Báo cáo khoa học: Recognition of DNA modified by trans-[PtCl2NH3(4hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex potx

Recognition of DNA modified by trans-[PtCl
2
NH
3
(4-
hydroxymethylpyridine)] by tumor suppressor protein p53
and character of DNA a dducts o f this cytotoxic complex
Kristy
´
na Stehlı
´
kova
´
1
*, Jana Kas
ˇ
pa
´
rkova
´
1
*, Olga Nova
´
kova
´
1
*, Alberto Martı
´
nez
2
, Virtudes Moreno

2
and Viktor Brabec
1
1 Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
2 Departament de Quı
´
mica Inorga
´
nica, Universitat de Barcelona, Barcelona, Spain
The importance of platinum drugs in cancer chemo-
therapy is underlined by the clinical success of cisplatin
[cis-diamminedichloroplatinum(II)] and its analogues,
and by clinical trials of other, less toxic platinum com-
plexes that are active against resistant tumors. In the
search for new platinum anticancer drugs that exhibit
improved pharmacological properties in comparison
with the platinum drugs already used clinically, several
new analogues of clinically inefficient transplatin have
been designed, synthesized and tested for biological
effects. These analogues exhibit cytostatic activity
including activity in tumor cells resistant to cisplatin.
Examples are analogues containing iminoether groups,
heterocyclic amine ligand or aliphatic ligands [1–4].
Keywords
antitumor; conformation; DNA; p53;
platinum drug
Correspondence
V. Brabec, Institute of Biophysics, Academy
of Sciences of the Czech Republic,
Kralovopolska 135, CZ-61265 Brno,

Czech Republic
Fax: +420 541240499
Tel: +420 541517148
E-mail:
URL: ⁄ BNAIAD
*The authors wish it to be known that, in
their opinion, the first three authors should
be regarded as joint first authors.
(Received 12 August 2005, revised 2
November 2005, accepted 14 November
2005)
doi:10.1111/j.1742-4658.2005.05061.x
trans-[PtCl
2
NH
3
(4-Hydroxymethylpyridine)] (trans-PtHMP) is an analogue
of clinically ineffective transplatin, which is cytotoxic in the human leuke-
mia cancer cell line. As DNA is a major pharmacological target of anti-
tumor platinum compounds, modifications of DNA by trans-PtHMP and
recognition of these modifications by active tumor suppressor protein p53
were studied in cell-free media using the methods of molecular biology and
biophysics. Our results demonstrate that the replacement of the NH
3
group
in transplatin by the 4-hydroxymethylpyridine ligand affects the character
of DNA adducts of parent transplatin. The binding of trans-PtHMP is
slower, although equally sequence-specific. This platinum complex also
forms on double-stranded DNA stable intrastrand and interstrand cross-
links, which distort DNA conformation in a unique way. The most pro-

nounced conformational alterations are associated with a local DNA
unwinding, which was considerably higher than those produced by other
bifunctional platinum compounds. DNA adducts of trans-PtHMP also
reduce the affinity of the p53 protein to its consensus DNA sequence.
Thus, downstream effects modulated by recognition and binding of p53
protein to DNA distorted by trans-PtHMP and transplatin are not likely
to be the same. It has been suggested that these different effects may
contribute to different antitumor effects of these two transplatinum com-
pounds.
Abbreviations
BBR3464, [{trans-PtCl(NH
3
)
2
]
2
l-trans-Pt(NH
3
)
2
{H
2
N(CH
2
)
6
NH
2
]
2

]
4+
; cisplatin, cis-diamminedichloroplatinum(II); CDRE, consensus DNA
response element; CL, cross-link; CT, calf thymus; DMS, dimethyl sulfate; DPP, differential pulse polarography; EtBr, ethidium bromide;
FAAS, flameless atomic absorption spectrophotometry; PAA, polyacrylamide; r
b
, the number of molecules of the platinum compound bound
per nucleotide residue; r
i
, the molar ratio of free platinum complex to nucleotide phosphates at the onset of incubation with DNA; TBE, Tris-
borate ⁄ EDTA; t
m
, melting temperature; transplatin, trans-diamminedichloroplatinum(II); trans-PtHMP, trans-[PtCl
2
NH
3
(4-hydroxy-
methylpyridine)].
FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 301
Quite recently, the new complex trans-[PtCl
2
NH
3
(4-hy-
droxymethylpyridine)] (trans-PtHMP) (Fig. 1c) was
synthesized and tested for toxicity in several tumor cell
lines [5,6]. The initial examinations of the cytotoxic
activity of trans-PtHMP revealed no cytotoxicity of
this complex in two human ovarian cell lines A2780
and CH1 [5]. However, later studies of the cytotoxicity

of trans-PtHMP in HL-60 cells demonstrated a signi-
ficant efficiency of this new analogue to inhibit the
growth of this human leukemia cancer cell line [6]. The
IC
50
values (the concentration of the compound that
afforded 50% cell killing) were comparable with those
obtained for cisplatin. Thus, it is of interest to reveal
at least some features of the mechanism underlying the
cytostatic effects of this new transplatin analogue.
The antitumor effect of platinum complexes is
believed to result from their ability to form various
types of adducts with DNA. The nature of these
adducts affects a number of transduction pathways
and triggers apoptosis or necrosis in tumor cells
[7–9]. Interestingly, trans-PtHMP was shown to be
also highly effective in inducing apoptosis in HL-60
cells [6].
One of the main pathways regulating cell survival
following DNA damage is the p53 pathway [10]. A
marked in vivo response to cisplatin can occur via p53-
dependent apoptosis or independently of p53 status in
human ovarian xenografts [11], so the role of p53 in
tumor cells response to platinum drugs is ambiguous
and evidently depends on the tumor type or context.
The tumor suppressor protein p53 is a nuclear phos-
phoprotein involved in the control of cell cycle, DNA
repair and apoptosis. Hence, p53 is a potent mediator
of cellular responses against genotoxic insults, such as
platinum drugs [12] and exerts its effect through tran-

scriptional regulation. Upon exposure to genotoxic
compounds, p53 protein levels increase due to several
post-transcriptional mechanisms.
The biological functions of the p53 protein are
closely related to its sequence-specific DNA binding
activity. Active p53 binds as a tetramer to response
elements naturally occurring in the human genome
[consensus DNA response element (CDRE)]. Import-
antly, DNA adducts of cisplatin specifically and mark-
edly reduce binding affinity of the consensus DNA
sequence to active wild-type human p53 protein,
whereas the adducts of clinically ineffective transplatin
do not [13]. Hence, there is strong experimental sup-
port for the view that cisplatin may also inhibit the
p53 pathway in some tumor cells via the ability of
its DNA adducts to reduce the binding affinity of
the p53 protein to its consensus DNA sequence [13].
Similarly, DNA adducts of the new antitumor tri-
nuclear platinum complex [{trans-PtCl(NH
3
)
2
]
2
l-trans-
Pt(NH
3
)
2
{H

2
N(CH
2
)
6
NH
2
]
2
]
4+
(BBR3464) reduce the
binding affinity of the modified DNA to p53 protein
even markedly more efficiently than the adducts of
cisplatin [14]. Interestingly, BBR3464 retains significant
activity in human tumor cells lines and xenografts
refractory or poorly responsive to cisplatin and dis-
plays high activity in human tumor cell lines character-
ized by both wild-type and mutant p53 gene. In
contrast, on average, cells with mutant p53 are more
resistant to the effect of cisplatin. It has been suggested
[14] that different structural perturbations induced in
DNA by the adducts of BBR3464 and cisplatin pro-
duce differential responses to p53 protein activation
and recognition. It is therefore of great interest to
examine whether DNA adducts of cytostatic analogues
of inefficient transplatin also reduce the binding affin-
ity of DNA to p53 protein similarly as the adducts
of other antitumor complexes, such as cisplatin or
BBR3464, or whether DNA adducts of these transplat-

in analogues rather retain those features of the parent
transplatin which are responsible for its inefficiency to
affect binding affinity of p53 to its consensus DNA
sequence. This study was undertaken to examine inter-
actions of active p53 protein with oligodeoxyribonucle-
otide duplexes modified by trans-PtHMP in a cell-free
medium and to compare these results with those pub-
lished earlier [13,14] describing interactions of this pro-
tein with DNA modified by cisplatin and transplatin.
Hence, the focus of this work is on the biochemical
and biophysical aspects of the mechanisms underly-
ing the biological effects of transition metal-based
Fig. 1. (A) Structures of platinum complexes. a, cisplatin; b, trans-
platin; c, trans-PtHMP. (B) The sequences of the oligonucleotides
used in the present work. The top and bottom strand of the pairs
of oligonucleotides in Fig. 1(B) are designated ‘top’ and ‘bottom’,
respectively, throughout. The boldface letter in the top strand of
the duplexes indicates the platinated residues.
DNA recognition by p53 protein K. Stehlı
´
kova
´
et al.
302 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS
complexes and on comparison of these results with
pharmacological data already published by others, and
not on an extensive pharmacological study.
Our results demonstrate that the replacement of the
NH
3

group in transplatin by the 4-hydroxymethylpyri-
dine ligand affects the character of DNA adducts of
transplatin so that they reduce the affinity of the p53
protein to its consensus DNA sequence. Hence, we
have also examined the DNA-binding properties of
trans-PtHMP and compared these binding properties
with those of parent transplatin and its antitumor cis
isomer.
Results
Recognition by the tumor suppressor protein p53
of platinated DNA
The short (20 basepair) oligodeoxyribonucleotide
duplex, oligo-CDRE (for its nucleotide sequence, see
Fig. 1B) whose sequence follows the consensus
sequence pattern [15], was globally modified by trans-
platin, cisplatin or trans-PtHMP to r
b
in the range of
0.0125–0.05 (r
b
is defined as the number of molecules
of the platinum compound bound per nucleotide resi-
due). The unplatinated PvuII fragment of pPGM,
2513 basepairs long (containing no CDRE), was added
as the nonspecific competitor. These mixtures were
incubated with active p53 at various p53 ⁄ duplex molar
ratios (0.1–3) and analyzed using native PAGE
(Fig. 2A). Incubation of the unplatinated oligo-CDRE
with increasing amount of active p53 resulted in the
appearance of the new, more slowly migrating species

with a concomitant decrease of the intensity of the
band corresponding to the 20-basepair duplex incuba-
ted in the absence of p53 (shown for p53 ⁄ duplex ratio
of 0.3 in Fig. 2A, lane 1). This result was in agreement
with the previously published reports and demonstra-
ted formation of a sequence-specific complex between
oligo-CDRE and active p53 protein [13,16,17]. Import-
antly, addition of DO-1 mAb (which maps to the
N-terminal domain of p53) produced supershifted
complexes that migrated still more slowly than the
p53–oligo-CDRE complex (not shown) confirming the
presence of p53 in the more slowly migrating species.
In contrast, the incubation of oligo-CDRE modified
by trans-PtHMP and cisplatin at r
b
¼ 0.0125–0.05 with
active p53 reduced the yield of the species migrating
more slowly in the gel, trans-PtHMP being almost as
effective as cisplatin (Fig. 2B). Oligo-CDRE was also
globally modified by transplatin and incubated with
p53. In accordance with the results published earlier
[13], no reduction of the intensity of the band corres-
ponding to the p53–oligo-CDRE complex was noticed
even at an r
b
value as high as 0.05 (Fig. 2A, lane 10),
i.e. under conditions when cisplatin and trans-PtHMP
adducts inhibited formation of the complex between
p53 and the duplex (Fig. 2A, lanes 4 and 7). Thus,
these experiments have confirmed that the replacement

of the NH
3
group in transplatin by the 4-hydroxy-
methylpyridine ligand affects the character of DNA
adducts of transplatin so that they become capable of
reducing the affinity of the p53 protein to its CDRE,
similarly to cisplatin.
DNA binding
In order to shed light on the specific character of
DNA adducts of trans-PtHMP, we further examined
the DNA binding properties of this new transplatin
analogue and compared these binding properties with
those of parent transplatin and its antitumor cis iso-
mer. The first experiments were aimed at quantifying
trans-PtHMP binding to mammalian DNA. Solutions
of double-helical calf thymus (CT) DNA at a concen-
tration of 0.032 mgÆmL
)1
were incubated with trans-
PtHMP at the value of r
i
of 0.05 in 10 mm NaClO
4
at
37 °C(r
i
is defined as the molar ratio of free platinum
BA
Fig. 2. Binding of active p53 protein to the 20-basepair duplex con-
taining CDRE (see Fig. 1B for its sequence). The duplex was unpla-

tinated (lane 1), globally modified by cisplatin (lanes 2–4), trans-
PtHMP (lanes 5–7) and transplatin (lanes 8–10). Gel mobility retar-
dation assay was performed in the presence of the unplatinated
2513-basepair nonspecific competitor (PvuII fragment of pPMG1
lacking CDRE) in 5% native PAA gel; concentrations of the oligo-
nucleotide duplex and 2513-basepair fragment were 1.6 and
10 lgÆmL
)1
(1.26 · 10
)7
and 6 · 10
)9
M), respectively, and concen-
tration of p53 was 3.9 · 10
)8
M. r
b
values: 0 (lane 1); 0.0125 (lanes
2,5,8); 0.025 (lanes 3,6,9); 0.05 (lanes 4,7,10). The oligonucleotide
duplex was radioactively labeled at the 5¢-end of the top strand. For
other details, see the experimental part. (A) Autoradiogram. (B) The
plot of the amount of the oligonucleotide duplex in the complex
with p53 protein on the amount of the platinum complex bound per
one molecule of the duplex. Cisplatin, filled squares; trans-PtHMP,
empty squares; transplatin, filled triangles.
K. Stehlı
´
kova
´
et al. DNA recognition by p53 protein

FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 303
complex to nucleotide phosphates at the onset of incu-
bation with DNA). At various time intervals, an ali-
quot of the reaction mixture was withdrawn and
assayed by differential pulse polarography (DPP) for
platinum not bound to DNA. The amount of platinum
bound to DNA (r
b
) was calculated by subtracting the
amount of free (unbound) platinum from the total
amount of platinum present in the reaction. No chan-
ges in the pH of the reaction mixture containing DNA
and platinum compounds were measured within 48 h
after mixing DNA with the platinum complex. The
amount of the platinum compounds bound to DNA
increased with time. In this binding reaction the time
at which the binding reached 50% (t
50%
) was 120 min.
This result indicates that the rate of binding of trans-
PtHMP to natural double-helical DNA is comparable
to those of cisplatin or transplatin [18]. In further
experiments, CT DNA was incubated with trans-
PtHMP at r
i
¼ 0.2 and essentially the same rates of
the binding were observed as at r
i
¼ 0.05. The binding
of this new platinum compound to CT DNA was also

quantified in the other way. Aliquots of the reaction
withdrawn at various time intervals were quickly
cooled on an ice bath and then exhaustively dialyzed
against 10 mm NaClO
4
at 4 °C to remove free
(unbound) platinum compound. The content of plat-
inum in these DNA samples was determined by flame-
less atomic absorption spectrophotometry (FAAS).
Results identical to those obtained using the DPP
assay were obtained. The binding experiments of the
present work indicate that the modification reactions
resulted in the irreversible coordination of the new
analogue of transplatin to polymeric double-helical
DNA, which also facilitates sample analysis. Hence, it
is possible to prepare easily and precisely the samples
of DNA modified by the platinum complex at a prese-
lected value of r
b
. The samples of DNA modified by
new platinum compound and analyzed further by bio-
physical or biochemical methods were prepared in
10 mm NaClO
4
at 37 °C. If not stated otherwise, after
24 h of the reaction of DNA with the complex the
samples were precipitated in ethanol, dissolved in the
medium necessary for a particular analysis and the r
b
value in an aliquot of this sample was checked by

FAAS. In this way, the analyses described in the pre-
sent paper were performed in the absence of unbound
(free) platinum complex.
Sequence specificity of platinum adducts
There are several main methods that can be used to
determine the preferential DNA-binding sites or
sequence specificity of a DNA-binding agent [19].
In order to determine the sequence specificity of
trans-PtHMP we used in the present work a method
which consists in RNA synthesis by T7 RNA poly-
merase in vitro in the same way as in several previous
studies of the sequence specificity of various DNA-dam-
aging agents including platinum drugs [20–27]. T7
RNA polymerase was chosen to initiate these investiga-
tions because it is well characterized, its promoter is
clearly defined, and the purified enzyme is commercially
available. RNA synthesis by various RNA polymerases
including T7 RNA polymerase on DNA templates con-
taining several types of bifunctional adducts of plat-
inum complexes can be prematurely terminated at the
level or in the proximity of adducts [20–26,28,29].
Importantly, monofunctional DNA adducts of several
platinum complexes including cisplatin and transplatin
are unable to terminate RNA synthesis [21,22].
Cutting of pSP73KB DNA [21] by NdeI and HpaI
restriction endonucleases yielded a 212-bp fragment (a
substantial part of its nucleotide sequence is shown in
Fig. 3B). This fragment contained T7 RNA poly-
merase promotor [in the upper strand close to its 3¢-
end (Fig. 3B)]. The first experiments were carried out

using this linear DNA fragment, randomly modified
by transplatin, its analogue trans-PtHMP or cisplatin
at r
b
¼ 0.01, for RNA synthesis by T7 RNA poly-
merase (Fig. 3A, lanes transPt, trans-PtHMP and cis-
Pt, respectively). RNA synthesis on the template
modified by the platinum complexes yielded fragments
of defined sizes, which indicates that RNA synthesis
on these templates was prematurely terminated. The
sequence analysis revealed that the major bands result-
ing from termination of RNA synthesis by the adducts
of transplatin and trans-PtHMP were similar, appeared
mainly at G and C sites and in a considerably less
extent also at adenine (A) sites (Fig. 3B). Importantly,
the sequence dependence of the inhibition of RNA
synthesis by the adducts of transplatin and trans-
PtHMP is considerably less regular than that by the
adducts of cisplatin, indicating that the trans com-
pounds form a greater variety of adducts with DNA
and less regularly than does cisplatin.
Characterization of DNA adducts by thiourea
Cisplatin, transplatin and analogous bifunctional plat-
inum compounds coordinate to DNA in a two-step
process, forming first monofunctional adducts, prefer-
entially at guanine residues, which subsequently close
to bifunctional lesions [18,30,31]. Thiourea is used to
labilize monofunctionally bound transplatin from
DNA [32]. The displacement of transplatin is initiated
by coordination of thiourea trans to the nucleobase.

DNA recognition by p53 protein K. Stehlı
´
kova
´
et al.
304 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS
Because of the strong trans effect of sulfur, the
nucleobase nitrogen–platinum bond is weakened and
thus becomes susceptible to further substitution reac-
tions. Consequently, transplatin in monofunctional
DNA adducts is effectively removed, whereas bifunc-
tional adducts of transplatin are resistant to thiourea
treatment [32].
The initial experiments, aimed at the characteriza-
tion of DNA adducts of trans-PtHMP, were conducted
employing thiourea as a probe for DNA monofunc-
tional adducts formed by trans-platinum compounds
[32]. Double-stranded DNA was incubated with
transplatin analogue at a drug to nucleotide ratio of
r
i
¼ 0.05 in 10 mm NaClO
4
at 37 °C. At various times
the aliquots were withdrawn, the reaction in these
aliquots was stopped by quick adjusting the NaCl
concentration to 0.2 m and by immediate cooling to
)20 °C. In parallel experiments, the reaction was
stopped by addition of 10 mm thiourea solutions.
These samples were incubated for 10 min at 37 °C and

then quickly cooled to )20 °C. The samples were then
exhaustively dialyzed against 0.2 m NaCl and subse-
quently against water at 4 °C, and the platinum con-
tent was determined by FAAS (Fig. 4).
The reaction of DNA with trans-PtHMP was com-
plete after 48 h (Fig. 4). Thiourea displaced c. 97%
trans-PtHMP molecules from DNA at early time inter-
vals of the reaction of DNA with the platinum complex
(1–2 h, Fig. 4). At longer incubation times (8–24 h),
thiourea was less efficient in removing trans-PtHMP
from DNA, it displaced c. 50% trans-PtHMP mole-
cules. However, after 48 h thiourea displaced only a
negligible amount of trans-PtHMP molecules from
DNA. It implies that after 48 h most of the monofunc-
tional adducts (reactive with thiourea) closed to
bifunctional adducts not reactive with thiourea so that
after 48 h only a small fraction of adducts remained
monofunctional. It was verified that 5–60 min incuba-
tions with 10 mm thiourea gave the same results as
those shown in Fig. 4. Hence, trans-PtHMP forms con-
siderably more bifunctional adducts than transplatin,
which forms for instance after 48 h only 60% bifunc-
tional adducts under similar experimental conditions
[33]. We have also verified, in the same way as in our
recent work [34], that the different amount of DNA
adducts of transplatin and its analogue removed from
DNA by thiourea is not due to a different efficiency of
thiourea to displace the monofunctional adducts of
these different trans compounds from DNA.
Fig. 4. Kinetics of reaction of trans-PtHMP with double-helical DNA

at r
i
¼ 0.05 in 10 mM NaClO
4
at 37 °C. DNA concentration was
0.15 mgÆmL
)1
. Reactions were stopped with (n) or without (m)
10 m
M thiourea (10 min), and platinum associated with DNA was
assessed by FAAS. Data points measured in triplicate varied ± 2%
from their mean.
A
B
Fig. 3. Inhibition of RNA synthesis by T7 RNA polymerases on the
NdeI ⁄ HpaI fragment of pSP73KB plasmid modified by platinum
complexes. (A) Autoradiograms of 6% PAA ⁄ 8
M urea sequencing
gels showing inhibition of RNA synthesis by T7 RNA polymerase
on the NdeI ⁄ HpaI fragment containing adducts of platinum com-
plexes. Lanes: control, unmodified template; transPt, cisPt, and
trans-PtHMP, the template modified by transplatin, cisplatin or
trans-PtHMP at r
b
¼ 0.01, respectively; C, G, U, and A, chain ter-
minated marker RNAs. (B) Schematic diagram showing the portion
of the sequence used to monitor inhibition of RNA synthesis by
platinum complexes. The arrows indicate the start of the T7 RNA
polymerase, which used as template the upper strand of NdeI ⁄
HpaI fragment of pSP73KB DNA. The numbers correspond to the

nucleotide numbering in the sequence map of pSP73KB plasmid.
K. Stehlı
´
kova
´
et al. DNA recognition by p53 protein
FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 305
Interstrand cross-linking
Bifunctional platinum compounds that covalently bind
to DNA form various types of interstrand and intra-
strand cross-links (CLs). Considerable evidence suggests
that the antitumor efficacy of bifunctional platinum
compounds is the result of the formation of these
lesions, but their relative efficacy remains unknown.
Therefore, we have decided to quantitate the interstrand
cross-linking efficiency of trans-PtHMP in linearized
pSP73KB plasmid (2455 basepairs). This plasmid DNA
was linearized by EcoRI (EcoRI cuts only once within
pSP73KB plasmid) and modified by the platinum com-
plexes. The samples were analyzed for the interstrand
CLs by agarose gel electrophoresis under denaturing
conditions [22]. Upon electrophoresis, 3¢-end labeled
strands of linearized pSP73KB plasmid containing no
interstrand CLs migrate as a 2455-base single strand,
whereas the interstrand cross-linked strands migrate
more slowly as a higher molecular mass species (Fig. 5).
The experiments were carried out with DNA sam-
ples that were modified by the trans-PtHMP for 48 h
at various r
b

values. The bands corresponding to more
slowly migrating interstrand cross-linked fragments
were seen for r
b
values as low as 1 · 10
)4
(Fig. 5, lane
6). The intensity of the more slowly migrating band
increased with the growing level of the modification.
The radioactivity associated with the individual bands
in each lane was measured to obtain estimates of the
fraction of noncross-linked or cross-linked DNA under
each condition. The frequency of interstrand CLs
(%ICL ⁄ Pt) was calculated using the Poisson distribu-
tion from the fraction of noncross-linked DNA in
combination with the r
b
values and the fragment size.
The DNA interstrand cross-linking efficiency of the
new analogue of transplatin was almost independent
of r
b
and was 26%. Interestingly, interstrand cross-
linking efficiency of parent transplatin was consider-
ably lower (12% [22]). The samples of linearized
DNA modified by the compounds tested in the present
work at r
b
¼ 0.001 and 0.01 were also analyzed in 1%
nondenaturing agarose gel (not shown). No new, more

slowly migrating bands were observed, which indicates
that no CLs between DNA strands belonging to differ-
ent duplexes are formed.
Stability of the 1,3-GNG intrastrand cross-links
The 1,3-intrastrand CL of transplatin (G ¼ guanine;
N ¼ any base) is stable within single-stranded DNA
under physiological conditions. Within double-helical
DNA, its stability in several nucleotide sequences is
markedly reduced. These unstable CLs rearrange into
the interstrand CLs (preferentially formed by this plat-
inum compound between guanine and complementary
cytosine residues [22]). Consequently, the pairing of
single-stranded DNA containing 1,3-GNG intrastrand
CL of transplatin with their complementary DNA
sequences results in a rearrangement of these intra-
strand adducts into interstrand CLs [35]. The stability
of 1,3-GTG intrastrand CLs (T ¼ thymine) of trans-
Pt-HMP was investigated, similarly to our recent work
[34], using 20-mer oligodeoxyribonucleotide (the top
strand of the duplex TGTGT shown in Fig. 1B), which
was radioactively labeled at its 5¢-end and platinated
so that it contained single and central, site-specific 1,3-
GTG intrastrand CL. The single-stranded oligonucleo-
tide containing either this CL or the corresponding
duplex was incubated in 0.2 m NaClO
4
at 37 °C. At
various time intervals, aliquots were withdrawn and
analyzed by gel electrophoresis under denaturing con-
ditions (Fig. 6A). The 1,3-GTG intrastrand adducts of

trans-Pt-HMP within the single-stranded oligonucleo-
tides were inert over a long period of time (> 5 days)
(not shown). It was verified by dimethyl sulfate (DMS)
footprinting that no rearrangement of the 1,3-intra-
strand CL occurred within this period. In contrast, this
adduct formed by trans-Pt-HMP after pairing the
platinated single-stranded oligonucleotide with its com-
plementary strand was somewhat labile being trans-
formed into the interstrand CL. After 24 h of
incubation of the duplex TGTGT containing the 1,3-
intrastrand CL of trans-Pt-HMP, 12% of the 1,3-
intrastrand CLs were transformed into the interstrand
CLs. Importantly, the yields of these rearrangement
reactions involving the 1,3-intrastrand CLs of parent
transplatin was markedly higher, after 24 h 70% of
the 1,3-intrastrand CLs were transformed into the
interstrand CLs [34].
Fig. 5. The formation of the interstrand CLs by platinum complexes
in pSP73KB plasmid linearized by EcoRI. Autoradiogram of denatur-
ing 1% agarose gels of linearized DNA which was 3¢-end labeled.
The interstrand cross-linked DNA appears as the top bands migra-
ting on the gel more slowly than the single-stranded DNA (con-
tained in the bottom bands). The fragment was nonplatinated
(control) (lane 1) or modified by trans-PtHMP at r
b
¼ 7.5 · 10
)4
,
5 · 10
)4

,2.5· 10
)4
or 1 · 10
)4
(lanes 3–6, respectively); and by
cisplatin at r
b
¼ 1 · 10
)3
(lane 2).
DNA recognition by p53 protein K. Stehlı
´
kova
´
et al.
306 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS
DNA unwinding
Electrophoresis in native agarose gel is used to deter-
mine the unwinding induced in negatively supercoiled
pSP73 plasmid by monitoring the degree of supercoil-
ing [36] (Fig. 7). A compound that unwinds the DNA
duplex reduces the number of supercoils in closed
circular DNA so that their number decreases. This
decrease upon binding of unwinding agents causes a
decrease in the rate of migration through agarose gel,
which makes it possible to observe and quantify the
mean value of unwinding per one adduct.
Figure 7 shows electrophoresis gel from the experi-
ment in which variable amounts of trans-PtHMP have
been bound to a mixture of relaxed and negatively

supercoiled pSP73 DNA. The mean unwinding angle is
given by F ¼ 18r ⁄ r
b
(c), where r is the superhelical
density and r
b
(c) is the value of r
b
at which the super-
coiled and nicked forms co-migrate [36]. Under the
present experimental conditions, r was calculated to
be )0.055 on the basis of the data of cisplatin for
which the r
b
(c) was determined in this study and F ¼
13° was assumed. Using this approach, the DNA
unwinding angle of 28 ± 2° was determined. This
value is markedly higher than that found for parent
transplatin (9° [36]).
DNA melting
CT DNA was modified by trans-PtHMP to the value
of r
b
¼ 0.05 in 10 mm NaClO
4
at 37 °C for 24 h. The
samples were divided into two parts and in one part
the salt concentration was further adjusted by addition
of NaClO
4

(0.05 m). Hence, the melting curves for
DNA modified by trans-PtHMP to the same level were
measured in the two different media, at low and high
salt concentrations. The effect on the melting tempera-
ture (t
m
) is dependent on the salt concentration. At
high salt concentration (0.05 m), modification of DNA
by trans-PtHMP affected t
m
only very slightly (t
m
was
increased by 1.7 °C). If the concentration of salt in the
medium in which the melting curves were measured
was low (0.01 m) the modification of DNA by trans-
PtHMP resulted in a more pronounced increase of t
m
(5.6 °C). Thus, melting behavior of DNA was affected
by trans-PtHMP in a way which was similar to that by
parent transplatin [37].
CD spectroscopy
CD spectral characteristics were compared for CT
DNA in the absence and in the presence of trans-
PtHMP at r
b
values in the range of 0.01–0.05 (Fig. 8).
Upon binding of this compound to CT DNA, the con-
servative CD spectrum normally found for DNA in
canonical B-conformation transforms at wavelengths

below 300 nm. There was a slight, but significant
decrease in the intensity of the positive band around
280 nm if DNA was modified by trans-PtHMP
(Fig. 8B). This decrease was similar to that observed if
DNA was under identical conditions modified by
transplatin [38]. Based on the analogy with the changes
A
B
Fig. 6. Rearrangement of the 1,3-intrastrand CLs formed by trans-
PtHMP in the duplex TGTGT. The samples of the 2 l
M duplexes
were incubated at 37 °C in 0.2
M NaClO
4
,5mM Tris ⁄ HCl buffer
(pH 7.5) and 0.1 m
M EDTA; at various time intervals, the aliquots
were withdrawn and analyzed by electrophoresis in 12% PAA ⁄ 8
M
urea gel. (A) Autoradiograms of the gels of the duplex modified by
trans-PtHMP radioactively labeled at the 5¢-end of its top strand.
Incubation times in minutes are indicated under each lane. Lanes 0
refer to the 5¢-end labeled single-stranded top (platinated) strand.
(B) Plot of the percentages of 1,3-intrastrand CL of trans-PtHMP
(solid line) or transplatin (dashed line) versus time. These percent-
ages were calculated from the ratio of the radioactivity in each lane
in (A) associated with the band corresponding to the lower bands
in (A) to the sum of the radioactivities associated with both bands
(multiplied by 100). The plot for transplatin was taken from [34].
For other details, see text.

Fig. 7. Unwinding of supercoiled pSP73 plasmid DNA modified by
trans-PtHMP. Lanes: 1 and 9, control, nonmodified DNA; 2–8, r
b
¼
0.009, 0.018, 0.026, 0.035, 0.044, 0.053, 0.089, respectively. The
top bands correspond to the form of nicked plasmid (oc) and the
bottom bands to closed negatively supercoiled plasmid (sc).
K. Stehlı
´
kova
´
et al. DNA recognition by p53 protein
FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 307
in the CD spectra of DNA modified by cisplatin and
clinically ineffective transplatin [38], it might be sug-
gested that the binding of trans-PtHMP results in the
conformational alterations in double-helical DNA of
denaturational character similar to those induced in
DNA by parent transplatin.
Discussion
Several classes of mononuclear diaminedichloroplati-
num(II) complexes with trans geometry have been
shown to be more potent than their cis-oriented ana-
logues, especially in cell lines that are resistant to cis-
platin [1,2,4]. These studies have confirmed the effects
of a sterically demanding group in modulation of the
cytotoxicity of the transplatinum structure. The DNA
binding properties of these trans-oriented complexes
have been described in detail for several analogues of
transplatin in which either one NH

3
group was
replaced by a heterocyclic or aliphatic ligand [34,39–
43] or both NH
3
groups were replaced by iminoeth er
ligands [44]. It has been demonstrated that these
DNA-binding properties are fundamentally different
from those of cisplatin or transplatin, triggering differ-
ent cellular responses to DNA distortions. This is in
an accord with the working hypothesis that new plat-
inum compounds, which bind to DNA and affect its
conformation in a different manner to cisplatin, might
overcome cisplatin resistance [45,46]. The replacement
of one ammine group in transplatin by 4-hydroxy-
methylpyridine leads to the radical improvement of
cytotoxicity in tumor cells as well. It would also be of
interest to compare the cytotoxic activity with the cis
counterpart complex, but this work does not aim to
synthesize and characterize a new compound and to
investigate its cytotoxicity. Thus, to expand the data-
base of biochemical ⁄ biophysical properties of DNA
adducts of cytotoxic analogues of transplatin, we des-
cribed in the present work some aspects of DNA
modification by trans-PtHMP and how this modifica-
tion affects recognition by tumor suppressor protein
p53 of the consensus nucleotide sequence to which this
protein specifically binds. p53 is a potent mediator of
cellular responses against genotoxic insults including
those due to the treatment with antitumor platinum

drugs [12] and exerts its effect through transcriptional
regulation. The results of the present work demon-
strate that the efficiency of the inhibition of binding of
active p53 to the DNA consensus sequence is markedly
more pronounced for adducts of trans-PtHMP than
the adducts of parent transplatin (Fig. 2). Sequence-
dependent conformational variability of response
elements plays a critical role in the sequence-specific
binding of p53 to DNA and the stability of the result-
ing complex. Extraordinary demands for this binding
specificity and selectivity of p53 are closely related to
its tetrameric association with CDRE in which the
precise steric fit is extremely important [47].
The consensus sequences investigated in the present
work contained several sites at which adducts of plat-
inum compounds, such as cisplatin, transplatin and
trans-PtHMP, can be formed. In the CDRE investi-
gated in the present work, cisplatin forms bifunctional
adducts which strongly disturb its secondary structure.
It has been proposed [13] that the result of these per-
turbances is that the precise steric fit required for the
formation and stability of the tetrameric complex of
p53 with the consensus nucleotide sequence cannot be
attained so that p53 binds to its CDRE with a reduced
affinity. In contrast, clinically ineffective transplatin
also forms in DNA various types of adducts, but these
lesions induce in DNA relatively subtle structural per-
turbations [48,49] which have apparently no substan-
tial effect on the formation of the tetrameric complex
of p53 with the CDRE. The adducts of trans-PtHMP

reduce the affinity of p53 protein to its consensus
sequence in the extent comparable to that exhibited by
the adducts of cisplatin, which may imply that these
Fig. 8. CD spectroscopy of calf thymus DNA modified by trans-
PtHMP. CD spectra were recorded for DNA in 10 m
M NaClO
4
.(A)
CD spectra; curves: dashed lines – control (nonmodified) DNA; 1,
r
b
¼ 0.01; 2, r
b
¼ 0.03; 4, r
b
¼ 0.05. (B) Changes in the CD spectra
of DNA at the maximum of the positive band (280 nm).
DNA recognition by p53 protein K. Stehlı
´
kova
´
et al.
308 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS
adducts disturb DNA conformation much more
strongly than the adducts of transplatin and in the
extent similar to that exhibited by the adducts of cis-
platin.
The DNA-binding of trans-PtHMP is summarized
and compared with that of cisplatin and transplatin in
Table 1. An increase of t

m
due to modification of
DNA by trans-PtHMP (Table 1) can be interpreted to
mean that under these conditions ‘stabilizing’ effects,
such as those of interstrand CLs and a positive charge
introduced by the adducts of trans-PtHMP dominate
over ‘destabilizing’ effects of conformational altera-
tions [37]. In this respect, trans-PtHMP might globally
affect DNA similarly to transplatin. Similarly, both
transplatin and trans-PtHMP decreased the intensity
of the positive CD band at 275 nm consistent with
denaturational changes in DNA. Thus, overall impact
of the replacement of the ammine group in transplatin
by 4-hydroxymethylpyridine does not appear to be
reflected by melting experiments and CD analysis.
In contrast, the results of DNA unwinding experi-
ments are consistent with markedly different conform-
ational perturbances induced in DNA by transplatin
and trans-PtHMP. The values of unwinding angles are
affected by the nature of the ligands in the coordina-
tion sphere of platinum and the stereochemistry at
the platinum center. It has been shown [36] that
platinum(II) compounds with the smallest unwinding
angles (3–6°) are those that can bind DNA only mono-
functionally {[PtCl(dien)]Cl or [PtCl(NH
3
)
3
]Cl}. The
observation that the analogue of transplatin tested in

the present work cannot be grouped with monofunc-
tional platinum(II) compounds is readily understood in
terms of adduct structures in which the complexes are
preferentially coordinated to DNA in a bifunctional
manner. Interestingly, the unwinding angle produced
by trans-PtHMP was considerably higher than that
produced by bifunctional cisplatin (Table 1). Similar
higher unwinding angles in the range of 17–30° have
been produced by the adducts of other antitumor
transplatin analogues in which one NH
3
group was
replaced by heterocyclic planar or nonplanar ligand,
such as piperidine, piperazine, 4-picoline, thiazole or
quinoline [34,40]. This observation can be explained,
as in our previous papers [34,40], by the additional
contribution to unwinding associated with the interac-
tion of the planar 4-hydroxymethylpyridine ligand with
the duplex upon covalent binding of platinum. In this
way, the planar moiety in DNA adducts of trans-
PtHMP could be geometrically well positioned to
interact with the double helix. This observation can be
interpreted to mean that the replacement of the
ammine group in transplatin by the 4-hydroxymethyl-
pyridine ligand allows positioning of the planar moiet-
ies in the adducts of this analogue that would be
favorable for its interaction with the double helix. In
aggregate, the results of unwinding experiments dem-
onstrate that DNA binding mode of trans-PtHMP is
different from that of the parent transplatin and that

this different DNA binding mode correlates with con-
siderably enhanced efficiency of DNA adducts of this
new complex to inhibit binding of p53 protein to its
consensus DNA recognition sequence.
In conclusion, cellular pathways that are activated
in response to antitumor platinum drugs also involve
those related to p53, although the role of p53 in the
mechanism underlying cytotoxicity of platinum com-
plexes depends on several factors. Among these factors
belong, for instance, tumor cell type, activation of spe-
cific signaling pathways and the presence of other gen-
etic alterations. It has been proposed that sensitivity or
resistance of tumor cells to platinum complexes might
also be associated with cell cycle control and repair
processes involving p53. DNA is a major pharmacolo-
gical target of platinum compounds and DNA binding
activity of p53 protein is crucial for its tumor suppres-
sor function. Hence, ‘downstream’ effects modulated
by recognition and binding of p53 to DNA distorted
by trans-PtHMP and transplatin are not likely to be
the same, which may contribute to different antitumor
Table 1. Summary of DNA binding characteristics of trans-
[PtCl
2
NH
3
(4-hydroxymethylpyridine)] (trans-PtHMP), cisplatin and
transplatin.
a
This work.

b
The time at which the binding reached
50%.
c
Bancroft et al. [18].
d
Brabec and Leng [22].
e
DNA modified
for 48 h at r
b
¼ 0.05.
f
Fichtinger-Schepman et al. [31].
g
Kasparkova
et al. [34].
h
Rearrangement of the 1,3-GTG intrastrand CLs in the
duplex TGTGT after 24 h.
i
Brabec et al. [38].
j
Keck and Lippard [36].
k
Dt
m
is defined as the difference between the t
m
-values of platinat-

ed and nonmodified DNAs obtained in the medium of 0.2
M NaClO
4
at r
b
¼ 0.05.
l
Zaludova et al. [37].
trans-PtH MP
a
Cisplatin Transplatin
DNA binding (t
50%
)
b
300 min 120 min
c
120 min
c
% interstrand
CLs ⁄ adduct
26 6
d
12
d
% monofunctional
lesions ⁄ adduct
e
10 2
f

40
g
% intrastrand
CLs ⁄ adduct
64 90
f
48
% rearrangement of
1,3-intrastrand CLs
h
12 0 70
g
CD band at 278 nm
e
Decrease Increase
h
Decrease
h
Unwinding
angle ⁄ adduct
28° 13°
i
9°
i
Melting temperature
(Dt
m
)
j
1.7 °C ) 2.0 °C

l
0.6 °C
l
K. Stehlı
´
kova
´
et al. DNA recognition by p53 protein
FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 309
effects of these two transplatinum compounds. The
present work also demonstrates for the first time the
efficiency of the bifunctional mononuclear platinum(II)
complex containing the leaving ligands in the trans
configuration to inhibit binding of the p53 protein to
its consensus DNA sequence. Thus, in this respect the
new transplatin analogue, trans-PtHMP, resembles to
antitumor cisplatin, although the reasons for this
resemblance may be different.
Experimental procedures
Starting material
Cisplatin and transplatin were purchased from Sigma (Pra-
gue, Czech Republic). trans-PtHMP was synthesized, puri-
fied and characterized as described [6]. CT DNA (42%
G + C, mean molecular mass c. 20 000 kDa) was also
prepared and characterized as described previously [50].
Plasmids pSP73 (2464 basepairs) and pSP73KB (2455 base-
pairs) were isolated according to standard procedures. The
synthetic oligodeoxyribonucleotides (Fig. 1B) were pur-
chased from IDT, Inc. (Coralville, IA, USA) and purified
as described previously [51,52]; in the present work their

molar concentrations are related to the whole duplexes.
The human active p53 protein was expressed in baculovi-
rus-infected recombinant Sf9 insect cells. The details of the
purification and characterization were described previously
[17,53]. The protein concentration was determined by the
Bradford method. In the present paper the concentration of
the p53 protein is related to tetrameric protein units.
Restriction endonucleases EcoRI, NdeI, HpaI and T4 poly-
nucleotide kinase were purchased from New England
Biolabs (Beverly, MA, USA). Klenow fragment of DNA
polymerase I was from Boehringer-Mannheim GmbH
(Mannheim, Germany). Acrylamide, agarose, bis(acryla-
mide), ethidium bromide (EtBr), urea, thiourea, ethanol
and NaCN were from Merck kgaA (Darmstadt, Germany).
DMS was from Sigma-Aldrich s.r.o. (Prague, Czech Repub-
lic). The radioactive products were from Amersham (Ar-
lington Heights, IL, USA).
Platination reactions
CT or plasmid DNAs were incubated with the platinum
complex in 10 mm NaClO
4
at 37 °C in the dark. After
48 h, the samples of plasmid DNA were precipitated by
ethanol and redissolved in the medium required for subse-
quent biochemical or biophysical analysis whereas the sam-
ples of CT DNA were exhaustively dialyzed against such a
medium. An aliquot of these samples was used to determine
the value of r
b
by FAAS or DPP [54]. The duplex oligo-

CDRE (Fig. 1B) was incubated with trans-PtHMP in
10 mm NaClO
4
at 37 °C for 48 h in the dark. The values of
r
b
were determined by FAAS or DPP [54]. The single-stran-
ded oligonucleotide TGTGT [the top strand of the duplex
TGTGT (for its sequence, see Fig. 1B)] was reacted in stoi-
chiometric amount of trans-PtHMP. The platinated oligo-
nucleotide was repurified by ion-exchange FPLC. It was
verified by platinum FAAS and by the measurements of the
optical density that the modified oligonucleotide contained
one platinum atom. Using DMS footprinting of platinum
on DNA [22,55], it was also verified that one platinum
molecule was coordinated to two guanines at their N7 posi-
tion in the top strand of the duplex TGTGT. The plati-
nated top strand was allowed to anneal with unplatinated
complementary strand (bottom strand, Fig. 1B) in 0.1 m
NaClO
4
. Other details are in the text, or have been des-
cribed previously [22,48,51].
Preparation of DNA–protein complexes
Formation of the complexes of p53 with the oligonucleotide
duplex was examined in a buffer containing 5 mm
Tris ⁄ HCl, pH 7.6, 0.5 mm Na
3
EDTA, 50 mm KCl, 0.01%
Triton X-100 in a total volume of 12 lL. The nonmodified

or platinated duplexes were mixed with the nonmodified
2513 basepair fragment of pPGM1. The final amounts of
the duplexes and long fragment in the reactions were 20
and 120 ng, respectively. The molar ratio p53 ⁄ duplex was
0–3. Samples with p53 were incubated in ice for 30 min.
After the incubation was completed 3 lL of the loading
buffer (50% glycerol, 50 mm Na
3
EDTA, 2% bromophenol
blue) was added, the samples loaded on the native 5%
polyacrylamide (PAA) gel [mono ⁄ bis(acrylamide) ratio ¼
29 : 1] precooled to 4 °Cin0.5· Tris-borate ⁄ EDTA (TBE)
buffer. The radioactivity associated with the bands was
quantified. The primary p53 mAb DO-1 (purified and char-
acterized as described in [56]) was also added to the
p53 ⁄ DNA complex (molar ratio of mAb ⁄ p53 tetramer was
3), the mixture was incubated for an additional 30 min at
20 °C and the resulting p53–DNA–MAb complexes were
loaded on the gels.
DNA transcription by RNA polymerase in vitro
Transcription of the (NdeI ⁄ HpaI) restriction fragment of
pSP73KB DNA with DNA-dependent T7 RNA polymerase
and electrophoretic analysis of transcripts were performed
according to the protocols recommended by Promega
[Promega Protocols and Applications, 43–46 (1989 ⁄ 90)] and
previously described in detail [21,22].
DNA interstrand cross-link assay
trans-PtHMP at varying concentrations was incubated
with 2 lg of pSP73KB DNA linearized by EcoRI. The plati-
nated samples were precipitated by ethanol and analyzed

DNA recognition by p53 protein K. Stehlı
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´
et al.
310 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS
for DNA interstrand CLs in the same way as described in
several recent papers [22,57]. The linear duplexes were first
3¢-end labeled by means of Klenow fragment of DNA po-
lymerase I in the presence of [ a -
32
P]dATP. The samples
were deproteinized by phenol, precipitated by ethanol and
the pellet was dissolved in 18 lL of a solution containing
30 mm NaOH, 1 mm EDTA, 6.6% sucrose and 0.04%
bromophenol blue. The amount of interstrand CLs was an-
alyzed by electrophoresis under denaturing conditions on
alkaline agarose gel (1%). After the electrophoresis was
completed, the intensities of the bands corresponding to
single strands of DNA and interstrand cross-linked duplex
were quantified.
Rearrangement of intrastrand cross-links
The platinated oligodeoxyribonucleotide (top strand of the
duplex TGTGT, for its sequence, see Fig. 1B) at 20 lm
was allowed to anneal with the unplatinated comple-
mentary strand in 0.2 m NaClO
4
⁄ 5mm Tris ⁄ HCl,
pH 7.5 ⁄ 0.1 mm EDTA at 20 °C for 30 min and then for
2 h at 4 °C. The resulting duplex at 2 lm was subsequently

incubated at 37 °C. At various time intervals, aliquots were
withdrawn and analyzed by electrophoresis in denaturing
12% PAA ⁄ 8 m urea gel. The bases involved in the inter-
strand CLs were determined by Maxam-Gilbert footprint-
ing [21].
Unwinding of negatively supercoiled plasmid
DNA
Unwinding of closed circular supercoiled pSP73 plasmid
DNA was assayed by an agarose gel mobility shift assay
[36]. The unwinding angle F, induced per platinum-DNA
adduct, was calculated upon the determination of the r
b
value at which the complete transformation of the super-
coiled to relaxed form of the plasmid was attained. Samples
of pSP73 plasmid were incubated with trans-PtHMP at
37 °C in the dark for 48 h. All samples were precipitated
by ethanol and redissolved in the TBE buffer. An aliquot
of the precipitated sample was subjected to electrophoresis
on 1% agarose gels running at 25 °C in the dark with TBE
buffer and the voltage set at 30 V. The gels were then
stained with EtBr, followed by photography on Polaroid
667 film with transilluminator. The other aliquot was used
for the determination of r
b
values by FAAS.
DNA melting
The melting curves of CT DNAs were recorded by measur-
ing the absorbance at 260 nm. The melting curves were
recorded in a medium containing 10 mm or 0.2 m NaClO
4

with 1 mm Tris ⁄ HCl ⁄ 0.1 mm EDTA, pH 7.4. The value of
t
m
was determined as the temperature corresponding to a
maximum on the first-derivation profile of the melting
curves. The t
m
values could be thus determined with an
accuracy of ±0.5 °C.
Other physical methods
Absorption spectra were measured with a Beckmann
DU-7400 spectrophotometer. FAAS measurements were
carried out on a Unicam 939 AA spectrometer with a
graphite furnace. For FAAS analysis, DNA was precipita-
ted with ethanol and dissolved in 0.1 m HCl. DPP curves
were recorded with the aid of an EG & C PARC Electro-
chemical Analyzer, Model 384B. FPLC purification was
carried out on a Pharmacia Biotech FPLC System with
MonoQ HR 5 ⁄ 5 column. CD spectra were recorded at
25 °C using a JASCO spectropolarimeter, Model J720. The
gels were dried and visualized by using the FUJIFILM
bioimaging analyzer BAS-2500, and the radioactivities asso-
ciated with bands were quantitated with the AIDA image
analyzer software, all equipment was from raytest Isotopen-
messgera
¨
te GmbH (Straubenhardt, Germany).
Acknowledgements
This research was supported by the Grant Agency of
the Czech Republic (Grants 305 ⁄ 05 ⁄ 2030 and

204 ⁄ 03 ⁄ H016) and the Grant Agency of the Ministry
of Health of the Czech Republic (NR8562-4 ⁄ 2005).
The authors also acknowledge that this work was also
carried out within the Institutional Research Plan
AVOZ50040507 and that their participation in the
European Commission Cooperation in the Field of
Scientific and Technical Research Chemistry Action
D20 enabled them to exchange regularly the most
recent ideas in the field of platinum anticancer drugs
with several European colleagues. J.K. is the interna-
tional research scholar of the Howard Hughes Medical
Institute.
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