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: 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-[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 ´ kova ´ et al. 310 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS [...]... for p53 in maintaining genomic integrity Cell Mol Life Sci 55, 12–27 13 Kasparkova J, Pospisilova S & Brabec V (2001) Different recognition of DNA modified by antitumor cisplatin and its clinically ineffective trans isomer by tumor suppressor protein p53 J Biol Chem 276, 16064–16069 14 Kasparkova J, Fojta M, Farrell N & Brabec V (2004) Differential recognition by the tumor suppressor protein p53 of DNA. .. 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Rev Oncol Hematol 35, 109–120 3 Brabec V (2002) DNA modifications by antitumor platinum and ruthenium compounds: their recognition and repair Prog Nucleic Acid Res Mol Biol 71, 1–68 FEBS Journal 273 (2006) 301–314 ª 2005 The Authors Journal compilation ª 2005 FEBS 311 ´ ´ K Stehlıkova et al DNA recognition by p53 protein 4 Natile G & Coluccia M (2004) Antitumor active transplatinum compounds In Metal... cis-diamminedichloroplatinum(II) with DNA: formation, identification, and quantitation Biochemistry 24, 707–713 32 Eastman A & Barry MA (1987) Interaction of transdiamminedichloroplatinum(II) with DNA: formation of monofunctional adducts and their reaction with glutathione Biochemistry 26, 3303–3307 33 Brabec V, Vrana O, Novakova O, Kleinwachter V, Intini FP, Coluccia M & Natile G (1996) DNA adducts of antitumor trans-[PtCl2(E-imino . 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. 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