of the structural basis
for the bending of DNA caused by cisplatin CLs, it is of considerable
interest to examine how frequent DNA adducts of BBR3464 (interstrand
CLs) affect conformational properties of DNA such as bending and
unwinding. In this work, we performed studies on the bending and
unwinding induced by single, site-specific 1,4-GG interstrand CLs of
BBR3464 formed in the oligodeoxyribonucleotide duplexes in the 3' 3' and 5' 5' directions using electrophoretic retardation as a
quantitative measure of the extent of planar curvature.
The oligodeoxyribonucleotide duplexes [1,4;3'-3'] and [1,4;5'-5']
(20-23 bp, whose sequences were identical (21 bp) or similar to those
of duplexes [1,4;3'-3'(21)] and [1,4;5'-5'(21)] shown in Fig.
1B; the 20-bp duplexes had one marginal C·G pair deleted, whereas one and two additional A·T pairs were added to the ends in
the 22- and 23-bp duplexes, respectively) were used for the bending and
unwinding studies in this work. All sequences were designed to leave a
one-nucleotide overhang at their 5'-ends in double-stranded form (shown
for duplexes [1,4;3'-3'(21)] and [1,4;5'-5'(21)] in Fig.
1B). These overhangs facilitate polymerization of the monomeric oligonucleotide duplexes by T4 DNA ligase in only one orientation and maintain a constant interadduct distance throughout the
resulting multimer. Other experimental details of these studies are
given in our recent report describing DNA intrastrand CLs of BBR3464
(12). Autoradiograms of electrophoresis gels revealing resolution of
the ligation products of unplatinated 20-23-bp duplexes [1,4;5'-5'(20-23)] or containing a unique 1,4-GG interstrand CL of
BBR3464 in the 5' 5' direction are shown in Fig.
5A. A small but significant
retardation was observed for the multimers of all platinated duplexes.
The K factor is defined as the ratio of calculated to actual
length. The variation of the K factor versus
sequence length obtained for multimers of the duplexes 20-23 bp long
and containing the unique 1,4-GG interstrand CL of BBR3464 in the 5'
5' direction is shown in Fig. 5B. Maximum retardation
was observed for the 22-bp cross-linked duplex. This observation
suggests that the natural 10.5-bp repeat of B-DNA and that of DNA
perturbed by the interstrand CL of BBR3464 are different as a
consequence of DNA unwinding (49). Similar results were also obtained
for the 1,4-GG interstrand CL of BBR3464 formed in the 3' 3'
direction in duplexes [1,4;3'-3'(20-23)] (data not shown).
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Fig. 5.
Mobility of the ligation products of duplexes
[1,4; 5'-5'(20-23)] containing a single, site-specific interstrand
CL of BBR3464 on 8% polyacrylamide gel. A,
autoradiogram of the ligation products. The duplexes contained a unique
1,4-interstrand CL formed by BBR3464 between the central G residue in
the top strand and the G residue in the bottom strand oriented in the
5' 5' direction (see "Results"). Pt lanes,
interstrand cross-linked duplexes; NoPt lane, unplatinated
duplexes. B, plots showing the relative mobility K
versus sequence length curves for oligomers 20-23 bp long denoted
as 20-mer, 21-mer, 22-mer, and
23-mer, respectively. C, plot showing the
relative mobility K versus interadduct distance in bp for
oligomers 20-23 bp long with a total length of 132 bp. The
experimental points represent the average of three
independent electrophoresis experiments. The curve
represents the best fit of these experimental points to the equation
K = ad2 + bd + c (49).
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The unwinding angles were calculated from the exact helical
repeat of the 1,4-interstrand cross-linked duplexes (49). The maxima of
these curves constructed for the interstrand cross-linked duplexes with
a total length of 150 bp (shown for the CL in the 5' 5' direction
in Fig. 5C) were determined to be 21.41 ± 0.04 and
21.42 ± 0.04 bp for the CLs in the 3' 3' and 5' 5'
directions, respectively. Total sequence lengths other than 150 bp were
examined and gave identical results. To convert the interadduct
distance in base pairs corresponding to the curve maximum into a duplex unwinding angle in degrees, the value was compared with that of the
helical repeat of B-DNA, which is 10.5 ± 0.05 bp (50, 51). The
difference between the helical repeat of B-DNA and DNA containing the
1,4-interstrand CL of BBR3464 formed in the 3' 3' direction is
therefore ((21.41 ± 0.04) 2(10.5 ± 0.05)) = 0.41 ± 0.09 bp. There are 360°/10.5 bp, so the DNA unwinding
due to one 1,4-interstrand adduct of BBR3464 formed in the 3' 3'
direction is 14 ± 3°. The same calculation for the
1,4-interstrand CL formed in the 5' 5' direction yielded the same
value for the unwinding angle. This unwinding angle is relatively very
small, in contrast to that found, for example, for the 1,2-GG
interstrand CL of cisplatin using the same experimental procedure
(79° (52) or ~90° (19)), but not markedly different from that
produced by the 1,3-GG interstrand CLs formed by the dinuclear
antitumor platinum compound
[(trans-PtCl(NH3)2)2(µ-H2N(CH2)nNH2)]Cl2 (n = 2-6) (9° (32)).
The evaluation of the relationship between interadduct distance and
phasing for self-ligated multimers composed of the identical number of
monomeric duplexes (bend units) resulted in a bell-shaped pattern
(shown for the CL in the 5' 5' direction in Fig. 5C) characteristic of bending. Quantitation of the bend angles of the
1,4-interstrand CLs of BBR3464 was performed as described previously
(19, 33, 38, 53, 54) utilizing the following empirical equation
(Equation 1),
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(Eq. 1)
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where L represents the length of a particular oligomer
with relative mobility K, and RC is the curvature relative
to DNA bending induced at the tract of A residues (A tract) (53, 55). Application of Equation 1 to the 110-, 132-, and 154-bp multimers of
the 22-bp oligomer containing the single 1,4-interstrand CL of BBR3464
leads to a mean curvature of 0.53 or 0.38, respectively, relative to the A tract. The average bend angle per helix turn can be
calculated by multiplying the relative curvature by the absolute value
of the A tract bend of 20° (38, 53, 55, 56). The results indicate
that the bends induced by the 1,4-GG interstrand CLs formed by BBR3464
in the 3' 3' and 5' 5' directions are 21° and 15°,
respectively. We assigned the bend direction by reference to an A
tract, which is bent by ~20° toward the minor groove (55), using
the same procedure described previously (19). Duplexes [1,4;3'-3'+(A/T)5(33)] and
[1,4;5'-5'+(A/T)5(33)] (Fig. 1B) were used,
which also contained, besides the single 1,4-interstrand CL of BBR3464
formed in the 3' 3' and 5' 5' directions, respectively, the A
tract located "in phase" from the CL (the center of the sequence
involved in the CL and the center of the A tract were separated by 11 bp) (Fig. 1B). In the cross-linked multimers, the CLs or the
A tracts were separated by 33 bp, corresponding to about three helical
turns after incorporation of the estimated 14° of unwinding at the
lesion (see above). The cross-linked multimers of duplexes
[1,4;3'-3'+(A/T)5(33)] and
[1,4;5'-5'+(A/T)5(33)] migrated on the gel in all cases
more rapidly than their unplatinated counterparts (data not shown).
Hence, the effective bend of the helix axis at the center of the
1,4-interstrand CLs of BBR3464 is in the opposite direction from that
at the center of the A tract, i.e. the 1,4-GG interstrand
CLs formed by BBR3464 in both the 3' 3' and 5' 5' directions
bend DNA toward the major groove. Other details of the calculations of
the unwinding and bending angles are given in previous studies (19, 31,
33, 38, 52-54).
Recognition by the Domains of the HMGB1 Protein--
The bending
of the helix axis induced in DNA by 1,2-intrastrand and
1,2-interstrand CLs of cisplatin and the altered structures attract HMG domain proteins and other proteins (8, 13, 22, 57). This
binding of HMG domain proteins to DNA modified by cisplatin has been
postulated to mediate its antitumor properties (14, 58). As
bifunctional BBR3464 and other polynuclear platinum compounds exhibit
antitumor activity different from that of cisplatin, it was of
considerable interest to examine how the interstrand CLs of BBR3464 are
recognized by HMG domain proteins. The interactions of the rat HMGB1
protein, which is the prototypical member of a family of these
proteins, and its domains A and B with 1,4-interstrand CLs of BBR3464
were investigated by gel mobility shift experiments (Fig.
6). In the experiments with the domains,
the 21-bp duplexes [1,4;3'-3'(21)] and [1,4;5'-5'(21)] with blunt
ends (see "Experimental Procedures" for their exact sequences) were
modified so that they contained a single, site-specific 1,4-GG
interstrand CL formed by BBR3464 in the 3' 3' and 5' 5'
directions, respectively; or for comparative purposes, also duplex
[TGGT] (Fig. 1B) was modified so that it contained a
single, site-specific 1,2-GG intrastrand CL of cisplatin. The binding
of HMGB1a and HMGB1b to these DNA probes was detected by retardation of
the migration of the radiolabeled 21-bp probes on the gel (Fig. 6) (14,
59, 60).
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Fig. 6.
Analysis of the binding affinity of 21-bp DNA
containing a single, site-specific 1,4-GG interstrand CL of BBR3464 or
the 1,2-GG intrastrand CL of cisplatin for HMGB1a (A),
HMGB1b (B), and the full-length HMGB1 protein
(C) on 6% polyacrylamide gel. Lanes
1-4, unplatinated duplex; lanes 5-8, duplex
containing the intrastrand CL of cisplatin; lanes 9-12
and lanes 13-16, duplex containing the interstrand CL
formed by BBR3464 in the 5' 5' or 3' 3' direction,
respectively. Lanes 1, 5, 9, and
13, no protein added; lanes 2, 6,
10, and 14, HMGB1a, HMGB1b, and full-length HMGB1
at 0.05, 0.54, and 0.48 µM, respectively; lanes
3, 7, 11, and 15, HMGB1a, HMGB1b,
and full-length HMGB1 at 0.10, 1.10, and 0.97 µM,
respectively; lanes 4, 8, 12, and
16, HMGB1a, HMGB1b, and full-length HMGB1 at 0.29, 4.30, and
3.50 µM, respectively.
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HMGB1a and HMGB1b exhibited negligible binding to the unmodified 21-bp
duplexes. As indicated by the presence of a shifted band whose
intensity increased with increasing protein concentration (Fig. 6,
A and B, lanes 5-8), both HMGB1a and
HMGB1b recognized and bound to the duplex containing the 1,2-GG
intrastrand CL of cisplatin. The results of the titration of the
duplexes containing the 1,4-GG interstrand CL of BBR3464 in the 3' 3' and 5' 5' directions with HMGB1a and HMGB1b are shown in Fig. 6
(A and B, respectively). These titration data
indicate that neither HMGB1a nor HMGB1b bound the probes containing the
interstrand CL of BBR3464 under conditions in which they exhibited a
high affinity for the 1,2-GG intrastrand CL of cisplatin. Hence, the
DNA interstrand CLs formed by BBR3464 in both directions are not
recognized by HMG domain proteins, or their affinity for these proteins
is markedly lower than that of the major 1,2-GG intrastrand CL of cisplatin.
To examine the affinity and specificity of the full-length HMGB1
protein for 1,4-interstrand CLs of BBR3464, 149-bp probes (see
"Experimental Procedures"), unmodified or containing a single, site-specific 1,4-GG interstrand CL formed by BBR3464 in the 3' 3'
or 5' 5' direction or the 1,2-GG intrastrand CL of cisplatin, were
used in electrophoretic mobility shift assays. Not surprisingly, the
HMGB1 protein exhibited affinity for the probe containing the 1,2-GG
intrastrand CL of cisplatin, as indicated by the presence of a shifted
band of the 149-bp probe containing the single 1,2-GG intrastrand CL of
cisplatin with the protein (the intensity of which rose with increasing
protein concentration) (Fig. 6C, lanes 5-8). The
interaction between the protein and the DNA intrastrand cross-linked by
cisplatin was not a result of general DNA affinity for the protein
because the protein failed to bind the unmodified 149-bp duplexes under
identical conditions (Fig. 6C, lanes 1-4). On
the other hand, under the same experimental conditions, the full-length
HMGB1 protein did not bind the 148-bp probes containing either of the
1,4-interstrand CLs of BBR3464 (Fig. 6C, lanes
9-16). Thus, the results obtained with the full-length HMGB1
protein were entirely consistent with those obtained with its isolated domains.
Nucleotide Excision Repair--
NER is a pathway used by human
cells for the removal of damaged nucleotides from DNA (61-63). In
mammalian cells, this repair pathway is an important mechanism for the
removal of bulky, helix-distorting DNA adducts such as those generated
by various chemotherapeutics, including cisplatin (64). Efficient
repair of 1,2-GG and 1,3-GTG intrastrand CLs of cisplatin has been
reported for various NER systems, including human and rodent
excinucleases (16-18, 65-67). Importantly, intrastrand CLs of
BBR3464 and the dinuclear platinum compound
[(trans-PtCl(NH3)2)2(µ-H2N(CH2)nNH2)]Cl2
are also readily repaired by the NER systems (12, 68). The results presented in Fig. 7B
(lanes 4 and 5) confirm these reports. Excision repair substrates containing a site-specific 1,4-GG interstrand CL of
BBR3464 formed either in the 3' 3' or 5' 5' direction were
prepared (Fig. 7A) so that the radioactive label was
introduced on both strands 5' to the platinated sites to facilitate
detection of cleavage sites on either strand. The interstrand
cross-linked substrates were incubated with human or rodent CFE. To
detect products arising from incision on only one strand, the DNA
sample was treated with NaCN after incubation with CFE to remove
platinum. The NaCN treatment was included to eliminate the effect of
the positively charged platinum complex bound to the excised fragments on their migration on the gel and also to see if the excision would take place only on one strand of the interstrand cross-linked substrate. The products were analyzed by electrophoresis on denaturing polyacrylamide-agarose gel (shown for rodent CFE in Fig.
7B). The substrate containing a single, site-specific
1,3-GTG intrastrand CL of cisplatin was run as a positive control to
ensure that the extract was active in NER (Fig. 7B,
lane 6). However, in contrast, no excision products
could be detected in the NER assay after NaCN treatment if the
substrates containing single, site-specific 1,4-GG interstrand CLs
formed by BBR3464 in both directions were analyzed using both human and
rodent excinucleases (shown for the CLs treated with rodent
excinuclease in Fig. 7B, lanes 8 and 10). Varying the incubation times in the range of
10-60 min and the amount of CFE in the range of 20-100 µg did not
alter this result (data not shown).
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Fig. 7.
Excision of the 1,4-GG interstrand of BBR3464
and 1,3-intrastrand CLs of BBR3464 and cisplatin by rodent
excinuclease. A, shown is a schematic
representation of the formation of the full-length duplex substrates
used in excision assay. The sites of the radiolabels are marked by
asterisks. For other details, see "Experimental
Procedures." B, the substrates were incubated with Chinese
hamster ovary AA8 CFE for 40 min at 30 °C and subsequently treated
overnight with NaCN prior to analysis on denaturing 8 M
urea and 10% polyacrylamide gel. The 20- and 30-nucleotide
(nt) markers are indicated. IAC, intrastrand CL;
ICL, interstrand CL.
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The mammalian CFEs containing the excinuclease hydrolyze the
phosphodiester bond located at a position five nucleotides to the
3'-site of the damage and 22-24 nucleotides to the 5'-site of the
damage (61-63). The substrates containing the 3' 3' or 5' 5' 1,4-interstrand CL used in this study possessed a radioactive label
at the 11th phosphodiester bond to the 5'-side of the damage on one
strand of the duplex and at the 18th or 12th phosphodiester bond to the
5'-side of the damage on the other strand, respectively (Fig.
7A). Thus, if NER of the 1,4-interstrand CLs of BBR3464 occurred in the normal manner, it should be detected by this assay.
The rate-determining step of NER is recognition of the damaged DNA (61,
69). This recognition process involves multiple protein components; and
for example, RPA is one of these proteins that belongs to the initial
damage-sensing factors of human excision nuclease initiating repair
(68, 70). These recognition proteins preferentially bind to damaged
DNA; so, for instance, binding of RPA to damaged DNA may be a sensitive
indicator to predict whether mammalian NER could be effective in the
removal of damaged nucleotides.
The binding of RPA is thought to proceed via the denaturation of the
DNA substrate and subsequent high affinity binding to the
single-stranded DNA generated (28, 68, 71). Hence, it appears
particularly interesting to examine whether RPA binds to the
interstrand cross-linked substrates because the interstrand CL would
prevent denaturation of DNA (which is a prerequisite for RPA binding).
To determine the effect of 1,4-GG interstrand CLs of BBR3464 on RPA
binding, the 21-bp duplexes were prepared so that they contained a
single, site-specific 1,4-interstrand CL formed in either the 3' 3'
or 5' 5' direction (the duplexes identical to those used in the
experiments of this work, in which recognition of the interstrand CLs
of BBR3464 by HMG domain proteins was examined (see above and Fig. 6)).
RPA binding to these duplexes was assessed in a gel mobility shift
assay (Fig. 8). The duplexes with blunt
ends were purified as described under "Experimental Procedures" so
that their samples contained no contaminating single-stranded DNA, as
evident from analysis on native polyacrylamide gel (data not shown).
The 21-bp duplex [TGGT] containing a single, site-specific 1,2-GG
intrastrand CL of cisplatin was run as a positive control (Fig.
8B, lanes 5-8). The results of the gel mobility
shift assay demonstrated that increasing RPA concentrations resulted in
an increasing amount of this protein bound to the 1,2-GG intrastrand CL
of cisplatin. On the other hand, the same analysis performed with the
undamaged substrate or the substrate containing a single, site-specific
1,4-interstrand CL formed by BBR3464 in either the 3' 3' or 5' 5' direction revealed that increasing RPA concentrations resulted in a
low level of RPA binding to the undamaged duplexes (Fig. 8A,
lanes 1-4 and 9-12) and to the duplexes
containing a single, 1,4-interstrand CL of BBR3464 in either direction
(lanes 5-8 and 13-16). Quantitation of these
results is shown in Fig. 8C. The inability of RPA to bind a
substrate containing an interstrand CL of BBR3464 is consistent with
RPA binding occurring via denaturation of DNA. A low level of
nonspecific RPA binding to undamaged control substrate was explained as
being the result of decreased stability of the relatively short
duplexes (71). Taken together, these results strongly support the view
(see above) that the efficiency of the mammalian NER pathway to remove
the interstrand CLs of BBR3464 is very low.
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Fig. 8.
Analysis of the recognition of platinated
21-bp DNA containing a single, site-specific 1,4-GG interstrand CL of
BBR3464 (A) or the 1,2-GG intrastrand CL of cisplatin
(B) by RPA protein and quantitative evaluation of two
independent experiments performed with cross-linked substrates
(C). A and B,
autoradiograms of electrophoretic shift mobility assay on native 6%
polyacrylamide gel. A: lanes 1, 5,
9, and 13, no protein added; lanes 2,
6, 10, and 14, 50 ng of RPA added;
lanes 3, 7, 11, and 15, 150 ng of RPA added; lanes 4, 8, 12, and
16, 300 ng of RPA added. B: lanes 1 and 5, no protein added; lanes 2 and
6, 50 ng of RPA added; lanes 3 and 7,
150 ng of RPA added; lanes 4 and 8, 300 ng of RPA
added. C: open and closed symbols,
unplatinated and cross-linked duplexes, respectively;  and ,
duplex containing the 1,4-interstrand CL of BBR3464 in the 3' 3'
direction;  and , duplex containing the 1,4-interstrand CL of
BBR3464 in the 5' 5' direction; ×, duplex containing the 1,2-GG
intrastrand CL of cisplatin.
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DISCUSSION |
The sequence specificity of the interstrand cross-linking in DNA
by BBR3464 has been assayed in this work by Maxam-Gilbert footprinting.
The experiments presented here (Figs. 2 and 3) clearly demonstrate that
the preferential DNA-binding sites in these lesions are G residues. The
CLs between G residues separated by 4 bp (1,6-interstrand CLs) were
formed at a pronouncedly slower rate compared with 1,2- and
1,4-interstrand CLs (Fig. 2B), so it is reasonable to
suggest that 1,6-GG interstrand CLs represent less frequent adducts of BBR3464. The CLs between G residues separated by >4 bp (1,7- and 1,8-interstrand CLs) were not formed under these conditions by BBR3464,
so the maximum length of the interstrand CL formed by BBR3464 in DNA is
likely to be that in which the cross-linked sites are separated by up
to 4 bp. Thus, the formation of long-range CLs by BBR3464 proposed in
our earlier reports (9, 72) and hence the ability of this trinuclear
platinum compound to target larger sequences of DNA are further
confirmed. Importantly, the results of this work have also confirmed
under competitive conditions that the interstrand CLs of BBR3464 can be
formed in both the 3' 3' and 5' 5' directions (Fig. 3). Whereas
the 1,2-interstrand CLs between G residues in neighboring base pairs
were formed exclusively in the 3' 3' direction, the 1,4-interstrand
CLs of BBR3464 were formed with approximately the same preference for
both the 3' 3' and 5' 5' directions. The 1,6-interstrand CLs
were also formed by BBR3464 in both directions, but with a clear
preference for the formation of the CL in the 5' 5' direction.
Thus, our results demonstrate that although the shortest
1,2-interstrand CL of BBR3464 is formed with a strong preference for
the 3' 3' direction, the growing length of these CLs reverses this
preference to the opposite 5' 5' direction. Interestingly, the
interstrand CLs of the antitumor dinuclear platinum compounds
[(trans-PtCl(NH3)2)2(µ-H2N(CH2)nNH2)]Cl2 and
[(cis-PtCl(NH3)2)H2N(CH2)nNH2]Cl2
(n = 4 and 6; which have leaving chloride
ligands cis to the linker) (32, 39) and cisplatin (30, 73)
are formed exclusively in the 5' 5' direction under the conditions
employed here, so the capability of BBR3464 to form interstrand CLs in
the 3' 3' direction is one of the unique features of the
DNA-binding mode of this novel platinum drug. The reasons for the
dependence of the orientation of DNA interstrand CLs of BBR3464 on
their length are unknown. In parallel NMR studies, binding of both
[(trans-PtCl(NH3)2)2(µ-H2N(CH2)6NH2)]Cl2 and BBR3464 to the 14-bp duplex sequence
5'-d(ATACATGGTACATA)-3'·5'-d(TATGTACCATGTAT)-3' in both the 5' 5'
and 3' 3' CL formations was
observed.3 Pre-association as
also observed by NMR studies may influence the direction of the CLs
formed, as the central platinum unit lies in the minor groove as a
consequence of the pre-association. In the study, we have also
described the conformational distortions induced in DNA by the most
frequent interstrand CLs of BBR3464, 1,4-GG interstrand CLs formed in
both the 3' 3' and 5' 5' directions. The bending experiments
were carried out with the double-stranded oligodeoxyribonucleotides
[1,4;3'-3'(20-23)] and [1,4;5'-5'(20-23)] containing the unique
interstrand CL in their central sequence. The phasing assay revealed
that the 1,4-GG interstrand CLs formed by BBR3464 in the 3' 3' and
5' 5' directions resulted in a directional bending of the helix
axis (21° and 15°, respectively, toward the major groove) and
duplex unwinding (14° for both types of the interstrand CL) (Fig. 5).
In addition to these bending and unwinding effects, the 1,4-interstrand
CLs formed by BBR3464 created local conformational distortions revealed
by the chemical probes (Fig. 4). These distortions were non-symmetrical
and extended mainly over ~4 base pairs (Fig. 4E). The
patterns of distorted sites were different for the CLs formed in the 3'
3' and 5' 5' directions, confirming a different character of
localized conformational distortions due to the different orientation
of these lesions. Interestingly, in the case of the 1,4-interstrand CL
formed in sequence (5'-ATGTACAT-3')2, the structure shows
unique delocalization of the adduct structure, including the presence of the syn-conformation of deoxyriboadenosines outside the
binding site (72).
It has been suggested that HMG domain proteins play a role in
sensitizing cells to cisplatin (14, 15). It has been shown that HMG
domain proteins recognize and bind to DNA CLs formed by cisplatin
between bases in neighboring base pairs (14, 15, 22). The molecular
basis for this recognition is still not entirely understood, although
several structural details of the 1:1 complex formed between the HMG
domain and the duplex containing the 1,2-GG intrastrand CL of cisplatin
were recently elucidated (14). The details of how the binding of HMG
domain proteins to cisplatin-modified DNA sensitizes tumor cells to
cisplatin are also still not completely resolved, but possibilities
such as shielding cisplatin-DNA adducts from excision repair or that
these proteins could be titrated away from their transcriptional
regulatory function have been suggested (8, 15, 58, 74) to explain how
these proteins are involved in the antitumor activity.
An important structural motif recognized by HMG domain proteins on DNA
containing the major 1,2-GG intrastrand CL of cisplatin is a stable,
directional bend of the helix axis toward the major groove (15). As
demonstrated in the present work (Fig. 5), the 1,4-GG interstrand CLs
of BBR3464 bend the helix axis less efficiently than the CLs of
cisplatin (for instance, gel retardation assays revealed that the
1,2-GG intrastrand CL of cisplatin produces a rigid, directed bend
32-34° into the major groove of DNA (38, 53)). No recognition of DNA
interstrand CLs of BBR3464 by HMGB1 proteins was observed in the
present work (Fig. 6). A plausible explanation of this observation may
be that the prebending due to the 1,4-interstrand CL of BBR3464 is too
small to be recognized by HMG domain proteins. Alternatively, this
result may also be associated with some structural features of the
1,4-interstrand CL of BBR3464 revealed recently by the NMR analysis of
the 8-bp duplex containing this adduct (72). This analysis has shown that the "central" tetraamine linker
trans-H2N(CH2)6NH2Pt(NH3)2(CH2)6NH2 of this CL is situated in or very close to the minor groove of DNA. It
is reasonable to expect that this location of the linker could
sterically block binding of the HMG domain proteins to DNA (because
they also bind to DNA from the minor groove (75, 76)). In addition, the
location of the linker in the minor groove could restrict the
additional DNA bending required for HMG domain binding (14, 15). Hence,
it is also possible that BBR3464 in the 1,4-interstrand CL restricts
the additional DNA bending required for binding of the HMGB1 protein.
Thus, from the results of this work, it is clear that the DNA
interstrand CLs of the antitumor compound BBR3464 may present a block
to DNA or RNA polymerase, but are not a substrate for recognition by
HMG domain proteins. From these considerations and from the fact that
also intrastrand CLs are not recognized by HMG domain proteins (12), we
conclude that the mechanism of antitumor activity of
bifunctional BBR3464 does not involve recognition by HMG domain
proteins as a crucial step, in contrast to the proposals for cisplatin
and its direct analogs.
Several reports have demonstrated that NER is a major mechanism
contributing to cisplatin resistance (16-18). The examination of
excision repair of 1,4-interstrand CLs of BBR3464 has revealed that
these adducts cannot be removed as readily by excision repair as
intrastrand adducts. Hence, the interstrand CLs of bifunctional trinuclear BBR3464 would not have to be shielded by damaged DNA recognition proteins, such as those containing HMG domains, to prevent their repair. Thus, it is reasonable to suggest that
interstrand CLs of BBR3464 could persist considerably longer than
intrastrand adducts (23, 24), which would potentiate the toxicity of
BBR3464 to tumor cells sensitive to this drug.
In conclusion, the results of this work provide additional strong
support for the hypothesis that platinum drugs that bind to DNA in a
fundamentally different manner compared with cisplatin have altered
pharmacological properties. Importantly, in contrast to cisplatin, the
mediation of antitumor properties of bifunctional trinuclear platinum
complexes by HMG domain proteins is unlikely, so polynuclear platinum
compounds represent a novel class of platinum anticancer drugs acting
by a different mechanism compared with cisplatin and its analogs. The
cytotoxic effects of BBR3464 may realistically be due to a cumulative
effect of the structurally heterogeneous adducts produced by this drug,
but the role of structurally unique interstrand CLs in the antitumor
effects of BBR3464 may predominate.
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ACKNOWLEDGEMENTS |
We thank J. T. Reardon and A. Sancar for
HeLa and Chinese hamster ovary cell extracts and J. J. Turchi for
replication protein A. We acknowledge that participation in European
Community COST Chemistry Actions D20 and D21 enabled us to exchange
regularly the most recent ideas in the field of platinum anticancer
drugs with several European colleagues.
| |
FOOTNOTES |
*
This work was supported by Grant 305/02/1552A from the Grant
Agency of the Czech Republic, by Grant A5004
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ABSTRACT
INTRODUCTION
