J Biol Inorg Chem (2014) 19:1203–1208 DOI 10.1007/s00775-014-1176-8
ORIGINAL PAPER
The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands Michaela Frybortova • Olga Novakova Viktor Brabec
•
Received: 9 April 2014 / Accepted: 14 June 2014 / Published online: 2 July 2014 Ó SBIC 2014
Abstract Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH3NH2)2Cl2], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand crosslinks. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced
frequency of intrastrand cross-links yielded by trans[Pt(CH3NH2)2Cl2] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH3NH2)2Cl2]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1176-8) contains supplementary material, which is available to authorized users.
Introduction
M. Frybortova V. Brabec Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, 77146 Olomouc, Czech Republic M. Frybortova O. Novakova V. Brabec (&) Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265 Brno, Czech Republic e-mail: [email protected]
Keywords Platinum drugs Antisense technology Anticancer DNA Cross-links Abbreviations ATP Adenosine triphosphate Bp Base pair CL Cross-link DEPC Diethyl pyrocarbonate DMS Dimethyl sulfate FAAS Flameless atomic absorption spectroscopy HPLC High-performance liquid chromatography PAA Polyacrylamide Transplatin trans-Diamminedichloridoplatinum(II)
Selective control of gene expression can be obtained using artificially created nucleic acids that are complementary to a RNA sequence (antisense approach) [1]. The advantage of the antisense oligonucleotides is their ability to recognize a given sequence of nucleic acid and subsequently to inhibit selectively the cellular machinery at a predetermined step [2–4]. One of the major difficulties in the clinical use of the antisense (or antigene) oligonucleotides is to avoid the dissociation of the hybrids by the cellular
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machinery. This can be achieved by cross-linking the oligonucleotides to their targets. It has been demonstrated [5, 6] that transplatin-modified oligonucleotides [transplatin = trans-diamminedichloridoplatinum(II)] are attractive tools in antisense or antigene approach because of the simplicity and efficiency of the interstrand cross-linking reaction. Clinically ineffective transplatin (the trans stereoisomer of antitumor cisplatin) forms in DNA 1,3-GNG intrastrand cross-links (CLs) which are stable in single-stranded oligonucleotides under physiological conditions. The pairing of the oligonucleotides containing these adducts with their complementary sequences within DNA or RNA triggers the rearrangement of these intrastrand CLs into interstrand CLs formed between 50 guanine contained in the original intrastrand CL and its complementary cytosine [7–9]. Thus, one of the major limitations of the clinical use of the antisense oligonucleotides can be overcome by covalent cross-linking the oligonucleotides containing intrastrand CL of transplatin to their targets by rearrangement of these intrastrand CLs to interstrand lesions. Interestingly, transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences, they readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable so that the rate of the rearrangement of the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA is dependent on choice of the targeted sequence [8–10] which represented a problem when platinated oligonucleotides were tested in cells [5]. Hence, the research continues to design and test transplatin derivatives for their use in antisense or antigene strategies with the goal to find a transplatinummodified oligonucleotide that would serve as a more promising agent in antisense or antigene approach in comparison with ‘classical’ transplatin. Very recently, we described DNA binding mode of the transplatin derivative, namely trans-[Pt(CH3NH2)2Cl2] (Fig. 1a), in which NH3 groups were replaced only by a small, non-bulky methylamine ligand [11]. Notably, this replacement in the molecule of ineffective transplatin resulted in a radical enhancement of its activity in tumor cell lines including cisplatin-resistant tumor cells. The results also indicated that the DNA interstrand cross-linking efficiency of trans-[Pt(CH3NH2)2Cl2] was similar to that of transplatin (12 % [7]), but in contrast to transplatin, trans-[Pt(CH3NH2)2Cl2] forms more intrastrand CLs, which, presumably, distort DNA more than monofunctional adducts known to induce no significant distortion of the double helix [12]. It has been shown [9] that in some sequences the 1,3GNG intrastrand adducts formed by transplatin in doublehelical DNA readily rearrange into interstrand CLs. On the
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Fig. 1 a Schematic representation of the PtII complexes used in the present work. b Sequences of the synthetic oligodeoxyribonucleotides used in this study with their abbreviations. The top and bottom strands of each pair are designated top and bottom, respectively, in the text. The bold letters in the top strands of the duplexes indicate the location of the 1,3-intrastrand cross-link after modification of the oligodeoxyribonucleotides by trans-[Pt(CH3NH2)2Cl2] in the way described in the experimental section; the bold letters in the bottom strands of these duplexes indicate the location of the sites that were involved in the interstrand CLs after the linkage isomerization reaction was completed (see the text)
other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] in the sequence contexts in which transplatin formed the relatively stable intrastrand CLs which did not readily rearranged into interstrand CLs. We have found that 1,3GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] even in such sequences readily rearrange into interstrand CLs.
Materials and methods Starting materials and reagents Complex trans-[Pt(CH3NH2)2Cl2] was synthesized and characterized as described in detail in our previously published article [11]. Transplatin (Fig. 1a) was obtained from Sigma (Prague, Czech Republic). The stock solutions of the platinum complexes were prepared in water and kept in the dark at 277 K. The concentrations of platinum in the stock solutions and after dilution by water were determined by flameless atomic absorption spectroscopy (FAAS). The synthetic oligodeoxyribonucleotides (Fig. 1b) were purchased from VBC-genomics (Vienna, Austria) or DNA
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Technology (Aarhus, Denmark). The purity of the oligonucleotides was verified by high-performance liquid chromatography (HPLC) or gel electrophoresis. T4 polynucleotide kinase was purchased from New England BioLabs (Beverly, MA, USA). Acrylamide and bis(acrylamide) were from Merck KgaA (Darmstadt, Germany). Dimethyl sulfate (DMS), KMnO4, diethyl pyrocarbonate (DEPC), KBr, KHSO5, NaCN and urea were from Sigma. [c-32P]-deoxyriboadenosine triphosphate ([c-32P]ATP) was from MP Biomedicals, LLC (Irvine, CA, USA). Platination reactions The single-stranded oligonucleotides (the top strands of the duplexes shown in Fig. 1b) were reacted in stoichiometric amounts with trans-[Pt(CH3NH2)2Cl2] or transplatin. The platinated oligonucleotides were repurified by ionexchange HPLC. It was verified by FAAS and by the measurements of the optical density that the modified oligonucleotides contained one platinum atom. It was also verified using DMS footprinting of platinum on DNA [7, 13] that one platinum molecule was coordinated to two guanines at their N7 position in the top strands of the duplexes shown in Fig. 1b. The platinated top strands were allowed to anneal with unplatinated complementary strands (the bottom strands shown in Fig. 1b) in NaClO4 (0.2 M). Other details are in the text (vide infra) or have been described previously [7, 14, 15]. Rearrangement of intrastrand cross-links of trans-[Pt(CH3NH2)2Cl2] and transplatin The platinated single-stranded oligodeoxyribonucleotides (the top strands of the 20-bp duplexes shown in Fig. 1b) (8 lM) were allowed to anneal with the unplatinated complementary (bottom) strands in NaClO4 (0.2 M), Tris–HCl buffer (5 mM, pH 7.5) and EDTA (0.1 mM) at 293 K for 30 min, and for 2 h at 277 K. Samples of these duplexes (2 lM) were further incubated at 310 K in the same medium; at various time intervals, aliquots were withdrawn and analyzed by electrophoresis in 12 % polyacrylamide (PAA)/8 M urea gel. The bases involved in the interstrand CLs were determined by hydroxyl radical footprinting [16, 17]. Chemical modifications Modifications by KMnO4, DEPC, and KBr/KHSO5 were carried out as described previously [14, 18, 19] at 288 K. The duplex strands were 50 -end labeled with [c-32P]ATP. In the case of the platinated oligonucleotides, the platinum complex was removed after reaction of the DNA with the probe by incubation with NaCN (0.2 M, pH 11) at 318 K for 10 h in the dark.
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Other physical methods Absorption spectra were measured with a Varian Cary 4000 UV–VIS spectrophotometer equipped with a thermoelectrically controlled cell holder and quartz cells with the pathlength of 1 cm. Purification of oligonucleotides with the aid of HPLC was carried out on a Waters HPLC system consisting of Waters 262 Pump, Waters 2487 UV detector, and Waters 600S Controller with MonoQHR 5/5 column. The FAAS measurements were carried out on a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer. The gels were visualized using a BAS 2500 FUJIFILM bioimaging analyzer, and the radioactivity associated with each band was quantitated with the AIDA image analyzer software (Raytest, Germany).
Results and discussion The stability of 1,3-GNG intrastrand CLs of trans[Pt(CH3NH2)2Cl2] or transplatin was investigated using 20-mer oligodeoxyribonucleotides (the top strands of the 20-bp duplexes shown in Fig. 1b) that were platinated and radioactively labeled at their 50 -ends so that they contained single and site-specific 1,3-GNG intrastrand adduct in the central sequences TGAGT, TGTGT, TGCGC and CGCGC. The sequences of these duplexes (Fig. 1b) were chosen so that the sequence contexts in which 1,3-GNG intrastrand adducts were formed represent those in which these adducts formed by transplatin in double-helical DNA readily rearrange into interstrand CLs (TGAGT and TGTGT) or are relatively stable (TGCGC and CGCGC) [9]. The single-stranded oligonucleotides containing this intrastrand CL or the corresponding duplexes (Fig. 1b) were incubated in 0.2 M NaClO4 at 310 K. At various time intervals, aliquots were withdrawn and analyzed by gel electrophoresis under denaturing conditions (Fig. 2). The 1,3-GNG intrastrand adducts of trans-[Pt(CH3NH2)2Cl2] or transplatin in all single-stranded oligonucleotides (the top strands of the duplexes shown in Fig. 1b) were inert over a long period of time (*5 days) (not shown). It was verified by DMS footprinting [7, 20] that no rearrangement of the 1,3-intrastrand adducts occurred within this period. In contrast, the intrastrand CL formed by trans[Pt(CH3NH2)2Cl2] after pairing both platinated singlestranded oligonucleotides with their complementary strands was labile. As a function of time, the radioactivity associated with the band corresponding to the 1,3-intrastrand adduct of trans-[Pt(CH3NH2)2Cl2] decreased with a concomitant increase of the radioactivity associated with the new, more slowly migrating species (Fig. 2a). This
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J Biol Inorg Chem (2014) 19:1203–1208 Table 1 Rearrangement of single, site-specific 1,3-GNG intrastrand cross-links of trans-[Pt(CH3NH2)2Cl2] and transplatin in 20-bp duplexes into interstrand cross-links Intrastrand crosslinked sequencea
trans-[Pt(CH3NH2)2Cl2]
Transplatin
% Interstrand CLs after 24 h at 310 K
t50 % (h)b
% Interstrand CLs after 24 h at 310 K
TGAGT
67
0.6
60
TGTGT
84
0.9
71
12
TGCGC
62
7
7
[24
CGCGC
67
3
26
[24
t50 % (h)b 13
The platinated duplexes (2 lM) were incubated in a medium of NaClO4 (0.2 M), Tris–HCl (5 mM, pH 7.5) at 310 K a
The central nucleotide sequence of the top strand of the 20-bp duplex at which the 1,3-GNG intrastrand cross-link of transplatin was formed. G’s in bold represent the platinated sites in the intrastrand cross-link
b
Fig. 2 Rearrangement of the 1,3-intrastrand CLs formed by trans[Pt(CH3NH2)2Cl2] or transplatin in the duplexes shown in Fig. 1b. a Autoradiograms of the gels of the duplex TGAGT (left) and TGCGC (right), both modified by trans-[Pt(CH3NH2)2Cl2] radioactively labeled at the 50 -end of its top strand. Incubation times in hours are indicated under each lane. b Plots of the percentages of 1,3interstrand CL of trans-[Pt(CH3NH2)2Cl2] (open symbols) or transplatin (full symbols) in the duplexes shown in Fig. 1b versus time. These percentages were calculated from the ratio of the radioactivity in the lane associated with the band corresponding to the lower bands to the sum of the radioactivities associated with both bands (multiplied by 100). For other details, see the text
new band migrated at approximately the same rate as the 20-bp duplexes containing a single interstrand CL (this CL resulted from the reaction between the oligonucleotide and trans-[Pt(CH3NH2)2Cl2] carried out as described [7]). No other bands were detected. Similar results were obtained at higher concentrations of the platinated duplex, which excluded an interduplex reaction. This result was interpreted to mean that the 1,3intrastrand adduct was transformed into an interstrand CL [21, 22]. After 24 h of incubation of the duplexes TGAGT, TGTGT, TGCGC and CGCGC containing a 1,3-intrastrand adduct of trans-[Pt(CH3NH2)2Cl2], 62–84 % of the 1,3intrastrand adducts were transformed into the interstrand CLs (Fig. 2; Table 1). In contrast, the yield of this rearrangement reaction involving the formation of a 1,3intrastrand CL of transplatin in the sequence TGCGC and
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The time at which the rearrangement reached 50 %
CGCGC was radically lower (only 7 and 26 %, respectively) in accordance with the previously obtained results [9]. Also in accordance with the previously obtained results [9] the yield of this rearrangement reaction involving the formation of a 1,3-intrastrand CL of transplatin in the sequences TGAGT and TGTGT was high (60–71 %). Hence, our results indicate that trans-[Pt(CH3NH2)2Cl2] forms in double-helical DNA 1,3-GNG intrastrand adducts, whose stability depends on the sequence context much less than that of the adducts of transplatin and that propensity of the intrastrand CLs of transplatin to rearrange into interstrand CLs is considerably enhanced if NH3 groups in transplatin are replaced by a small, non-bulky methylamine ligands. The bases in the interstrand CLs resulting from the rearrangement of the 1,3-intrastrand adducts of trans[Pt(CH3NH2)2Cl2] were identified from hydroxyl radical footprinting in the same way as described in previous articles [16, 17] (not shown). The results indicated that the interstrand CLs of trans-[Pt(CH3NH2)2Cl2] in both duplexes tested in this work were always formed between the platinated 50 G of the 1,3-GNG intrastrand adduct and the complementary C. We showed in our previous report [9] that a driving force behind the process involving rearrangement of the 1,3-GNG intrastrand CLs formed by transplatin in doublehelical DNA to interstrand CLs was extent of conformational distortion induced in double-helical DNA by the intrastrand CL in a respective nucleotide sequence context. It has been shown [9] that, for instance, the markedly reduced propensity of the 1,3-GNG intrastrand CL of transplatin in the CGCGC sequence to rearrange into an interstrand CL (Table 1) is associated with markedly
J Biol Inorg Chem (2014) 19:1203–1208
lowered efficiency of this CL to distort DNA. Thus, we tested hypothesis that a similar driving force is behind the process involving rearrangement of the 1,3-GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] in double-helical DNA to interstrand CLs. In accord with this hypothesis, the 1,3-GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] should markedly distort DNA also in the sequences in which the 1,3-GNG intrastrand CLs of transplatin have a markedly lowered inherent ability to undergo linkage isomerization reactions. We characterized conformational alterations, induced by the central, site-specific 1,3 GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] in the 20-bp duplexes TGAGT or TGCGC, treating these duplexes with several chemical agents that are used as tools for monitoring the existence of conformations other than canonical B-DNA. The duplexes TGAGT and TGCGC were chosen to represent duplexes in which the yield of the linkage isomerization reaction is high and very low, respectively, in the case of the intrastrand CLs of transplatin, but high in both sequences in the case of the intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] (Table 1). In addition, to minimize the rearrangement of intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] to interstrand CLs during reactions of intrastrand crosslinked duplexes with chemical probes, which could obscure analysis focused on intrastrand CLs, the intrastrand crosslinked duplexes were kept at 277 K for 12 h; at this temperature, rearrangement of the 1,3-GNG intrastrand CLs is reduced markedly. It was verified by gel electrophoresis (vide supra) that the 1,3-GTG intrastrand CL formed by trans[Pt(CH3NH2)2Cl2] in the duplexes TGAGT and TGCGC was stable at 277 K so that only ca. 1 % intrastrand crosslinked duplexes TGAGT and TGCGC were transformed into interstrand crosslinked within 12 h (not shown). Reactions with chemical probes were carried out at 288 K, but the times of these reactions were only 5–30 min (see ‘‘Materials and methods’’). Therefore, it implies that only a negligible fraction of duplexes containing a 1,3GTG intrastrand CL formed in the duplexes TGAGT or TGCGC rearranged to interstrand crosslinked duplexes within 30 min. KMnO4, bromine, or DEPC are used as chemical probes of DNA conformation [23]. These chemical probes react, under the conditions used, with thymine, cytosine, and adenine/guanine residues, respectively, in single-stranded DNA and distorted double-stranded DNA, but not with these base residues in intact, double-stranded DNA [14, 18, 19]. For this analysis, we used exactly the same methodology as in our recent studies dealing with DNA adducts of various antitumor platinum drugs. Thus, the details of this experiment can be found in those articles [14, 24], and representative gels showing piperidine-induced specific strand cleavage at KMnO4-modified, KBr/KHSO5-
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Fig. 3 Summary of the reactivity of chemical probes with the duplexes TGAGT and TGCGC containing a 1,3-GNG intrastrand cross-link of trans-[Pt(CH3NH2)2Cl2]. Solid, half-solid, and open circles designate strong, medium, or weak reactivity, respectively
modified, and DEPC-modified bases in the TGAGT and TGCGC duplexes unplatinated or containing a single 1,3GNG intrastrand CL of trans-[Pt(CH3NH2)2Cl2] are shown in the Supplementary Material, Fig. S1. The results are schematically summarized in Fig. 3. The pattern and degree of reactivity toward the chemical probes were similar for the 1,3-GNG intrastrand CLs formed by trans-[Pt(CH3NH2)2Cl2] in both sequences TGAGT and TGCGC. The extent of the distortion was large (extended over ca. 7 bp) and comparable with that observed for the 1,3-GNG intrastrand CL formed by transplatin in the TGTGT sequence (see Fig. 4 in Ref. [9]) in which this CL readily isomerized into the interstrand CL. This was in contrast to the less extensive and markedly less pronounced distortion induced in the sequence CGCGC in which the 1,3-GNG intrastrand CL of transplatin was stable (see Fig. 4 in Ref. [9]). Thus, this result confirms that the character of the conformational distortion induced by the 1,3-GNG intrastrand CL of trans-[Pt(CH3NH2)2Cl2] is less sequence-dependent than that induced by the CL of transplatin. It implies that rearrangement of the 1,3-intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] into interstrand CLs is facilitated in more sequences than the rearrangement of the 1,3-intrastrand CLs of transplatin.
Conclusions The results of the present work on the formation of DNA adducts by a novel transplatinum compound suggests novel applications related to antisense technology. It is demonstrated that a simple analog of transplatin trans[Pt(CH3NH2)2Cl2] also forms in DNA 1,3-intrastrand CLs, which are, however, only stable in single-stranded oligonucleotides. This is in contrast with several 1,3-intrastrand adducts of ‘parental’, clinically ineffective transplatin, which are stable also if formed in double-helical DNA. Importantly,
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our results indicate that the hybridization of the oligonucleotides containing intrastrand CLs of novel transplatinum compound trans-[Pt(CH3NH2)2Cl2] with the complementary sequence in DNA may trigger the linkage isomerization reaction resulting in the formation of interstrand CLs, which is, however, much faster than that observed in the case of ‘classical’ transplatin. This work also suggests that an enhanced frequency of intrastrand CLs yielded by the new transplatinum complex trans-[Pt(CH3NH2)2Cl2] is a consequence of the fact that intrastrand CLs of this derivative of transplatin considerably distort double-helical DNA in far more sequence contexts than inefficient parent transplatin. Hence, much less limitations, as regards target nucleotide sequences in which this isomerization can take place, can be expected in comparison with ‘classical’ transplatin. Accordingly, trans-[Pt(CH3NH2)2Cl2]-modified oligonucleotides may serve as promising candidates for antisense or antigene approach. Acknowledgments This research was supported by the Czech Science Foundation (Grants 13-08273S) and the Ministry of Education of the CR (Grant LH14317). Research of M. F. was also supported by the student project of the Palacky University in Olomouc (Grant IGAPrF 2014 029). The authors acknowledge that their participation in the EU COST Action CM1105 enabled them to exchange regularly the most recent ideas in the field of metallodrugs with several European colleagues. The authors also thank to Prof. Dan Gibson for a kind gift of trans-[Pt(CH3NH2)2Cl2].
References 1. Weiss B (1997) Antisense oligodeoxynucleotides and antisense RNA novel pharmacological and therapeutic agents. CRC Press, Boca Raton 2. Dean NM, Cooper SR, Shanahan W, Taylor J, Myers K (2000) J Clin Ligand Assay 23:43–49 3. Galderisi U, Cascino A, Giordano A (1999) J Cell Physiol 181:251–257
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J Biol Inorg Chem (2014) 19:1203–1208 4. Fabbro D, Muller M, Geiger T (1998). In: Crooke ST (ed) Antisense research and application. Springer, Berlin, Heidelberg, Platz 3/W-1000 Berlin 33/Germany, pp 395–425 5. Giraud-Panis M-J, Leng M (2000) Pharmacol Ther 85:175–181 6. Aupeix-Scheidler K, Chabas S, Bidou L, Rousset J-P, Leng M, Toulme J–J (2000) Nucleic Acids Res 28:438–445 7. Brabec V, Leng M (1993) Proc Natl Acad Sci USA 90:5345–5349 8. Boudvillain M, Guerin M, Dalbies R, Saison-Behmoaras T, Leng M (1997) Biochemistry 36:2925–2931 9. Kasparkova J, Marini V, Bursova V, Brabec V (2008) Biophys J 95:4361–4371 10. Colombier C, Boudvillain M, Leng M (1997) Antisense Nucleic Acid Drug Dev 7:397–402 11. Frybortova M, Novakova O, Stepankova J, Novohradsky V, Gibson D, Kasparkova J, Brabec V (2013) J Inorg Biochem 126:46–54 12. Park GY, Wilson JJ, Song Y, Lippard SJ (2012) Proc Natl Acad Sci USA 109:11987–11992 13. Comess KM, Costello CE, Lippard SJ (1990) Biochemistry 29:2102–2110 14. Brabec V, Sip M, Leng M (1993) Biochemistry 32:11676–11681 15. Brabec V, Reedijk J, Leng M (1992) Biochemistry 31:12397–12402 16. Leng M, Locker D, Giraud-Panis MJ, Schwartz A, Intini FP, Natile G, Pisano C, Boccarelli A, Giordano D, Coluccia M (2000) Mol Pharmacol 58:1525–1535 17. Kasparkova J, Vojtiskova M, Natile G, Brabec V (2008) Chem Eur J 14:1330–1341 18. Bailly C, Waring MJ (1997). In: Fox KR (ed) Drug-DNA interaction protocols. Humana Press Inc, 999 Riverview Dr/Ste 208/Totowa/NJ 07512-1165, pp 51–79 19. Ross SA, Burrows CJ (1996) Nucleic Acids Res 24:5062–5063 20. Brabec V, Neplechova K, Kasparkova J, Farrell N (2000) J Biol Inorg Chem 5:364–368 21. Boudvillain M, Dalbies R, Aussourd C, Leng M (1995) Nucleic Acids Res 23:2381–2388 22. Dalbies R, Boudvillain M, Leng M (1995) Nucleic Acids Res 23:949–953 23. Nielsen PE (1990) J Mol Recogn 3:1–24 24. Kasparkova J, Farrell N, Brabec V (2000) J Biol Chem 275:15789–15798
ORIGINAL PAPER
The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands Michaela Frybortova • Olga Novakova Viktor Brabec
•
Received: 9 April 2014 / Accepted: 14 June 2014 / Published online: 2 July 2014 Ó SBIC 2014
Abstract Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH3NH2)2Cl2], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand crosslinks. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced
frequency of intrastrand cross-links yielded by trans[Pt(CH3NH2)2Cl2] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH3NH2)2Cl2]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1176-8) contains supplementary material, which is available to authorized users.
Introduction
M. Frybortova V. Brabec Department of Biophysics, Faculty of Science, Palacky University, 17. listopadu 12, 77146 Olomouc, Czech Republic M. Frybortova O. Novakova V. Brabec (&) Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265 Brno, Czech Republic e-mail: [email protected]
Keywords Platinum drugs Antisense technology Anticancer DNA Cross-links Abbreviations ATP Adenosine triphosphate Bp Base pair CL Cross-link DEPC Diethyl pyrocarbonate DMS Dimethyl sulfate FAAS Flameless atomic absorption spectroscopy HPLC High-performance liquid chromatography PAA Polyacrylamide Transplatin trans-Diamminedichloridoplatinum(II)
Selective control of gene expression can be obtained using artificially created nucleic acids that are complementary to a RNA sequence (antisense approach) [1]. The advantage of the antisense oligonucleotides is their ability to recognize a given sequence of nucleic acid and subsequently to inhibit selectively the cellular machinery at a predetermined step [2–4]. One of the major difficulties in the clinical use of the antisense (or antigene) oligonucleotides is to avoid the dissociation of the hybrids by the cellular
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machinery. This can be achieved by cross-linking the oligonucleotides to their targets. It has been demonstrated [5, 6] that transplatin-modified oligonucleotides [transplatin = trans-diamminedichloridoplatinum(II)] are attractive tools in antisense or antigene approach because of the simplicity and efficiency of the interstrand cross-linking reaction. Clinically ineffective transplatin (the trans stereoisomer of antitumor cisplatin) forms in DNA 1,3-GNG intrastrand cross-links (CLs) which are stable in single-stranded oligonucleotides under physiological conditions. The pairing of the oligonucleotides containing these adducts with their complementary sequences within DNA or RNA triggers the rearrangement of these intrastrand CLs into interstrand CLs formed between 50 guanine contained in the original intrastrand CL and its complementary cytosine [7–9]. Thus, one of the major limitations of the clinical use of the antisense oligonucleotides can be overcome by covalent cross-linking the oligonucleotides containing intrastrand CL of transplatin to their targets by rearrangement of these intrastrand CLs to interstrand lesions. Interestingly, transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences, they readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable so that the rate of the rearrangement of the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA is dependent on choice of the targeted sequence [8–10] which represented a problem when platinated oligonucleotides were tested in cells [5]. Hence, the research continues to design and test transplatin derivatives for their use in antisense or antigene strategies with the goal to find a transplatinummodified oligonucleotide that would serve as a more promising agent in antisense or antigene approach in comparison with ‘classical’ transplatin. Very recently, we described DNA binding mode of the transplatin derivative, namely trans-[Pt(CH3NH2)2Cl2] (Fig. 1a), in which NH3 groups were replaced only by a small, non-bulky methylamine ligand [11]. Notably, this replacement in the molecule of ineffective transplatin resulted in a radical enhancement of its activity in tumor cell lines including cisplatin-resistant tumor cells. The results also indicated that the DNA interstrand cross-linking efficiency of trans-[Pt(CH3NH2)2Cl2] was similar to that of transplatin (12 % [7]), but in contrast to transplatin, trans-[Pt(CH3NH2)2Cl2] forms more intrastrand CLs, which, presumably, distort DNA more than monofunctional adducts known to induce no significant distortion of the double helix [12]. It has been shown [9] that in some sequences the 1,3GNG intrastrand adducts formed by transplatin in doublehelical DNA readily rearrange into interstrand CLs. On the
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Fig. 1 a Schematic representation of the PtII complexes used in the present work. b Sequences of the synthetic oligodeoxyribonucleotides used in this study with their abbreviations. The top and bottom strands of each pair are designated top and bottom, respectively, in the text. The bold letters in the top strands of the duplexes indicate the location of the 1,3-intrastrand cross-link after modification of the oligodeoxyribonucleotides by trans-[Pt(CH3NH2)2Cl2] in the way described in the experimental section; the bold letters in the bottom strands of these duplexes indicate the location of the sites that were involved in the interstrand CLs after the linkage isomerization reaction was completed (see the text)
other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] in the sequence contexts in which transplatin formed the relatively stable intrastrand CLs which did not readily rearranged into interstrand CLs. We have found that 1,3GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] even in such sequences readily rearrange into interstrand CLs.
Materials and methods Starting materials and reagents Complex trans-[Pt(CH3NH2)2Cl2] was synthesized and characterized as described in detail in our previously published article [11]. Transplatin (Fig. 1a) was obtained from Sigma (Prague, Czech Republic). The stock solutions of the platinum complexes were prepared in water and kept in the dark at 277 K. The concentrations of platinum in the stock solutions and after dilution by water were determined by flameless atomic absorption spectroscopy (FAAS). The synthetic oligodeoxyribonucleotides (Fig. 1b) were purchased from VBC-genomics (Vienna, Austria) or DNA
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Technology (Aarhus, Denmark). The purity of the oligonucleotides was verified by high-performance liquid chromatography (HPLC) or gel electrophoresis. T4 polynucleotide kinase was purchased from New England BioLabs (Beverly, MA, USA). Acrylamide and bis(acrylamide) were from Merck KgaA (Darmstadt, Germany). Dimethyl sulfate (DMS), KMnO4, diethyl pyrocarbonate (DEPC), KBr, KHSO5, NaCN and urea were from Sigma. [c-32P]-deoxyriboadenosine triphosphate ([c-32P]ATP) was from MP Biomedicals, LLC (Irvine, CA, USA). Platination reactions The single-stranded oligonucleotides (the top strands of the duplexes shown in Fig. 1b) were reacted in stoichiometric amounts with trans-[Pt(CH3NH2)2Cl2] or transplatin. The platinated oligonucleotides were repurified by ionexchange HPLC. It was verified by FAAS and by the measurements of the optical density that the modified oligonucleotides contained one platinum atom. It was also verified using DMS footprinting of platinum on DNA [7, 13] that one platinum molecule was coordinated to two guanines at their N7 position in the top strands of the duplexes shown in Fig. 1b. The platinated top strands were allowed to anneal with unplatinated complementary strands (the bottom strands shown in Fig. 1b) in NaClO4 (0.2 M). Other details are in the text (vide infra) or have been described previously [7, 14, 15]. Rearrangement of intrastrand cross-links of trans-[Pt(CH3NH2)2Cl2] and transplatin The platinated single-stranded oligodeoxyribonucleotides (the top strands of the 20-bp duplexes shown in Fig. 1b) (8 lM) were allowed to anneal with the unplatinated complementary (bottom) strands in NaClO4 (0.2 M), Tris–HCl buffer (5 mM, pH 7.5) and EDTA (0.1 mM) at 293 K for 30 min, and for 2 h at 277 K. Samples of these duplexes (2 lM) were further incubated at 310 K in the same medium; at various time intervals, aliquots were withdrawn and analyzed by electrophoresis in 12 % polyacrylamide (PAA)/8 M urea gel. The bases involved in the interstrand CLs were determined by hydroxyl radical footprinting [16, 17]. Chemical modifications Modifications by KMnO4, DEPC, and KBr/KHSO5 were carried out as described previously [14, 18, 19] at 288 K. The duplex strands were 50 -end labeled with [c-32P]ATP. In the case of the platinated oligonucleotides, the platinum complex was removed after reaction of the DNA with the probe by incubation with NaCN (0.2 M, pH 11) at 318 K for 10 h in the dark.
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Other physical methods Absorption spectra were measured with a Varian Cary 4000 UV–VIS spectrophotometer equipped with a thermoelectrically controlled cell holder and quartz cells with the pathlength of 1 cm. Purification of oligonucleotides with the aid of HPLC was carried out on a Waters HPLC system consisting of Waters 262 Pump, Waters 2487 UV detector, and Waters 600S Controller with MonoQHR 5/5 column. The FAAS measurements were carried out on a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer. The gels were visualized using a BAS 2500 FUJIFILM bioimaging analyzer, and the radioactivity associated with each band was quantitated with the AIDA image analyzer software (Raytest, Germany).
Results and discussion The stability of 1,3-GNG intrastrand CLs of trans[Pt(CH3NH2)2Cl2] or transplatin was investigated using 20-mer oligodeoxyribonucleotides (the top strands of the 20-bp duplexes shown in Fig. 1b) that were platinated and radioactively labeled at their 50 -ends so that they contained single and site-specific 1,3-GNG intrastrand adduct in the central sequences TGAGT, TGTGT, TGCGC and CGCGC. The sequences of these duplexes (Fig. 1b) were chosen so that the sequence contexts in which 1,3-GNG intrastrand adducts were formed represent those in which these adducts formed by transplatin in double-helical DNA readily rearrange into interstrand CLs (TGAGT and TGTGT) or are relatively stable (TGCGC and CGCGC) [9]. The single-stranded oligonucleotides containing this intrastrand CL or the corresponding duplexes (Fig. 1b) were incubated in 0.2 M NaClO4 at 310 K. At various time intervals, aliquots were withdrawn and analyzed by gel electrophoresis under denaturing conditions (Fig. 2). The 1,3-GNG intrastrand adducts of trans-[Pt(CH3NH2)2Cl2] or transplatin in all single-stranded oligonucleotides (the top strands of the duplexes shown in Fig. 1b) were inert over a long period of time (*5 days) (not shown). It was verified by DMS footprinting [7, 20] that no rearrangement of the 1,3-intrastrand adducts occurred within this period. In contrast, the intrastrand CL formed by trans[Pt(CH3NH2)2Cl2] after pairing both platinated singlestranded oligonucleotides with their complementary strands was labile. As a function of time, the radioactivity associated with the band corresponding to the 1,3-intrastrand adduct of trans-[Pt(CH3NH2)2Cl2] decreased with a concomitant increase of the radioactivity associated with the new, more slowly migrating species (Fig. 2a). This
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J Biol Inorg Chem (2014) 19:1203–1208 Table 1 Rearrangement of single, site-specific 1,3-GNG intrastrand cross-links of trans-[Pt(CH3NH2)2Cl2] and transplatin in 20-bp duplexes into interstrand cross-links Intrastrand crosslinked sequencea
trans-[Pt(CH3NH2)2Cl2]
Transplatin
% Interstrand CLs after 24 h at 310 K
t50 % (h)b
% Interstrand CLs after 24 h at 310 K
TGAGT
67
0.6
60
TGTGT
84
0.9
71
12
TGCGC
62
7
7
[24
CGCGC
67
3
26
[24
t50 % (h)b 13
The platinated duplexes (2 lM) were incubated in a medium of NaClO4 (0.2 M), Tris–HCl (5 mM, pH 7.5) at 310 K a
The central nucleotide sequence of the top strand of the 20-bp duplex at which the 1,3-GNG intrastrand cross-link of transplatin was formed. G’s in bold represent the platinated sites in the intrastrand cross-link
b
Fig. 2 Rearrangement of the 1,3-intrastrand CLs formed by trans[Pt(CH3NH2)2Cl2] or transplatin in the duplexes shown in Fig. 1b. a Autoradiograms of the gels of the duplex TGAGT (left) and TGCGC (right), both modified by trans-[Pt(CH3NH2)2Cl2] radioactively labeled at the 50 -end of its top strand. Incubation times in hours are indicated under each lane. b Plots of the percentages of 1,3interstrand CL of trans-[Pt(CH3NH2)2Cl2] (open symbols) or transplatin (full symbols) in the duplexes shown in Fig. 1b versus time. These percentages were calculated from the ratio of the radioactivity in the lane associated with the band corresponding to the lower bands to the sum of the radioactivities associated with both bands (multiplied by 100). For other details, see the text
new band migrated at approximately the same rate as the 20-bp duplexes containing a single interstrand CL (this CL resulted from the reaction between the oligonucleotide and trans-[Pt(CH3NH2)2Cl2] carried out as described [7]). No other bands were detected. Similar results were obtained at higher concentrations of the platinated duplex, which excluded an interduplex reaction. This result was interpreted to mean that the 1,3intrastrand adduct was transformed into an interstrand CL [21, 22]. After 24 h of incubation of the duplexes TGAGT, TGTGT, TGCGC and CGCGC containing a 1,3-intrastrand adduct of trans-[Pt(CH3NH2)2Cl2], 62–84 % of the 1,3intrastrand adducts were transformed into the interstrand CLs (Fig. 2; Table 1). In contrast, the yield of this rearrangement reaction involving the formation of a 1,3intrastrand CL of transplatin in the sequence TGCGC and
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The time at which the rearrangement reached 50 %
CGCGC was radically lower (only 7 and 26 %, respectively) in accordance with the previously obtained results [9]. Also in accordance with the previously obtained results [9] the yield of this rearrangement reaction involving the formation of a 1,3-intrastrand CL of transplatin in the sequences TGAGT and TGTGT was high (60–71 %). Hence, our results indicate that trans-[Pt(CH3NH2)2Cl2] forms in double-helical DNA 1,3-GNG intrastrand adducts, whose stability depends on the sequence context much less than that of the adducts of transplatin and that propensity of the intrastrand CLs of transplatin to rearrange into interstrand CLs is considerably enhanced if NH3 groups in transplatin are replaced by a small, non-bulky methylamine ligands. The bases in the interstrand CLs resulting from the rearrangement of the 1,3-intrastrand adducts of trans[Pt(CH3NH2)2Cl2] were identified from hydroxyl radical footprinting in the same way as described in previous articles [16, 17] (not shown). The results indicated that the interstrand CLs of trans-[Pt(CH3NH2)2Cl2] in both duplexes tested in this work were always formed between the platinated 50 G of the 1,3-GNG intrastrand adduct and the complementary C. We showed in our previous report [9] that a driving force behind the process involving rearrangement of the 1,3-GNG intrastrand CLs formed by transplatin in doublehelical DNA to interstrand CLs was extent of conformational distortion induced in double-helical DNA by the intrastrand CL in a respective nucleotide sequence context. It has been shown [9] that, for instance, the markedly reduced propensity of the 1,3-GNG intrastrand CL of transplatin in the CGCGC sequence to rearrange into an interstrand CL (Table 1) is associated with markedly
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lowered efficiency of this CL to distort DNA. Thus, we tested hypothesis that a similar driving force is behind the process involving rearrangement of the 1,3-GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] in double-helical DNA to interstrand CLs. In accord with this hypothesis, the 1,3-GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] should markedly distort DNA also in the sequences in which the 1,3-GNG intrastrand CLs of transplatin have a markedly lowered inherent ability to undergo linkage isomerization reactions. We characterized conformational alterations, induced by the central, site-specific 1,3 GNG intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] in the 20-bp duplexes TGAGT or TGCGC, treating these duplexes with several chemical agents that are used as tools for monitoring the existence of conformations other than canonical B-DNA. The duplexes TGAGT and TGCGC were chosen to represent duplexes in which the yield of the linkage isomerization reaction is high and very low, respectively, in the case of the intrastrand CLs of transplatin, but high in both sequences in the case of the intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] (Table 1). In addition, to minimize the rearrangement of intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] to interstrand CLs during reactions of intrastrand crosslinked duplexes with chemical probes, which could obscure analysis focused on intrastrand CLs, the intrastrand crosslinked duplexes were kept at 277 K for 12 h; at this temperature, rearrangement of the 1,3-GNG intrastrand CLs is reduced markedly. It was verified by gel electrophoresis (vide supra) that the 1,3-GTG intrastrand CL formed by trans[Pt(CH3NH2)2Cl2] in the duplexes TGAGT and TGCGC was stable at 277 K so that only ca. 1 % intrastrand crosslinked duplexes TGAGT and TGCGC were transformed into interstrand crosslinked within 12 h (not shown). Reactions with chemical probes were carried out at 288 K, but the times of these reactions were only 5–30 min (see ‘‘Materials and methods’’). Therefore, it implies that only a negligible fraction of duplexes containing a 1,3GTG intrastrand CL formed in the duplexes TGAGT or TGCGC rearranged to interstrand crosslinked duplexes within 30 min. KMnO4, bromine, or DEPC are used as chemical probes of DNA conformation [23]. These chemical probes react, under the conditions used, with thymine, cytosine, and adenine/guanine residues, respectively, in single-stranded DNA and distorted double-stranded DNA, but not with these base residues in intact, double-stranded DNA [14, 18, 19]. For this analysis, we used exactly the same methodology as in our recent studies dealing with DNA adducts of various antitumor platinum drugs. Thus, the details of this experiment can be found in those articles [14, 24], and representative gels showing piperidine-induced specific strand cleavage at KMnO4-modified, KBr/KHSO5-
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Fig. 3 Summary of the reactivity of chemical probes with the duplexes TGAGT and TGCGC containing a 1,3-GNG intrastrand cross-link of trans-[Pt(CH3NH2)2Cl2]. Solid, half-solid, and open circles designate strong, medium, or weak reactivity, respectively
modified, and DEPC-modified bases in the TGAGT and TGCGC duplexes unplatinated or containing a single 1,3GNG intrastrand CL of trans-[Pt(CH3NH2)2Cl2] are shown in the Supplementary Material, Fig. S1. The results are schematically summarized in Fig. 3. The pattern and degree of reactivity toward the chemical probes were similar for the 1,3-GNG intrastrand CLs formed by trans-[Pt(CH3NH2)2Cl2] in both sequences TGAGT and TGCGC. The extent of the distortion was large (extended over ca. 7 bp) and comparable with that observed for the 1,3-GNG intrastrand CL formed by transplatin in the TGTGT sequence (see Fig. 4 in Ref. [9]) in which this CL readily isomerized into the interstrand CL. This was in contrast to the less extensive and markedly less pronounced distortion induced in the sequence CGCGC in which the 1,3-GNG intrastrand CL of transplatin was stable (see Fig. 4 in Ref. [9]). Thus, this result confirms that the character of the conformational distortion induced by the 1,3-GNG intrastrand CL of trans-[Pt(CH3NH2)2Cl2] is less sequence-dependent than that induced by the CL of transplatin. It implies that rearrangement of the 1,3-intrastrand CLs of trans-[Pt(CH3NH2)2Cl2] into interstrand CLs is facilitated in more sequences than the rearrangement of the 1,3-intrastrand CLs of transplatin.
Conclusions The results of the present work on the formation of DNA adducts by a novel transplatinum compound suggests novel applications related to antisense technology. It is demonstrated that a simple analog of transplatin trans[Pt(CH3NH2)2Cl2] also forms in DNA 1,3-intrastrand CLs, which are, however, only stable in single-stranded oligonucleotides. This is in contrast with several 1,3-intrastrand adducts of ‘parental’, clinically ineffective transplatin, which are stable also if formed in double-helical DNA. Importantly,
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our results indicate that the hybridization of the oligonucleotides containing intrastrand CLs of novel transplatinum compound trans-[Pt(CH3NH2)2Cl2] with the complementary sequence in DNA may trigger the linkage isomerization reaction resulting in the formation of interstrand CLs, which is, however, much faster than that observed in the case of ‘classical’ transplatin. This work also suggests that an enhanced frequency of intrastrand CLs yielded by the new transplatinum complex trans-[Pt(CH3NH2)2Cl2] is a consequence of the fact that intrastrand CLs of this derivative of transplatin considerably distort double-helical DNA in far more sequence contexts than inefficient parent transplatin. Hence, much less limitations, as regards target nucleotide sequences in which this isomerization can take place, can be expected in comparison with ‘classical’ transplatin. Accordingly, trans-[Pt(CH3NH2)2Cl2]-modified oligonucleotides may serve as promising candidates for antisense or antigene approach. Acknowledgments This research was supported by the Czech Science Foundation (Grants 13-08273S) and the Ministry of Education of the CR (Grant LH14317). Research of M. F. was also supported by the student project of the Palacky University in Olomouc (Grant IGAPrF 2014 029). The authors acknowledge that their participation in the EU COST Action CM1105 enabled them to exchange regularly the most recent ideas in the field of metallodrugs with several European colleagues. The authors also thank to Prof. Dan Gibson for a kind gift of trans-[Pt(CH3NH2)2Cl2].
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