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, April 9, 2001
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ABSTRACT |
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The new antitumor trinuclear
platinum compound
[{trans-PtCl(NH3)2}2µ-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+
(designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is
characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various
types of the intrastrand cross-links of BBR3464 affect conformational
properties of DNA, and how these adducts are recognized by high
mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have
revealed that intrastrand cross-links of BBR3464 create a local
conformational distortion, but none of these cross-links results in a
stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be
relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.
The trinuclear compound
[{trans-PtCl(NH3)2}2µ-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+
(Fig. 1) is currently in phase II clinical trials. The compound, designated as BBR3464, is the lead representative of an entirely new
structural class of DNA-modifying anticancer agents based on the
poly(di,tri)nuclear platinum structural motif (1-3). In phase I
trials, objective partial responses in pancreatic and lung cancers as
well as melanoma were observed (4, 5). These results suggest the
potential for genuinely complementary clinical anticancer activity of
BBR3464 in comparison to cis-diamminedichloroplatinum(II) (cisplatin).1 Cisplatin
has a major role in combination chemotherapy for several solid tumors,
such as germ cell tumors, lung cancer, head and neck cancer, ovarian
cancer, and bladder cancer (6-9). The specific choice of BBR3464 as
clinical candidate comes from preclinical studies showing cytotoxicity
at 10-fold lower concentration than cisplatin and collateral
sensitivity in cisplatin-resistant cell lines (1, 4, 10). Importantly,
BBR3464 also displays consistently high antitumor activity in human
tumor xenografts characterized as mutant p53 (1, 11). This important
feature suggests that the new agent may find utility in the over 60%
of cancer cases where mutant p53 status has been indicated. DNA damage
by chemotherapeutic agents is in many cases mediated through the p53
pathway (12). Consistently, cytotoxicity displayed in mutant cell lines
would suggest an ability to bypass this pathway (11).
DNA is generally considered the major pharmacological target of
platinum drugs (13). As such it is of considerable interest to
understand the patterns of DNA damage that may lead to differential cell signaling induced by polynuclear platinum complexes in comparison to those induced by mononuclear agents such as cisplatin and
carboplatin. Previous results have indicated a unique DNA binding
profile for BBR3464, strengthening the original hypothesis that
modification of DNA binding in manners distinct from that of cisplatin
will also lead to a distinct and unique profile of antitumor activity (14). The bifunctional DNA binding of BBR3464 is characterized by the
rapid formation of long range intra- and interstrand cross-links (CLs).
Since the central platinum unit does not contribute to covalent binding
to DNA, it is not surprising that the DNA binding profile of BBR3464 is
shared by dinuclear platinum compounds with simple diamine and
polyamine (spermidine, spermine) linkers (14, 15).2 The incorporation into
the linker backbone of charge and hydrogen-bonding capacity
dramatically increases the DNA affinity and affects the charge/lipophilicity balance as well as increasing the distance between
the two platinum-DNA binding coordination spheres. All of these
features may contribute to contribute to differentiating DNA binding,
cellular uptake, and antitumor activity within the polynuclear platinum
family itself (1, 17, 18).
The high charge on BBR3464 facilitates rapid binding to DNA, which is
significantly faster than that of the neutral cisplatin. This feature
is also manifested in rapid binding to single-stranded DNA (19).
Bifunctional binding to duplex DNA preferentially involves guanine (G)
residues. Quantitation of interstrand DNA cross-linking in natural and
linear DNA indicated ~20% of the DNA to be interstrand cross-linked.
This value is significantly higher than that for cisplatin; on the
other hand, an intriguing aspect of BBR3464 is that long range
delocalized CLs in which the platinated sites are separated by one or
more base pairs are equally or even more probable than interstrand adducts.
The (platinum,platinum) intrastrand CLs of BBR3464 are thus analogues
of the major adducts of cisplatin, which forms on DNA ~90%
bifunctional intrastrand adducts between neighboring purine residues,
affording an unwound duplex with a directional fixed kink and a
widened, shallow minor groove (20). The structure of these adducts
determined by phasing assay based on gel electrophoresis and by
chemical probes of DNA conformation has revealed (21-24) that these
adducts induce the overall helix bend of 32-34° toward major groove,
DNA unwinding of 13°, 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. Similarly, the minor 1,3-intrastrand CL of
cisplatin also bends the helix axis toward the major groove by ~35°
and locally unwinds DNA by ~23° (24, 25). 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 (23, 26), in
contrast to the case of the 1,2-intrastrand adduct. Given the recent
advances in our understanding of the structural basis for the
conformational alteration caused by intrastrand CLs of cisplatin, it is
of considerable interest to examine how the structures of the various
types of the intrastrand CLs of BBR3464 affect conformational
properties of DNA.
Some structures altered by platinum adducts, such as stable directional
bending and unwinding, attract various damaged DNA-binding proteins
such as those containing high mobility group (HMG) domain (27-29).
This binding of these proteins has been postulated to mediate the
antitumor properties of the platinum drugs (28, 29). In addition,
several reports have demonstrated (30-32) that intrastrand CLs of
cisplatin are removed from DNA during nucleotide excision repair (NER)
reactions and that NER is also a major mechanism contributing to
cisplatin resistance. Therefore, in addition to examining the
structural alterations induced in DNA by the intrastrand CLs of
BBR3464, we also investigated in the present work how these adducts are
recognized by HMG1 protein and removed from DNA during in
vitro NER reactions.
Chemicals--
BBR3464 (Fig. 1) was prepared by standard
methods. Cisplatin was obtained from Sigma (Prague, Czech Republic).
The stock solutions of platinum compounds were prepared at the
concentration of 1 × 10 Platinations of Oligonucleotides--
The single-stranded
oligonucleotides (the top strands of the duplexes in Fig. 1) were
reacted in stoichiometric amounts with BBR3464. The platinated
oligonucleotides were repurified by ion-exchange fast protein liquid
chromatography (FPLC). It was verified by platinum flameless atomic
absorption spectrophotometry and by the measurements of the optical
density that the modified oligonucleotides contained three platinum
atoms. It was also verified using DMS footprinting of platinum on DNA
(35-37) that in the platinated top strands of all duplexes the N7
position of the two guanine (G) residues was not accessible for
reaction with DMS. Briefly, platinated and nonmodified top strands (5'
end-labeled with 32P) were reacted with DMS. DMS methylates
the N7 position of G residues in DNA, producing alkali-labile sites
(38). However, if N7 is coordinated to platinum, it cannot be
methylated. The oligonucleotides were then treated with hot piperidine
and analyzed by denaturing 24% polyacrylamide gel electrophoresis. For
the nonmodified oligonucleotides, shortened fragments due to the
cleavage of the strand at one methylated G were observed in the gel.
However, no such bands were detected for the oligonucleotides modified by BBR3464. These results indicate that one BBR3464 molecule was coordinated to both G resides in the top strands of all duplexes. If
not stated otherwise, the platinated top strands were allowed to anneal
with unplatinated complementary strands (bottom
strands in Fig. 1) in 50 mM NaCl plus 10 mM Tris-HCl (pH 7.4) and used immediately in further
experiments. This annealing procedure included a rapid heating of the
mixture of the complementary oligonucleotides to 60 °C followed by
the incubation at 25 °C for 2 h. It was verified that under
these conditions the intrastrand CLs of BBR3464 were stable for at
least 24 h. FPLC purification and flameless atomic absorption
spectrophotometry measurements were carried out on an Amersham
Pharmacia Biotech FPLC system with MonoQ HR 5/5 column and a Unicam 939 AA spectrometer equipped with a graphite furnace, respectively. Other
details have been described previously (33, 35, 39).
Chemical Modifications--
The modification by
KMnO4, DEPC, and KBr/KHSO5 were performed as
described previously (39-42). The strands of the duplexes (22 bp shown
in Fig. 1B) were 5' end-labeled with
[ Ligation and Electrophoresis of
Oligonucleotides--
Unplatinated 15- and 19-22-mer single strands
(bottom strands in Fig. 1B) were 5'
end-labeled with [ Gel Mobility Shift Assay--
The 20-mer oligonucleotides
5'-d(AGAAGAAGACCAGAGAGAGG), 5'-d(AGAAGAACACAAGAGAGAGG), or
5'-d(AGAAGAACAACAGAGAGAGG) were 5' end-labeled and annealed (see above)
to their complementary strands 5'-d(CCTCTCTCTG*G*TCTTCTTCT),
5'-d(CCTCTCTCTTG*TG*TTCTTCT), or 5'-d(CCTCTCTCTG*TTG*TTCTTCT)
respectively, where the asterisks represent a platinum CL. The duplexes
(0.4 nM) were incubated with increasing concentrations of
proteins 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 BSA,
and 0.05% Nonidet P40. Samples were incubated on ice for 30 min and
then made 7% in sucrose and 0.017% in xylene cyanol prior loading on
prerun, precooled (4 °C) 6% native polyacrylamide gels
(mono:bis(acrylamide) ratio = 29:1). Gels were
electrophoresed for 3 h, visualized by using a Molecular
Dynamics PhosphorImager (Storm 860 system), and the bands were
quantitated with the ImageQuant software.
Nucleotide Excision Assay--
The 20-mer
oligonucleotides 5'd(CCTCTCTCTTG*G*TTCTTCTT),
5'd(CCTCTCTCTTG*TG*TTCTTCT), and 5'd(CCTCTCTCTTG*TTTG*TTCTCT), where the asterisks represent a BBR3464 CL, were used for preparation of
linear 148-bp duplexes with centrally located 1,2-, 1,3-, or 1,5-intrastrand CL of BBR3464 at nucleotides 75 and 76, 75 and 77, or 75 and 78, respectively. Uniquely modified 20-mers were end-labeled
to introduce a radiolabel at the 11th phosphodiester bond 5' to the CL,
annealed with a set of five complementary and partially overlapping
oligonucleotides, and ligated with T4 DNA ligase. Full-length
substrates were separated from unligated products in a 6%
denaturing polyacrylamide gel, purified by electroelution, reannealed,
and stored in annealing buffer (50 mM Tris-HCl (pH 7.9),
100 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol) at
Oligonucleotide excision reactions were performed in cell-free extracts
(CFEs) prepared from the HeLa S3 and CHO AA8 cell lines as described
(31, 46). These extracts were kindly provided by J. T. Reardon and
A. Sancar from the University of North Carolina (Chapel Hill, NC).
In vitro repair of intrastrand CLs of BBR3464 was measured
with excision assay using these CFEs and 148-bp linear DNA substrates
(see above) in the same way as described previously (31) with small
modifications. The reaction mixtures (25 µl) contained 10 fmol of
radiolabeled DNA, 50 µg of CFE, 20 µM each of dATP,
dCTP, dGTP, and TTP in the 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 BSA, and 2.5% glycerol) and were
incubated at 30 °C for 40 min. DNA was deproteinized and
precipitated by ethanol. The excision products were separated on 10%
denaturing polyacrylamide gels and visualized by using a Molecular
Dynamics PhosphorImager (Storm 860 system), and the bands were
quantitated with the ImageQuant software.
Mapping of incision sites was performed as described in a previous
report (31) with small modifications. Briefly, the major excision
product (gel-purified) was further incubated for 10 min at 30 °C
with T4 DNA polymerase (0.15 units) in 10 µl of buffer composed of 50 mM Tris-HCl (pH 8.8), 15 mM
(NH4)2SO4, 7 mM
MgCl2, 0.1 mM EDTA, 50 mM
Chemical Probes of DNA Conformation--
We demonstrated in our
previous paper (14) that preferential G binding of BBR3464 results in
various types of adducts including long range intrastrand and
interstrand CLs. Quantitation of cross-linking revealed that
intrastrand CLs are equally or even more probable than interstrand
adducts. Considering these facts we have designed a series of synthetic
oligodeoxyribonucleotide duplexes, TGGT, TGTGT, and TGTTTGT, whose
sequences are shown in Fig. 1. The
pyrimidine-rich top strands of these duplexes only contained two G
residues in the sequences TGGT, TGTGT, and TGTTGT in the center (Fig.
1, bold). These top strands were modified by BBR3464 so that
they contained a single 1,2-, 1,3-, and 1,5-intrastrand adduct of this
platinum complex between two G residues at these central sequences. The 1,2-intrastrand CL is formed between neighboring G sites, whereas, in
1,3- and 1,5-intrastrand CLs, the platinated G sites are separated by
one or three nucleotides, respectively.
The cross-linked top strands of the duplexes TGGT, TGTGT, or TGTTTGT
were hybridized with their complementary strands. The samples of the
platinated TGGT, TGTGT, or TGTTTGT duplexes in which the upper strand
was only 5' end-labeled with 32P were reacted with DMS,
which does not react with platinated G because the N7 position is no
longer accessible (35). The adducts were removed by NaCN (36, 47), and
then the sample was treated with piperidine. In the unplatinated
duplexes, the central G residues in the top strands were reactive with
DMS (data not shown). They were no longer reactive in all three
cross-linked duplexes. This observation confirms that the two G
residues in the upper strands remained platinated even after the duplex
was formed and were involved in the intrastrand CL (36, 47).
The oligonucleotide duplexes containing a site-specific 1,2-, 1,3-, or
1,5-intrastrand CL between G residues were further analyzed by chemical
probes of DNA conformation. The intrastrand cross-linked duplexes
(22-bp, shown in Fig. 1B) 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 (39-42, 48). The
results of the analysis by chemical probes of the TGGT(22), TGTGT(22),
or TGTTTGT(22) duplexes containing intrastrand CLs of BBR3464 are
summarized in Fig. 2B.
KMnO4 is hyperreactive with thymine (T) residues in
single-stranded nucleic acids and in distorted DNA as compared with
B-DNA (40, 42, 49, 50). KMnO4 reacted with no residue
within the unplatinated duplexes (shown for the TGGT(22) duplex in Fig. 2A (left side, lane ds)).
All T residues were strongly reactive in the unplatinated
single-stranded top oligonucleotide (shown for TGGT duplex in Fig.
2A (left side, lane ss)).
The intrastrand cross-linked duplexes showed strong reactivity of the
5' T residue adjacent to the adduct (shown for the TGGT(22) duplex in
Fig. 2A (left side, lane
IAC)). A strong or somewhat weaker reactivity was also
observed for the second 5' T adjacent to the 1,3- or 1,2-CL,
respectively. Similarly, a weaker reactivity was observed for the T
residues between the platinated G residues in the TGTGT or TGTTTGT
cross-linked duplexes.
DEPC carbetoxylates purines at the N7 position. It is hyperreactive
with unpaired and distorted adenine (A) residues in DNA and with
left-handed Z-DNA (40, 42, 51, 52). A and G residues within the
unplatinated single-stranded oligonucleotide (top and bottom) readily reacted with DEPC (shown for the bottom
strand of the 1,2 duplex in Fig. 2A (center,
lane ss)). No reactivity of A and G residues was
observed within the unplatinated duplex (shown for the bottom strand of
the TGGT(22) duplex in Fig. 2A (center,
lane ds)). Within the double-stranded
oligonucleotides containing either intrastrand CL, other A residues in
the bottom strand became reactive (shown for the TGGT(22) duplex in
Fig. 2A (center, lane
IAC)). These are readily identified as the A residues
complementary to the reactive T residues of the top strand. Importantly, A residues complementary to strongly reactive T residues also reacted with DEPC strongly whereas those A residues complementary to more weakly reactive T residues also reacted with DEPC only more weakly.
Bromination of cytosine (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 (41). All C residues within the unplatinated
single-stranded top or bottom strands of the TGGT(22), TGTGT(22), or
TGTTTGT(22) duplexes were strongly reactive (shown for the bottom
strand of the TGGT(22) duplex in Fig. 2A (right
side, lane ss)). No reactivity of these
residues was observed within the unplatinated duplexes (shown for the
bottom strand of the TGGT duplex in Fig. 2A (right
side, lane ds)). Within the double-stranded
duplexes containing the intrastrand CL, no C residue in the top strand
was reactive (data not shown). In contrast, the only two C residues in
the bottom strand of the platinated TGGT(22) duplex (complementary to
the platinated G residues in the top strand of the TGGT(22) duplex)
(Fig. 2A, right side, lane IAC) and only one C residue in the bottom strands of the
platinated TGTGT(22) or TGTTTGT(22) duplexes (complementary to the
platinated 5' G residue in the top strands of these duplexes) were
reactive (data not shown).
DNA Bending--
Among the alterations of secondary and tertiary
structure of DNA to which it may be subject, the role of intrinsic
bending of DNA is increasingly recognized as of potential importance in regulating replication, transcription and repair 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 (28, 29). 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 intrastrand DNA adducts of BBR3464 affect conformational
properties of DNA such as bending. In this work we performed further
studies on the bending induced by single, site-specific 1,2-, 1,3-, and
1,5-intrastrand CL of BBR3464 formed in the oligodeoxyribonucleotide
duplexes using electrophoretic retardation as a quantitative measure of
the extent of planar curvature.
The oligodeoxyribonucleotide duplexes TGGT(15,19-22),
TGTGT(15,19-22), and TGTTTGT(15,19-22) (15 and 19-22 bp long, for
their sequences see Fig. 1B) were used for the bending
studies of the present work. All sequences were designed to leave a one
nucleotide overhang at their 5' ends in double-stranded form. 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.
Autoradiograms of electrophoresis gels revealing resolution of the
ligation products of unplatinated TGGT(15,19-22) duplexes or
containing a unique 1,2 GG intrastrand CL of BBR3464 are shown in Fig.
3A. A significant retardation
was observed for the multimers of all platinated duplexes. Decreased
gel electrophoretic mobility may result from a decrease in the DNA
end-to-end distance (53). Various platinum(II) complexes have been
shown to form DNA adducts, which decrease gel mobility of DNA fragments
due to either stable curvature of the helix axis or increased isotropic
flexibility (23, 39, 54-56). DNA multimers of identical length and
number of stable bend units, but with differently phased bends, have
different end-to-end distances. The DNA bends of a multimer must be,
therefore, spaced evenly and phased with the DNA helical repeat in
order to add constructively. Such constructively phased bends add in
plane, yielding short end-to-end distances and the most retarded gel
migration. In other words, gel electrophoresis of multimers of
oligonucleotide duplexes that only differ in length and contain a
stable curvature induced by the same platinum adduct should exhibit a
phase effect, i.e. the maximum retardation should be
observed for the multimers having the bends in phase with helix screw.
In contrast, the normal electrophoretic mobility should be observed for
the multimers having the bends separated by a half-integral number of
DNA turns. Importantly, a gel mobility retardation of multimers due to
the platinum adducts introducing isotropic flexibility rather than
stable curvature is not expected to display a phase dependence (55).
The K factor is defined as the ratio of calculated to actual
length. The calculated length is based on a multimer's mobility and is
obtained from a calibration curve constructed from the mobilities of
unplatinated multimers. The variation of the K factor
versus sequence length obtained for multimers of the TGGT
15,19-22 bp long and containing the unique 1,2-GG intrastrand CL
of BBR3464 are shown in Fig. 3B. No significant change in
the slopes of these plots was observed in a broad range of the
lengths of the monomeric TGGT duplexes (15 and 19-22 bp). Similar
results were obtained (data not shown) for the multimers of the TGTGT
and TGTTTGT duplexes containing intrastrand CL of BBR3464. These
results suggest that conformational distortion induced in
double-stranded DNA by 1,2-, 1,3-, and 1,5-intrastrand CLs between G
residues by BBR3464 does not result in a stable curvature (rigid
directional bending).
Recognition by HMG1 Domains A and B--
The bending of the helix
axis induced by DNA intrastrand and interstrand CLs of cisplatin and
the altered structure attract HMG domain and other proteins (27, 57).
This binding of HMG domain proteins to cisplatin-modified DNA has been
postulated to mediate the antitumor properties of this drug (28, 29). As bifunctional BBR3464 exhibits antitumor activity different from
cisplatin, it was of considerable interest to examine how the
intrastrand CLs of BBR3464 are recognized by HMG domain proteins. The
interactions of the HMG1domA and HMG1domB with 1,2, 1,3 and 1,5 DNA
intrastrand CLs of BBR3464 were investigated by means of gel mobility
shift experiments. In these experiments, the 20-bp duplexes (see "Gel
Mobility Shift Assay" for their sequences) were modified so that they
contained a single, defined 1,2-, 1,3-, or 1,5-intrastrand CL of
BBR3464. The HMG1domA and HMG1domB were found to bind the probes
similar to those used in the present work, but containing
1,2-d(GpG)-intrastrand CL of cisplatin (58, 59).
The binding of the HMG1domA and HMG1domB to these DNA probes was
detected by retardation of the migration of the radiolabeled 20 bp-probes through the gel (28, 58, 59) (Fig.
4). There is no binding of the HMG1domA
and HMG1domB to the DNA probe containing the 1,2-, 1,3-, or
1,5-intrastrand CL of BBR3464 (shown in Fig. 4 (lanes
6-8 and 11-13) for the 1,3-intrastrand CL of
BBR3464) that would be evidenced by the presence of slower migrating
band. Importantly, this more slowly migrating band was clearly seen for
the probe containing 1,2-intrastrand CL of cisplatin analyzed in the
presence of the HMG1domA and HMG1domB, even at concentrations 5-6
times lower than was their maximum concentration used in the experiments with the probes containing the CLs of BBR3464 (Fig. 4,
lanes 4 and 10). Also importantly, no
binding of the proteins occurred under identical experimental
conditions in the cases where the same 20-bp DNA probes were not
platinated (shown in Fig. 4 (lane 1) for the
duplex containing in its top strand the central TGGT sequence). From
these results it is clear that in contrast to 1,2-d(GpG) intrastrand
adducts of cisplatin the intrastrand CLs of BBR3464 are not recognized
by HMG1 domain proteins.
Nucleotide Excision Repair--
NER is a major pathway used by
human cells for the removal of damaged nucleotides from DNA (60-62).
In mammalian cells, this repair pathway is the only known mechanism for
the removal of bulky, helix-distorting DNA adducts, such as those
generated by various chemotherapeutics including cisplatin (63).
Efficient repair of 1,2-d(GpG) and 1,3-d(GTG) intrastrand CLs of
cisplatin has been reported by various NER systems including human and
rodent excinucleases (30-32, 64-66). The results presented in Fig.
5A (lanes 4 and 8) confirm these reports. Importantly,
1,2-, 1,3-, and 1,5-intrastrand CLs of BBR3464 were also repaired with
a similar efficiency as cisplatin 1,3-intrastrand adduct, but with a
considerably higher efficiency than 1,2-intrastrand CL of cisplatin by
both human and rodent excinucleases (shown in Fig. 5 (A and
B) for the CLs repaired by rodent excinuclease). The
excision repair assay detects radiolabeled fragments resulting from
dual incisions both 5' and 3' to the lesion. The mobility of these
fragments is, however, considerably affected by the 4+ charge of the
platinum complex moiety (Fig. 5C), which complicates
determination of their length by comparing their migration in the gel
with that of the unplatinated marker oligonucleotides. The decreased
mobility of the excised platinated fragments was, therefore, reversed
by NaCN treatment (0.2 M, pH 10-11, 45 °C overnight),
which removes platinum from DNA. Thus, for the substrate containing
intrastrand adducts of BBR3464, the excised fragments were primarily
23-28 nucleotides in length, although 22-31-nucleotide-long fragments
were also observed (Fig. 5, A (lane 6)
and C (lane 2)). This range of product sizes reflects variability at both the 3' and 5' incision sites (31,
67); smaller excision products are due to degradation of the primary
excision products by exonucleases present in the extracts (31).
Incubation of the 20-mer containing single 1,3-intrastrand CL of
BBR3464 (used in the nucleotide excision assay) with T4 DNA polymerase
in the absence of deoxyribonucleotide triphosphates (i.e.
exploiting 3' This paper describes the conformational distortions induced in
duplex DNA containing the unique 1,2-, 1,3-, and 1,5-intrastrand CLs of BBR3464 (Fig. 1). The phasing assay (Fig. 3) has revealed that
none of three intrastrand CLs of BBR3464 results in a stable curvature
(directional bending). Lack of phase dependence of the retardation of
gel mobility of DNA containing the intrastrand CLs of the trinuclear
BBR3464 is consistent with the view that these adducts increase
flexibility of the duplex. An increased flexibility introduced to the
helix in this manner is sustained by the observation that these lesions
create a local conformational distortion revealed by the chemical
probes (Fig. 2). This distortion mainly occurs on the 5' side of the
CLs and in the case of the 1,3 and 1,5 adducts also on the base pairs
between the platinated G residues. The chemical probes allow detection
of the sites where the distortion is localized and its extent, but they
do not provide all details about the character or nature of the
distortion. The 1,2-intrastrand CL is formally the structural analog of
the most prominent adduct formed by cisplatin. Because of the long
distance between the two platinating centers of BBR3464, the formation of this CL is unlikely to be favored over the longer range intrastrand and, indeed, interstrand CLs possible. The formation of the
1,2-intrastrand CL has been observed for the dinuclear compounds such
as
[{trans-[PtCl(NH3)2}2H2N(CH2)4,6NH2]Cl2, with the relatively short bridging butane- and hexanediamines. The
chemical probes and phasing assays suggest that the structures of the
1,2-intrastrand adduct in the present case are similar to those formed
by the dinuclear compound (68, 69). Thus, the principal feature
introducing conformational flexibility is reasonably inferred to be the
presence of the monofunctional coordination spheres with only one
purine base bound. The greater length of the BBR3464 linker and its
charge and hydrogen-bonding capacity may, respectively, produce a
bulkier adduct and enhance initial DNA recognition but do not appear to
affect greatly the major structural feature of conformational
flexibility. It is noted that this fundamental feature of
monofunctional coordination spheres also leads to considerable
conformational flexibility in interstrand CL structure (70).
It has been suggested (28, 29) that HMG domain proteins play a role in
sensitizing cells to cisplatin. It has been shown that HMG domain
proteins recognize and bind to DNA CLs formed by cisplatin between
bases in neighboring base pairs (28, 29). The molecular basis for this
recognition is still not entirely understood, although several
structural details of the 1:1 complex formed between HMG domain
proteins and the duplex containing 1,2 d(GpG) intrastrand CL were
recently elucidated (28, 59). The details of how the binding of HMG
domain proteins to cisplatin-modified DNA sensitize 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 (29, 71-73) as clues for how
these proteins are involved in the antitumor activity.
An important structural motif recognized by HMG domain proteins on DNA
modified by cisplatin is a stable, directional bend of the helix axis
(29, 57, 74). Therefore, it is not surprising that we have observed in
the present work (Fig. 4) no recognition of DNA intrastrand CLs of
BBR3464 by HMG1domA and HMG1domB consistent with the assumption that an
important structural motif recognized by HMG domain proteins is bent or
kinked duplex axis. Thus, it is clear from the results of the present
work that the intrastrand DNA adducts of antitumor BBR3464 may present
a block to DNA or RNA polymerases (14) but are not a substrate for
recognition by HMG domain proteins. These results parallel our previous
findings on the dinuclear compound (68). From these considerations we could conclude that the mechanism of antitumor activity of bifunctional polynuclear BBR3464 does not involve recognition of its intrastrand CLs
by HMG domain proteins as a crucial step, in contrast to the proposals
for cisplatin and its direct analogues.
One possible role for binding of HMG domain proteins to DNA modified by
cisplatin is that these proteins shield damaged DNA from intracellular
excision repair (29, 30, 72, 73). The examinations of excision repair
of DNA containing various intrastrand CLs of BBR3464 revealed that
these adducts were readily removed from DNA by NER (Fig. 5). These
results suggest that the processing of the intrastrand CLs of BBR3464
in tumor cells sensitive to this drug may not be relevant to its
antitumor effects despite the fact that the trinuclear platinum
compound forms on DNA intrastrand CLs with a relatively high frequency.
An interesting feature of the repair assay is that a greater range of
excised fragments is seen for the conformationally flexible adducts
produced by BBR3464 than the sterically rigid adducts produced by
cisplatin (Figs. 5 and 6). Indeed, the flexible 1,3-intrastrand adduct
of cisplatin also appears to produce more variability in cutting site
than the corresponding 1,2-adduct. It is tempting to speculate that
delocalized lesions formed by long range adducts of polynuclear platinum compounds may still represent unique challenges for
recognition and excision by repair enzymes.
On the other hand, BBR3464 also forms on DNA interstrand CLs with a
considerably higher frequency than cisplatin (14). 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 antitumor effects of BBR3464. In addition, the interstrand CLs pose
a special challenge to repair enzymes because they involve both strands
of DNA and cannot be repaired using the information in the
complementary strand for resynthesis. The fact that interstrand CLs
cannot be removed so readily by excision repair as intrastrand lesions
is also corroborated by the observation that excision repair of the
interstrand CL formed by cisplatin was not detected under condition
when intrastrand CLs of this drug were readily removed by a
reconstituted system containing highly purified nucleotide excision
repair factors (30). Hence, the interstrand CLs, which are rather
frequent DNA adducts of BBR3464, would not even have to be shielded by damaged DNA recognition proteins to prevent their repair. 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 (16, 18). Data on conformation, recognition by HMG domain proteins and NER of DNA interstrand CLs of
BBR3464 will provide more insights into which DNA adduct of BBR3464 is
a more likely lesion responsible for antitumor effects of this
polynuclear platinum drug. The cytotoxic effects may also be due to a
cumulative effect of the structurally heterogeneous adducts produced by
polynuclear platinum drugs.
In conclusion, the results of the present work provide additional
strong support for the hypothesis that platinum drugs that bind to DNA
in a fundamentally different manner to that of cisplatin have altered
pharmacological properties. Importantly, in contrast to cisplatin, the
mediation of antitumor properties of bifunctional trinuclear platinum
complex BBR3464 by shielding its intrastrand adducts by HMG domain
proteins is unlikely so that polynuclear platinum compounds apparently
represent a novel class of platinum anticancer drugs acting by a
different mechanism than cisplatin and its analogues. A further
understanding of how bifunctional polynuclear platinum compounds modify
DNA and how these modifications are further processed in cells should
provide a rational basis for the design of new platinum antitumor drugs
and chemotherapeutic strategies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 M
in 10 mM NaClO4 and stored at 4 °C in the
dark. The synthetic oligodeoxyribonucleotides (Fig. 1) were synthesized
and purified as described previously (33). HMG1 domain A (HMG1domA) and
HMG1 domain B (HMG1domB) (residues 1-84 and 85-180, respectively)
were prepared by M. Stros as described previously (34); their sequences (34) were derived from rat HMG1 cDNA. T4 DNA ligase, T4
polynucleotide kinase, and T4 DNA polymerase were purchased from New
England Biolabs (Beverly, MA). Acrylamide, bis(acrylamide), urea, and NaCN were from Merck KgaA (Darmstadt, Germany). Dimethyl sulfate (DMS),
KMnO4, diethyl pyrocarbonate (DEPC), KBr, and
KHSO5 were from Sigma (Prague, Czech Republic).
[
-32P]ATP was from Amersham Pharmacia Biotech.
ATP and deoxyribonucleoside triphosphates were from Roche Molecular
Biochemicals (Mannheim, Germany).
-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.
-32P]ATP by using T4 polynucleotide
kinase. Then they were annealed (see above) with their phosphorylated
complementary strands (unplatinated or containing intrastrand CL of
BBR3464 between G residues). Unplatinated and intrastrand CL-containing
duplexes were allowed to react with T4 DNA ligase. The resulting
samples along with ligated unplatinated duplexes were subsequently
examined on 8% native polyacrylamide (mono:bis(acrylamide) ratio = 29:1) electrophoresis gels. Other details of these experiments were
as described in previously published papers (23, 43).
20 °C. Other details of
the purification of DNA substrates for NER were the same as described
previously (44, 45).
-mercaptoethanol, and 20 µg/ml BSA, supplemented with 0.5 µg of
SmaI-digested pBluescript DNA, and visualized by autoradiography following resolution in 10% denaturing polyacrylamide gel. Similar analyses using radiolabeled, platinated 20-mers (used in
the nucleotide excision assays) were also used to identify the
nucleotide(s) at which the exonuclease activity of T4 DNA polymerase is
blocked 3' to the lesion. The location of the 5' incision site made by
the excinuclease was determined by comparison with the length of
excision products observed in the absence of T4 DNA polymerase digestion.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
[in a new window]
Fig. 1.
Structures of platinum complexes
(A) and sequences of the synthetic
oligodeoxyribonucleotides with their abbreviations
(B). The sequences of the duplexes used in the
present study for the chemical probing of DNA conformation and in
phasing assays are only shown in this figure. 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
all duplexes indicate the location of the intrastrand CL after
modification of the oligonucleotides by BBR3464 in the manner described
under "Experimental Procedures."
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[in a new window]
Fig. 2.
Chemical probes of DNA conformation.
Piperidine-induced specific strand cleavage at
KMnO4-modified (A, left),
DEPC-modified (A, center), and
KBr/KHSO5-modified (A, right) bases
in the duplex TGGT(22) unplatinated or containing single, 1,2-d(GpG)
intrastrand CL of BBR3464. The oligomers were 5' end-labeled at
their top or bottom strands. Lanes in panel
A, left (KMnO4, only top strand
end-labeled): ss, the unplatinated top strand;
ds, the unplatinated duplex; IAC, the duplex
intrastrand cross-linked by BBR3464; G, a Maxam-Gilbert
specific reaction for the unplatinated duplex. Lanes in
panel A, center (DEPC, only bottom
strand end-labeled), ss, the unplatinated bottom strand;
ds, the unplatinated duplex; IAC, the duplex
intrastrand cross-linked by BBR3464; G, a Maxam-Gilbert
specific reaction for the unplatinated duplex. Lanes in
panel A, right (KBr/KHSO5,
only bottom strand end-labeled), ss, the unplatinated bottom
strand; ds, the unplatinated duplex; IAC, the
duplex intrastrand cross-linked by BBR3464; G, a
Maxam-Gilbert specific reaction for the unplatinated duplex.
B, summary of the reactivity of chemical probes in the
duplexes TGGT(22), TGTGT(22), and TGTTTGT(22) containing single, 1,2-, 1,3-, and 1,5-intrastrand CL of BBR3464, respectively.
Closed and open circles designate
strong or weak reactivity, respectively.
View larger version (27K):
[in a new window]
Fig. 3.
Analysis of the mobility of the ligation
products of duplexes TGGT(15,19-22) unplatinated or containing a
single, 1,2-d(GpG) intrastrand CL of BBR3464 in an 8% polyacrylamide
gel. A, autoradiogram of the ligation products of the
TGGT duplexes. Lane NoPt, unplatinated duplexes;
lane Pt, the duplexes containing the CL.
B, plots showing the relative mobility K versus
sequence length curves for the oligomers containing the CL: ,
15-mer;
, 19-mer;
, 20-mer;
, 21-mer;
, 22-mer.
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Fig. 4.
Analysis of the binding affinity of
platinated 20-bp DNA containing intrastrand CL of BBR3464 or cisplatin
to HMG1 domain proteins in a 6% polyacrylamide gel.
Lanes 2, 4, and 6-8,
HMGdomA; lanes 9-13, HMGdomB; lanes
1, 3, and 5, no protein.
Lanes 1-4, 9, and 10 contain the duplex with the central sequence TG*G*T/ACCA, and
lanes 5-8 and 11-13 contain the
duplex with the central sequence TG*TG*T/ACACA (for the sequences,
where the asterisks represent a platinum CL, see "Gel Mobility Shift
Assay" under "Experimental Procedures"). Lanes
3, 4, and 10 contain 1,2-d(GpG)
intrastrand CL of cisplatin; lanes 5-8 and
11-13 contain 1,3-d(GpTpG) intrastrand CL of BBR3464.
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Fig. 5.
Excision of intrastrand cross-links of
BBR3464 and cisplatin by rodent excinuclease. A,
substrates (148 bp) containing central and unique 1,3-intrastrand CL of
BBR3464 (lane 6) or 1,2- or 1,3-intrastrand CL of
cisplatin (lanes 4 and 8,
respectively) were incubated with CHO CFE and resolved in 10%
denaturing polyacrylamide gel. Excision products released during the
reaction are primarily 23-29 nucleotides in length and are not
observed in the absence of CFE (lanes 1,
3, 5, and 7). In this experiment, the
percentages of excision by CHO CFE were 1.8, 6.6, and 7.8 for 1,2- and
1,3-intrastrand CLs of cisplatin and 1,3-intrastrand CL of BBR3464,
respectively. Degradation of primary excision products
(lanes 4, 6, and 8) is
discussed in the text. B, quanititative analysis of
removal of intrastrand CLs of BBR3464 and cisplatin by rodent
excinuclease. The columns marked 1,2-GG cisPt,
1,3-GTG cisPt, and 1,3-GTG BBR3464 are for
1,2-d(GpG) intrastrand CL of cisplatin, 1,3-d(GpTpG) intrastrand CL of
cisplatin, and 1,3-d(GpTpG) intrastrand CL of BBR3464. Data are the
average of three independent experiments done under the same
conditions; bars indicate range of excision. C,
after the excision reaction in which DNA containing 1,3-d(GpTpG)
intrastrand CL of BBR3464 was used as the substrate, the reaction
mixture was resolved in the 10% polyacrylamide denaturing gel to
separate excision products from substrate DNA, visualized by
autoradiography, and the mixture of excision products was gel-purified.
One half of this mixture was further treated with NaCN to remove
platinum. The mixtures (untreated and treated with NaCN) were again
analyzed on 10% polyacrylamide denaturing gel. Lane
1 contains the mixture of excision products not treated with
NaCN; lane 2 contains the mixture of excision
products treated with NaCN; lane 3 contains the
mixture of single-stranded oligodeoxyribonucleotides 21-30 nucleotides
long as the markers. For other details, see "Experimental
Procedures."
5' exonuclease activity of T4 DNA polymerase) was
used to determine the nucleotide at which the exonuclease activity is
inhibited. After incubation (10 min) with T4 DNA polymerase under these
conditions, the resulting products migrated as a species 5 nucleotides
shorter than the starting material (Fig.
6A), indicating that the
exonuclease activity is blocked at the second nucleotide 3' to the
intrastrand CL. When the 25-mer excision product, generated by
repair of the intrastrand CL of BBR3464, was treated in the same
manner, it was shortened by 6 nucleotides (Fig. 6B). Because the data with the 20-mer show that T4 DNA polymerase 3'
5'
exonuclease activity is stopped two nucleotides 3' to the CL, it
implies that the 3' incision site is 8 nucleotides (or at the 9th
phosphodiester bond) 3' to the CL. This in turn places the other
incision site at the 15th phosphodiester bond 5' to the
1,3-intrastrand CL of BBR3464.
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Fig. 6.
Mapping of incision sites. A,
time-course analysis with T4 DNA polymerase and 20-mer containing
1,3-intrastrand CL of BBR3464 (for its sequence, see "Nucleotide
Excision Assay" or the sequence in panel A) was
used to identify site of inhibition of T4 DNA polymerase exonuclease
activity. At all time points, this exonuclease activity was primarily
blocked at the second nucleotide 3' to the intrastrand CL, resulting in
migration of platinated 20-mers as platinated 15-mers. B,
limited (10 min) T4 DNA polymerase digestion was used to identify the
3' incision site of gel-purified oligomers released during the excision
repair reaction (Fig. 5A, lane 6). The
excised 25-mer (lane 1) migrated as a 19-mer
(lane 2) after treatment with T4 DNA polymerase.
Thus, one incision occurs at the 9th phosphodiester bond 3' to the CL,
and the second incision occurs at the 15th bond on the 5' side to
generate a 25-mer excision product. For other details, see
"Experimental Procedure."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank J. T. Reardon and A. Sancar for HeLa and CHO cell extracts and M. Stros for HMG1 domains A and B.
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FOOTNOTES |
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* This work was supported by the Grant Agency of the Czech Republic Grant 305/99/0695, 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 research of J. K. and V. B. was supported in part by an International Research Scholar's award from the Howard Hughes Medical Institute and the Wellcome Trust. J. Z. is supported by a doctoral fellowship from the Faculty of Sciences, Masaryk University, Brno. This work was also supported by National Institutes of Health Grant RO1-CA78754 and the American Cancer Society Grant RPG89-002-11-CDD.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 by a doctoral fellowship from the Faculty of Sciences, Masaryk University, Brno, Czech Republic.
To whom correspondence may be addressed. Tel.: 804-828-6320;
Fax: 804-828-8599; E-mail: nfarrell@mail1.vcu.edu.
** To whom correspondence may be addressed. Tel.: 420-5-41517148; Fax: 420-5-41240499; E-mail: brabec@ibp.cz.
Published, JBC Papers in Press, April 12, 2001, DOI 10.1074/jbc.M103118200
2 T. D. McGregor, J. Kasparkova, K. Neplechova, O. Novakova, H. Penazova, O. Vrana, V. Brabec, and N. Farrell, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); CL, cross-link; HMG, high mobility group; NER, nucleotide excision repair; bp, base pair(s); HMG1domA, high mobility group 1 domain A; HMG1domB, high mobility group 1 domain B; DMS, dimethyl sulfate; DEPC, diethyl pyrocarbonate; FPLC, fast protein liquid chromatography; CFE, cell-free extract; CHO, Chinese hamster ovary; BSA, bovine serum albumin.
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