Abbreviations BBR3464, [{trans-PtClNH32]2l-trans-PtNH 3 2{H2NCH26NH2]2] 4+ ; cisplatin, cis-diamminedichloroplatinumII; CDRE, consensus DNA response element; CL, cross-link; CT, calf thy
Trang 1hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex
Kristy´na Stehlı´kova´1*, Jana Kasˇpa´rkova´1*, Olga Nova´kova´1*, Alberto Martı´nez2, Virtudes Moreno2 and Viktor Brabec1
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: brabec@ibp.cz
URL: http://www.ibp.cz/labs⁄ 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-[PtCl2NH3(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 NH3group
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(NH3)2]2l-trans-Pt(NH 3 )2{H2N(CH2)6NH2]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-[PtCl2NH3
(4-hydroxy-methylpyridine)].
Trang 2Quite recently, the new complex trans-[PtCl2NH3
(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
IC50 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 inp53-dependently 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(NH3)2]2 l-trans-Pt(NH3)2{H2N(CH2)6NH2]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 retatransplat-in 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.
Trang 3complexes 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
NH3 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 rb in the range of
0.0125–0.05 (rb 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 rb¼ 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 rb 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 NH3 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 riof 0.05 in 10 mm NaClO4at
37C (riis defined as the molar ratio of free platinum
B A
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)7and 6 · 10)9M ), respectively, and concen-tration of p53 was 3.9 · 10)8M rbvalues: 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.
Trang 4complex 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 (rb) 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% (t50%) 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 ri¼ 0.2 and essentially the same rates of
the binding were observed as at ri¼ 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 NaClO4 at 4C 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 rb The samples of DNA modified by
new platinum compound and analyzed further by
bio-physical or biochemical methods were prepared in
10 mm NaClO4 at 37C 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 rb
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 rb¼ 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
Trang 5Because 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
ri¼ 0.05 in 10 mm NaClO4 at 37C 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 37C and then quickly cooled to )20 C The samples were then exhaustively dialyzed against 0.2 m NaCl and subse-quently against water at 4C, 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 only60% 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 ri¼ 0.05 in 10 m M NaClO4 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 rb¼ 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.
Trang 6Interstrand 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 rbvalues The bands corresponding to more
slowly migrating interstrand cross-linked fragments
were seen for rbvalues 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 fracdistribu-tion of noncross-linked DNA in
combination with the rb values and the fragment size
The DNA interstrand cross-linking efficiency of the
new analogue of transplatin was almost independent
of rb 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 rb¼ 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 (G¼ guanine;
N¼ 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 interintra-strand 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 NaClO4 at 37C 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 rb¼ 7.5 · 10)4,
5 · 10)4, 2.5 · 10)4or 1 · 10)4 (lanes 3–6, respectively); and by
cisplatin at r b ¼ 1 · 10)3(lane 2).
Trang 7DNA 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 ⁄ rb(c), where r is the superhelical density and rb(c) is the value of rb 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 rb(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 rb¼ 0.05 in 10 mm NaClO4 at 37C for 24 h The samples were divided into two parts and in one part the salt concentration was further adjusted by addition
of NaClO4 (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 (tm) is dependent on the salt concentration At high salt concentration (0.05 m), modification of DNA
by trans-PtHMP affected tmonly very slightly (tmwas increased by 1.7C) 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 tm (5.6C) 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 rbvalues 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 NaClO4, 5 m M 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, rb¼
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).
Trang 8in 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 NH3 group was
replaced by a heterocyclic or aliphatic ligand [34,39–
43] or both NH3 groups were replaced by iminoether
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 recognimodifica-tion 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 bindresult-ing 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,
rb¼ 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).
Trang 9adducts 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 tm 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(NH3)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 NH3 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-[PtCl2NH3(4-hydroxymethylpyridine)] (trans-PtHMP), cisplatin and
transplatin. aThis work. bThe time at which the binding reached
50% c Bancroft et al [18] d Brabec and Leng [22] e DNA modified
for 48 h at rb¼ 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 Dtmis defined as the difference between the tm-values of
platinat-ed and nonmodifiplatinat-ed DNAs obtainplatinat-ed in the mplatinat-edium of 0.2 M NaClO 4
at r b ¼ 0.05 l
Zaludova et al [37].
trans-PtH MPa Cisplatin Transplatin DNA binding (t50%) b 300 min 120 min c 120 min c
% interstrand
CLs ⁄ adduct
% monofunctional
lesions ⁄ adduct e
% intrastrand
CLs ⁄ adduct
% rearrangement of
1,3-intrastrand CLsh
CD band at 278 nm e Decrease Increase h Decrease h
Unwinding
angle ⁄ adduct
Melting temperature
(Dtm) j
Trang 10effects 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 NaClO4 at 37C 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 rb by FAAS or DPP [54] The duplex
oligo-CDRE (Fig 1B) was incubated with trans-PtHMP in
10 mm NaClO4at 37C for 48 h in the dark The values of
rbwere 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 unplatiplati-nated complementary strand (bottom strand, Fig 1B) in 0.1 m NaClO4 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 Na3EDTA, 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 Na3EDTA, 2% bromophenol blue) was added, the samples loaded on the native 5% polyacrylamide (PAA) gel [mono⁄ bis(acrylamide) ratio ¼
29 : 1] precooled to 4C in 0.5 · 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
20C 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