J Biol Inorg Chem DOI 10.1007/s00775-014-1144-3

ORIGINAL PAPER

Characterising the atypical 50 -CG DNA sequence specificity of 9-aminoacridine carboxamide Pt complexes Hieronimus W. Kava • Anne M. Galea • Farhana Md. Jamil • Yue Feng • Vincent Murray

Received: 30 January 2014 / Accepted: 27 April 2014 Ó SBIC 2014

Abstract In this study, the DNA sequence specificity of four DNA-targeted 9-aminoacridine carboxamide Pt complexes was compared with cisplatin, using two specially constructed plasmid templates. One plasmid contained 50 CG and 50 -GA insert sequences while the other plasmid contained a G-rich transferrin receptor gene promoter insert sequence. The damage profiles of each compound on the different DNA templates were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. With the plasmid that contained 50 -CG and 50 -GA dinucleotides, the four 9-aminoacridine carboxamide Pt complexes produced distinctly different damage profiles as compared with cisplatin. These 9-aminoacridine complexes had greatly increased levels of DNA damage at CG and GA dinucleotides as compared with cisplatin. It was shown that the presence of a CG or GA dinucleotide was sufficient to reveal the altered DNA sequence selectivity of the 9-aminoacridine carboxamide Pt analogues. The DNA sequence specificity of the Pt complexes was also found to be similarly altered utilising the transferrin receptor DNA sequence.

Abbreviations CE-LIF

Keywords 9-Aminoacridine  Anticancer drug  Cisplatin  Cisplatin analogues  DNA sequence specificity

DMF NER TFRC

Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1144-3) contains supplementary material, which is available to authorized users. H. W. Kava  A. M. Galea  F. Md. Jamil  Y. Feng  V. Murray (&) School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia e-mail: [email protected]

Cisplatin 9AmAcPtCl2

7-Methoxy-9AmAcPtCl2

7-Fluoro-9AmAcPtCl2

9-Ethanolamine-AcPtCl2

Capillary electrophoresis with laser-induced-fluorescence detection cisDiamminedichloridoplatinum(II) Dichlorido(N-2-[(2aminoethyl)amino]-ethyl)-9aminoacridine-4carboxamide)platinum(II) Dichlorido(N-2-[(2aminoethyl)amino]-ethyl)-7methoxy-9-aminoacridine-4carboxamide)platinum(II) Dichlorido(N-2-[(2aminoethyl)amino]-ethyl)-7fluoro-9-aminoacridine-4carboxamide)platinum(II) Dichlorido(N-2-[(2aminoethyl)amino]-ethyl)-9ethanolamine-acridine-4carboxamide)platinum(II) Dimethylformamide Nucleotide excision repair Transferrin receptor

Introduction Cisplatin (cis-diamminedichloridoplatinum(II)) is one of the most successful cancer chemotherapeutic agents [1]. Cisplatin has shown most effectiveness in the treatment of testicular and ovarian cancers, especially when used in

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J Biol Inorg Chem Fig. 1 The chemical structures of cisplatin and the four 9-aminoacridine carboxamide Pt analogues used in this study

conjunction with vinblastine, bleomycin, cyclophosphamide, doxorubicin and hexamethylamine [2]. Other cancers which are treated using cisplatin include genitourinary neoplasms, lung cancer, head and neck cancer, sarcomas and lymphomas. Cisplatin consists of a central Pt atom linked to two chlorine and two ammine groups in cis formation (Fig. 1). The mechanism of action of cisplatin involves the formation of intrastrand DNA adducts [3] that result in the inhibition of the activity of DNA polymerase and cell death [4, 5]. Approximately 62 % of these intrastrand DNA adducts are formed by cross-linking cisplatin at the N7 of adjacent guanine nucleotides, while 22 % are formed at ApG motifs [6]. These intrastrand crosslinks bend the DNA and prevent the progress of DNA polymerase [7, 8]. The ApG DNA adducts have been shown to affect polymerase activity at a lower rate than GpG DNA adducts [9], suggesting cross-links formed at GpG are responsible for the cytotoxic activity of cisplatin. The capacity of cisplatin and cisplatin analogues to damage DNA has been shown to correlate with their cytotoxic ability [10]. DNA sequence specificity studies have shown that cisplatin has a strong preference for binding to runs of consecutive guanine nucleotides [11–14]. The human telomeric hexamer of 50 -TTAGGG is thus expected to be a major target of cisplatin [15]. CpG islands and other G-rich regions of gene promoter elements may also be preferentially targeted by cisplatin and this could have implications with regard to the different levels of gene expression in

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tumour cells as compared to healthy cells. Microarray studies have identified specific genes which are up-regulated or down-regulated in response to treatment by cisplatin, but not in response to its clinically ineffective isomer transplatin [16]. Among these, the transferrin receptor (TFRC) gene was shown to be among the most significantly down-regulated by cisplatin treatment. Transferrin is a glycoprotein responsible for sequestering iron into cells, although it has been shown to bind with many other heavy metals such as ruthenium and titanium [17]. Many tumour cells have increased metal ion requirements, requiring an increase in the number of cell surface transferrin receptors [17]. Cisplatin has been determined to bind to transferrin, which may have implications regarding the cellular uptake of the drug [17, 18]. Despite its success over many decades of treatment, cisplatin is hampered by serious clinical shortcomings including toxic side effects and cellular resistance. Cisplatin’s side effects include neurotoxicity, gastrointestinal toxicity, nephrotoxicity, and hematologic toxicity [19]. The primary cause for concern during ongoing treatment is the development of resistance by tumour cells, which renders treatment ineffective [20]. Cisplatin is thought to interact with cellular molecules such as metallothionein and glutathione, which compete with DNA binding activity [21, 22]. However, once cisplatin is bound to DNA, the innate repair mechanisms of the cell, particularly nucleotide excision repair (NER), are capable of removing Pt–DNA adducts to restore DNA integrity, allowing polymerase

J Biol Inorg Chem

activity to continue [23, 24]. These factors necessitate the development of new anti-tumour agents based on cisplatin that have higher efficacy with less harmful side effects. Several analogues of cisplatin, with reduced toxic side effects, are currently used in clinical treatment. Carboplatin is an analogue with reduced toxic side effects, especially nephrotoxicity, although its mechanism of action still makes it prone to NER once bound to DNA [25]. Another analogue which is used worldwide is oxaliplatin, while nedaplatin is currently only approved for use in Japan [26]. Cisplatin has no innate affinity for DNA and hence a cisplatin analogue with an attached DNA binding moiety has properties that could allow the analogue to overcome the shortcomings faced by cisplatin [27]. The attachment of a DNA binding group to a Pt complex will place the Pt atom in close proximity to the DNA and increases the rate of adduct formation to the biological target, DNA [27–32]. This faster reaction with DNA could reduce the reaction with cellular thiols [32, 33]. The attachment of a DNA binding agent could also result in an altered DNA sequence specificity for the Pt complex. This changed DNA sequence selectivity could give rise to a different spectrum of DNA adducts that may evade cellular repair processes [32]. A number of cisplatin analogues with an attached DNA binding moiety have been developed [32] and some of the most interesting contain a 9-aminoacridine component that can intercalate into DNA. One such analogue is dichlorido(N-2-((2-aminoethyl)amino)-ethyl)-9-aminoacridine-4-carboxamide)platinum(II) (9AmAcPtCl2) (Fig. 1), with a 9-aminoacridine component, attached via a carboxamide linker, behaving as an intercalating moiety. It has been investigated in intact human HeLa cells, where it has a more rapid rate of DNA damage [31] and a lower IC50 value as compared with cisplatin [34]. In this study, 9AmAcPtCl2, with an attached 9-aminoacridine, is the lead compound, and several variations have been synthesised with differing side-chains that were developed to optimise the DNA-binding effectiveness of the analogue [34]. These are dichlorido(N-2-[(2-aminoethyl)amino]-ethyl)-7-methoxy-9-aminoacridine-4-carboxa mide)platinum(II) (7-methoxy-9AmAcPtCl2), dichlorido (N-2-[(2-aminoethyl)amino]-ethyl)-7-fluoro-9-aminoacridi ne-4-carboxamide)platinum(II) (7-fluoro-9AmAcPtCl2) and dichlorido(N-2-[(2-aminoethyl)amino]-ethyl)-9-ethanolamine-acridine-4-carboxamide)platinum(II) (9-ethanolamine-AcPtCl2) (Fig. 1). The 7-methoxy-9AmAcPtCl2 possesses an electron-donating methoxy group, while the 7-fluoro-9AmAcPtCl2 has an electron-accepting fluoro group. The 9-ethanolamine analogue contains an ethanolamine group in place of the 9-amino group. The IC50 values of these analogues were determined in HeLa cells, where the value for cisplatin was found to be 10 lM, whereas the value for 9AmAcPtCl2 was 0.4 lM [34]. The

7-methoxy-9AmAcPtCl2 had an IC50 value equivalent to that for cisplatin, while 7-fluoro-9AmAcPtCl2 and 9-ethanolamine-AcPtCl2 had IC50 values of 1.3 lM and 1.1 lM, respectively [34]. The 9-aminoacridine carboxamide Pt complexes possess several properties which could potentially be beneficial for anti-tumour activity. The binding of the intercalating group to the DNA places the Pt at closer proximity to its DNA target, which promotes a more efficient reaction. These Pt complexes have an altered DNA sequence specificity as compared with cisplatin in both purified DNA [15, 30] and intact human cells [31], with an atypical affinity for 50 -CG and 50 -GA sequences as compared with the usual preference for consecutive guanines by cisplatin [15, 32]; they have an increased rate of reaction with both purified and cellular DNA [30, 31]; the cellular IC50 values are lower as compared with cisplatin [34–36]; the complexes have activity against cisplatin-resistant cells in vitro [35, 36]; and have been shown to have activity in tumour bearing mice containing cisplatin-resistant cells [35]. Binding to alternative DNA sequences would produce a different spectrum of Pt adducts that may evade Pt adduct repair mechanisms, resulting in a greater cytotoxic effect [36]. Compared with cisplatin, the 9-aminoacridine analogues were more able to kill cisplatin-resistant cells that had an increased DNA repair ability, but not those cells that were resistant due to increased intracellular thiols or reduced cellular uptake [36]. This investigation utilised a polymerase stop assay (linear amplification procedure) that permitted the DNA sequence specificity of cisplatin and analogues to be obtained at nucleotide resolution [37, 38]. The process utilises Taq DNA polymerase to extend a fluorescentlylabelled primer along a DNA template, which terminates at the sites of DNA adduct formation. The fragments are analysed using capillary electrophoresis with laser-induced fluorescence (CE-LIF) in an ABI 3730 capillary sequencer [15, 39]. This study aimed to use these techniques to quantitatively compare the DNA adduct forming activity of cisplatin and the four DNA-targeted analogues using constructed plasmids with defined DNA sequences of interest. Previous studies have examined the DNA sequence specificity of cisplatin and analogues using plasmid constructs containing telomeric repeats and runs of guanine nucleotides of various lengths [15, 40, 41]. The main aim of this study was to explore the 50 -CG and 0 5 -GA DNA sequence specificity of the 9-aminoacridine Pt analogues as compared with cisplatin. The 9-aminoacridine Pt class of analogues are one of a small number of cisplatin analogues that have an atypical DNA sequence specificity as compared with cisplatin. Hence a deeper understanding of the DNA sequence specificity parameters that lead to this altered sequence selectivity, would be beneficial. To

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J Biol Inorg Chem Fig. 2 The DNA sequences of the T7.CG.G10 and the T7.TFRC.G10 plasmid constructs. The RevII oligonucleotide was used as primer for the linear amplification procedure and is in purple text. The template, on the bottom strand, is shown 30 – 50 while the top strand is 50 –30 . The template contains the insert sequence in red, as well as the seven telomeric repeats (T1–T7) in blue and the ten consecutive guanine bases (G10) in green. The individual sequences of the major DNA damage sites in the insert are in bold and underlined. Other sequences where the drugs are expected to bind, are also underlined. a The T7.CG.G10 plasmid construct and b the T7.TFRC.G10 plasmid construct

A

B

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this end, a plasmid was constructed (T7.CG.G10) that contained variations in 50 -CG and 50 -GA DNA sequences (Fig. 2a). In this way, it was hoped to precisely define the DNA sequence parameters required for the altered DNA sequence specificity. Also, the GC-rich TFRC promoter sequence was included in the T7.TFRC.G10 plasmid to explore this potential cisplatin DNA target (Fig. 2b). The information obtained in this study provided a more detailed evaluation of the DNA sequence specificity of the 9-aminoacridine analogues. The use of 9AmAcPtCl2 and three other analogues enabled the structural requirements of the 9-aminoacridine intercalator to be explored. This study investigated the potential of the DNA-targeted hypothesis in the development of alternative analogues of cisplatin that are more effective as anti-tumour agents than cisplatin.

Materials and methods The cisplatin was purchased from Sigma-Aldrich. The 9AmAcPtCl2, 7-methoxy-9AmAcPtCl2, 7-fluoro-9AmAcPtCl2 and 9-ethanolamine-AcPtCl2 analogues were obtained from the laboratory of David McFadyen, University of Melbourne, where they were synthesised [34]. Cisplatin and the analogues were dissolved in dimethylformamide (DMF). The DNA templates used as a target for cisplatin and analogue treatment were based on a plasmid construct derived from the pUC19 vector named T7.G10, consisting of seven telomeric repeats of the TTAGGG sequence (T7) and ten adjacent guanine nucleotides (G10) [40]. A SacI restriction enzyme site between the T7 and G10 elements existed to allow sequences of interest to be inserted into the construct. Variations of the construct were prepared, named T7.CG.G10 and T7.TFRC.G10, which contain sequence elements of interest inserted into the Sac I site. Their sequences, highlighting the primer site and regions of interest, are listed in Fig. 2a, b. The CG plasmid contains two 50 -CGG, two 50 -CGA and two 50 -TGA trinucleotides, as well several other 50 -CG DNA sequences. The TFRC plasmid contained a TFRC promoter sequence that also contained 50 -CG sequences. The DNA damage assays used 240 ng of PvuII-digested plasmids of T7.CG.G10 and T7.TFRC.G10. Each damage tube contained 20 ll of reaction mixture including 2 mM N-(2-hydroxyethyl)piperazine-N’-ethanesulfonic acid (pH 7.8), 10 mM NaCl, 10 lM EDTA, and varying concentrations of cisplatin or the analogues. These were incubated at 37 °C for 6.75 h in the dark, and recovered by ethanol precipitation and dissolved in 10 ll of 10 mM Tris–HCl (pH 8.8) and 0.1 mM Na2EDTA. The concentration of cisplatin used was 1.0 lM. For the analogues 9AmAcPtCl2, 7-fluoro-9AmAcPtCl2, and 9-ethanolamine-

AcPtCl2, 0.1 lM of the analogues was used while for 7-methoxy-9AmAcPtCl2 it was 3.0 lM. A negative control with the same reagents and DNA template was used, with DMF solvent in place of the Pt compound. The damaged DNA was used as a template for a linear amplification procedure to identify the sites of Pt DNA adduct formation. Each 20 ll reaction volume contained 1 pmol of the fluorescently labelled primer, RevII-FAM (50 -ATTGTGAGCGGATAAC) and 48 ng of cisplatin- or analogue-treated DNA. The reaction mixture also included 16.6 mM (NH4)2SO4, 67 mM Tris–HCl (pH 8.8), 6.7 mM MgCl2, 0.3 mM deoxynucleoside triphosphates and 1 U of Taq DNA polymerase. This was subjected to 20 cycles of 95 °C for 45 s, 55 °C for 1 min, 72 °C for 1 min 15 s and a final step of 72 °C for 5 min in a Bio-Rad DNA Engine Dyad Peltier thermal cycler. For the dideoxy sequencing size standard, the same reaction mixture was used, but included an additional 1 mM of the appropriate dideoxynucleoside triphosphate and the deoxynucleoside triphosphates concentration was reduced to 0.1 mM. These tubes were put through the thermal cycler with 15 cycles of 95 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min 30 s. The DNA was recovered via ethanol precipitation and dissolved in 10 ll 10 mM Tris–HCl (pH 7.8), 0.1 mM EDTA. A 2 ll aliquot was analysed using an Applied Biosystems ABI 3730 capillary sequencer via the CE-LIF method [39]. The GeneMapper v3.7 program (Applied Biosystems) was used to analyse the results. For each selected damage region, the area under the peak in the resulting electropherogram was calculated after subtraction of the corresponding value for the DMF negative control. The average values across three experimental repeats were obtained and modified with a correction algorithm to normalise the bias towards shorter damage fragments [39, 42]. The values for each damage region were divided by the total sum of the areas of all damage peaks, including the full length fragment peak, and presented as a percentage. A statistical comparison of these values was performed via a two-tailed, unpaired t test between cisplatin and each analogue in turn, with the null hypothesis being that DNA damage is the same for cisplatin and each analogue.

Results The cisplatin and analogue damaged DNA was subjected to the linear amplification procedure, analysed by CE-LIF, and the output was viewed using the Genemapper v3.7 software as electropherograms (Fig. 3, Supplementary Figure 1). DNA damage is depicted as peaks of varying intensity corresponding to the relative frequency at which Pt DNA adducts were formed at that site. The DMF control

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J Biol Inorg Chem b Fig. 3 Electropherograms showing DNA adduct formation caused by

cisplatin and the four analogues with the T7.CG.G10 plasmid. The relative fluorescence intensity is shown on the y-axis while the DNA fragment size (in nucleotides) is on the x-axis. The peaks indicate the intensity of DNA adduct binding of the Pt compound at the indicated DNA sequence sites on the DNA template. The telomere T7 region is shown T1-T7, left to right. The CG insert region includes two 50 TGA, 50 -CGA and 50 -CGG sequence elements. A) The DMF negative control; B) Treatment with 1.0 lM cisplatin; C) 0.1 lM 9AmAcPtCl2; D) 3.0 lM 7-methoxy-9AmAcPtCl2; E) 0.1 lM 7-fluoro9AmAcPtCl2 and F) 0.1 lM 9-ethanolamine-9AcPtCl2

electropherogram showed a low level of background damage intensity. The dideoxy DNA sequencing reactions provided the precise base-pair resolution molecular weight markers. The two plasmids, T7.CG.G10 and T7.TFRC.G10, share the seven telomeric repeats (50 -TTAGGG), ten consecutive guanine nucleotides (G10), 50 -CGGGG and 50 -CGG motifs. The T7.CG.G10 plasmid additionally had a unique insert region that consisted of duplicated 50 -TGA, 50 -CGA and 50 CGG motifs; while the T7.TFRC.G10 plasmid contained a TFRC promoter sequence (Fig. 2). Damage assays using 1.0 lM cisplatin produced clear damage peaks and were used as a reference to be compared with the analogue damage reactions (Fig. 3). A higher concentration of the 7-methoxy-9AmAcPtCl2 analogue was required to produce a comparable level of damage to cisplatin and the other three analogues. Quantitative assessment after application of the correction algorithm is shown in the column graphs in Fig. 4. A total of 20 major damage sites were selected for analysis on the T7.CG.G10 template, while T7.TFRC.G10 had 16. The G10 region was the most highly damaged site by cisplatin on both templates, accounting for 6.6 % of damage on T7.CG.G10 and 6.5 % for T7.TFRC.G10 (Fig. 4). The G10 site is least damaged by 9AmAcPtCl2, accounting for 2.3 % and 1.5 % of damage on T7.CG.G10 and T7.TFRC.G10, respectively, while the other analogues showed intermediate amounts of damage. The DNA damage profiles in the insert region of the T7.CG.G10 template demonstrated the different sequence specificity for the aminoacridine Pt analogues as compared with cisplatin. While virtually no damage was made by cisplatin at the TGA and CGA motifs (\0.2 %) (Figs. 3, 4), each of the analogues showed considerable damage at these sites. The 9AmAcPtCl2 analogue had increased damage at TGA (1.1 %) and CGA (1.5 %), but had almost identical damage to cisplatin at CGG (0.78 % for cisplatin, 0.74 % for 9AmAcPtCl2). For the CG insert region, the analogues generally showed the most damage at CGA, followed by TGA and CGG that were approximately equal. This was expected as the acridine

intercalator has preference for both 50 -CG and 50 -GA motifs [32], and hence the increased damage at CGA. The 9-ethanolamine-AcPtCl2 damage was similar to 9AmAcPtCl2 for TGA and CGA, but was higher for CGG (Fig. 4). The 7-methoxy-9AmAcPtCl2 also appears most similar to cisplatin, damaging the G10 site to a similar level, whereas the other analogues have significantly reduced damage at this site. The TFRC promoter insert region (Fig. 2) is very guanine-rich, containing a guanine dinucleotide, a CGGGG motif, CGGGGG and G5 motifs. The damage level for the CGGGGG motif was similar between cisplatin and the analogues except for 7-methoxy-9AmAcPtCl2, which was significantly higher at 7.2 % as compared with 3.4 % for cisplatin (Supplementary Figure 1, Fig. 4). The 7-methoxy-9AmAcPtCl2 analogue showed higher damage at G10 than any other analogue. The 9-ethanolamine-AcPtCl2 analogue showed significantly higher damage at the GG motif in the TFRC insert region, accounting for 3.6 % of damage, as compared with 1.2 % for cisplatin and 1.9 % for 9AmAcPtCl2. The DMF negative control for the T7.TFRC.G10 template exhibited consistently high artefact peaks at the CGGGG motif in the promoter insert region and this prevented any analysis from being performed for this CGGGG region. A two-tailed, unpaired t test was performed on each of the damage regions to ascertain whether significant differences were present. The null hypothesis was that the damage patterns elicited by cisplatin and each analogue were the same, and the hypothesis was rejected for a p value \0.05. Table 1 gives the p values for each DNA motif across the different analogues for the different templates. Out of 80 p values for the T7.CG.G10 template comparing cisplatin damage with each analogue, 56 (70 %) of them show a significant difference. For T7.TFRC.G10, 34 out of 64 (53 %) p values show significance. This discrepancy is mainly due to the TFRC gene promoter insert having three DNA damage sites, whereas the CG plasmid has six damage sites in the insert. The damage by 9AmAcPtCl2 is significantly different to cisplatin at more sites than any other analogue, with 75 % of sites being significantly different on both T7.CG.G10 and T7.TFRC.G10. 7-fluoro-9AmAcPtCl2 is second, with 75 % significantly different sites on T7.CG.G10 and 63 % on T7.TFRC.G10. 7-methoxy-9AmAcPtCl2 and 9-ethanolamine-AcPtCl2 each have 65 % significantly different sites on T7.CG.G10 and 38 % on T7.TFRC.G10 (Table 1). On both templates, the G10 region was damaged to a significant level of difference as compared with cisplatin, by all analogues except 7-methoxy-9AmAcPtCl2. Analysis of the Pt damage sites with the higher levels of significance (\0.01 and \0.001) over both

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A

B

Fig. 4 Percentage DNA damage levels for cisplatin and the four analogues with the T7.CG.G10 and T7.TFRC.G10 plasmid. This bar chart shows the average percentage of total damage for each of the major DNA damage sites for cisplatin and the four analogues. The DNA damage sites are shown in 30 -50 order, left to right, on the template strand. The percentage of total damage has been corrected (by subtraction of the DMF blank) and normalised by use of the

correction algorithm. Error bars (standard error of the mean) are also shown. Treatment with 1.0 lM cisplatin is in blue; 0.1 lM 9AmAcPtCl2 is red; 3.0 lM 7-methoxy-9AmAcPtCl2 is green; 0.1 lM 7-fluoro-9AmAcPtCl2 is purple; and 0.1 lM 9-ethanolamine-9AcPtCl2 is turquoise. A) the T7.CG.G10 plasmid construct and B) the T7.TFRC.G10 plasmid construct

templates was performed. The 9AmAcPtCl2 compound has the largest number of significant differences as compared with cisplatin at 69 %. The 7-fluoro-

9AmAcPtCl2 is next with 42 %, followed by 9-ethanolamine-AcPtCl2 with 33 % and finally 7-methoxy9AmAcPtCl2 with 17 %.

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J Biol Inorg Chem Table 1 p values for a two-tailed, unpaired t test between DNA damage values for cisplatin and each analogue with the T7.CG.G10 and T7.TFRC.G10 DNA templates T7.CG.G10 Site

T7.TFRC.G10 9Am

7-M

7-F

9-Et

Site

9Am

7-M

7-F

9-Et

T1

0.21

0.99

0.062

0.0024

T1

0.050

0.48

0.079

0.82

T2

0.0021

0.082

0.015

0.87

T2

0.0041

0.035

0.022

0.18

T3 T4

0.0003 0.0001

0.048 0.045

0.0011 0.013

0.55 0.87

T3 T4

0.0025 0.0006

0.085 0.014

0.0074 0.024

0.12 0.12

T5

0.0001

0.0088

0.0075

0.42

T5

0.0004

0.031

0.0087

0.051

T6

0.0001

0.0064

0.0044

0.25

T6

0.0018

0.070

0.0044

0.11

T7

0.0078

0.019

0.025

0.22

T7

0.0068

0.027

0.016

0.16

CGGGG

0.032

0.76

0.15

0.0067

CGGGG

0.0066

0.95

0.0081

0.26

CGG

0.19

0.039

0.15

0.0001

CGG

0.80

0.42

0.27

0.0029

GG

0.25

0.094

0.28

0.026

CGGGGG

0.084

0.048

0.32

0.59

G5

0.0044

0.29

0.10

0.61

TGA

0.0003

0.0073

0.084

0.0000

TGA

0.0009

0.0001

0.040

0.0014

CGA

0.0008

0.0014

0.012

0.017

CGA

0.0011

0.023

0.0002

0.0019

CGG

0.18

0.23

0.0180

0.0110

CGG

0.30

0.017

0.0036

0.1800

CGA

0.0003

0.0002

0.0073

0.0007

G10

0.0000

0.41

0.0000

0.0001

G10

0.0000

0.12

0.0001

0.0003

G3

0.52

0.57

0.50

0.0032

G3

0.012

0.10

0.18

0.010

G3

0.0000

0.015

0.0021

0.092

G3

0.093

0.66

0.0039

0.033

G5

0.0000

0.59

0.014

0.045

G5

0.0000

0.021

0.0001

0.0010

P values are highlighted according to \0.001 (italics), \0.01 (bold) and \0.05 (bold italics), for each of the compounds 9AmAcPtCl2 (9Am), 7-methoxy-AmAcPtCl2 (7-M), 7-fluoro-AmAcPtCl2 (7-F) and 9-ethanolamine-AcPtCl2 (9-Et). The seven telomere DNA sequences are written T1-T7. The DNA sequence sites are written 50 to 30 . The DNA damage sites are shown in the order that they occur on the DNA template

Discussion In this study, the DNA sequence specificity of 9-aminoacridine carboxamide Pt analogues was compared with cisplatin, utilising a specially designed plasmid template that contained various 50 -CG and 50 -GA dinucleotides. It was shown that the presence of a 50 -CG or 50 -GA dinucleotide was sufficient to reveal the DNA sequence selectivity of the 9-aminoacridine carboxamide Pt analogues as compared with cisplatin. In this way it was possible to precisely define the DNA sequence requirements for the altered DNA sequence specificity. The altered DNA sequence specificity of the 9-aminoacridine Pt complexes is atypical for cisplatin analogues, since a very small number of Pt analogues have an altered DNA sequence specificity as compared with cisplatin [27, 32]. Across the different 9-aminoacridine analogue compounds, the general trend was an increase in DNA damage at the 50 -CG and 50 -GA motifs and a decrease at consecutive guanine repeats as compared with cisplatin. The

telomeric region was damaged to a greater level than the different motifs of CG insert sequence, except by 9AmAcPtCl2, which showed similar damage levels between CGA and the telomeres. This supports the hypothesis that the telomeres are important targets for damage resulting in cytotoxicity by cisplatin, as well as its cisplatin analogues [15]. Damage levels to the TFRC promoter by the cisplatin analogues were uniformly high as compared to other regions on the T7.TFRC.G10 template, and overall were similar to the levels produced by cisplatin. In a previous study, the TFRC promoter was found to be the most significantly down-regulated gene following cisplatin treatment [16]. This could be correlated to its GC-rich promoter, making it an attractive target for damage by cisplatin and cisplatin analogues. Preferential binding to GC-rich promoters may be an important parameter in the anti-tumour activity of cisplatin [43]. The damage to the TFRC promoter by cisplatin in the cell may result in the reduction of transcription of this gene as previously shown

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[16]. Since there is evidence that the transferrin receptor may be involved in cisplatin transport [17], damage to the TFRC promoter may affect cisplatin and analogue cytotoxicity. There is also evidence from previous studies that cisplatin-transferrin interaction led to spermatogenic damage in rat testes [44]. Since tumour cells are known to require increased metal ion intake [17] and thus more transferrin receptors on the cell surface, drugs that target transferrin or transferrin receptor production may result in improved cytotoxicity. Statistical t tests were applied to analyse the significance of differences between levels of damage by cisplatin and the analogues, and confirmed our expectations that the analogue compounds differentially targeted alternative DNA motifs. 9AmAcPtCl2 was previously shown to target 50 -CG and 50 -GA motifs [15]. The DNA damage at 50 -CGA and TGA trinucleotide motifs in the CG insert was strongly elevated for the 9-aminoacridine Pt complexes as compared with cisplatin and showed that the presence of 50 -CG and 50 -GA dinucleotides was sufficient for this altered selectivity. The duplication of the motifs further increased confidence in this interpretation. The 50 -CGA motif had the highest level of DNA damage as compared to the TGA and CGG motifs. This indicated that a combination of the preferred 50 -CG and GA dinucleotides had an additive effect on the level of DNA damage. Another major alteration in DNA sequence specificity was the reduction in DNA damage intensity at runs of consecutive guanines, e.g. G10, for 9-aminoacridine Pt complexes as compared with cisplatin. This was particularly apparent for the 9AmAcPtCl2 complex while the DNA damage intensity for the 7-methoxy-9AmAcPtCl2 complex was close to the value for cisplatin. If the run of consecutive guanines began with a 50 -CG dinucleotide, e.g. 50 -CGGGG, then the difference between the analogues and cisplatin was very low. The reason for the altered DNA sequence specificity of the 9-aminoacridine carboxamide Pt complexes as compared with cisplatin was the presence of the 9-aminoacridine moiety. This 9-aminoacridine carboxamide has a preference for binding to 50 -CG DNA sequences as determined by X-ray diffraction studies [45]. It is assumed that the intercalative binding preference of the 9-aminoacridine carboxamide is also present for the 9-aminoacridine carboxamide Pt complexes. This binding preference directs the Pt away from runs of consecutive guanines by locating the Pt in close proximity to 50 -CG sequences, with the result that 9-aminoacridine carboxamide Pt complexes can more readily form adducts at 50 -CG DNA sequences than runs of consecutive guanines. Presumably, although not formally documented, the 9-aminoacridine carboxamide intercalating group also has a preference for 50 -GA DNA sequences.

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The data from this study demonstrate that the 9-aminoacridine Pt complexes have a significantly different DNA sequence specificity as compared with cisplatin. The 9-aminoacridine Pt complexes also have an altered DNA sequence specificity in human cells [31]. Hence this altered sequence selectivity could have beneficial consequences for these complexes in tumour cells. This altered DNA sequence specificity of the analogue compounds could evade the site-specific NER process that removes cisplatin DNA adducts [46]. Since increased NER is a major factor in the development of resistance to cisplatin treatment by patients [20], bypassing NER with a DNA-targeted analogue may be able to alleviate some of the problems associated with clinical treatment. In support of this hypothesis, a comparison of three cisplatin-resistant cell lines indicated that 9-aminoacridine Pt complexes were able to overcome cisplatin-resistant cells that had an increased DNA repair capacity, but were ineffective against cell lines that were cisplatin-resistant due to increased intracellular thiols or reduced cellular uptake [32, 36]. This indicates that the altered DNA sequence specificity of the 9-aminoacridine Pt complexes is crucial for their anti-tumour activity against cisplatin-resistant cells. Modifications to the 9AmAcPtCl2 based lead compound did not improve the effectiveness of the analogues in forming DNA adducts at alternative sites since the 9AmAcPtCl2 compound showed the largest number of significant differences between its damage levels on the DNA construct templates as compared with cisplatin (Table 1). The order, from most to least significant differences, was: 9AmAcPtCl2, 7-fluoro-9AmAcPtCl2, 9-ethanolamine-AcPtCl2, and finally 7-methoxy-9AmAcPtCl2. This order correlates with IC50 values that have been previously determined for these complexes in HeLa cells. For cisplatin, the IC50 is 10 lM, while the value for 9AmAcPtCl2 is much lower at 0.4 lM [34]. The 7-methoxy9AmAcPtCl2, 7-fluoro-9AmAcPtCl2 and 9-ethanolamineAcPtCl2 analogues have IC50 values of 10, 1.3 and 1.1 lM, respectively [34]. This supports the relationship between a compound’s ability to damage alternate DNA sequences in purified DNA with its cytotoxic capability. The larger size of the methoxy substituent as compared with the fluoro compound is expected to be important since the major determinant of the properties of the complex is the steric conformation of the intercalator. The larger size of the methoxy group prevents efficient intercalation and produces a pattern of damage most similar to cisplatin as compared to the other analogues [15, 32, 45]. The 9-ethanolamine modification was synthesised because of the importance of the 9-amino substituent on the acridine moiety in producing an altered sequence specificity [15, 30]. However, this did not have the expected effect since it produced only minor alterations in the sequence specificity

J Biol Inorg Chem

of the complex as compared with the 9AmAcPtCl2 compound. The DNA sequence specificity of other acridine Pt complexes has been investigated [38, 47]. The acridine Pt complexes investigated by Bierbach and colleagues showed that damage occurs at 50 -CG, TA and GA dinucleotides as well as runs of consecutive guanines [47, 48]. It should be noted that the Pt DNA adducts might isomerise from 1,2-intrastrand crosslinks to other structures, including 1,2-interstrand crosslinks and 1,3-intrastrand crosslinks [49, 50], during the linear amplification procedure. However, the nature of isomerisation of Pt complexes means that the Pt adduct remains in approximately the same position on the DNA and hence should not appreciably affect the DNA sequence specificity of the Pt complex. In conclusion, this study has shown that addition of 50 CG and 50 -GA motifs is sufficient to direct the formation of atypical Pt adducts caused by the 9AmAcPtCl2 complexes away from consecutive guanine nucleotides. This altered DNA sequence specificity is directed by the 9-aminoacridine carboxamine that has preference for 50 -CG (and presumably 50 -GA) motifs. This results in a Pt complex that has an altered DNA sequence selectivity and can evade cellular DNA repair pathways that can be elevated in cisplatin resistant cells. This results in a greater cytotoxic effect. The 9AmAcPtCl2 complexes also have been shown to have activity in tumour bearing mice containing cisplatin-resistant cells [35]. Hence these complexes could form the basis for clinically effective cancer chemotherapeutic agents and are deserving of further investigation. Acknowledgments Support of this work by the University of New South Wales, Science Faculty Research Grant Scheme, is gratefully acknowledged.

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Characterising the atypical 5'-CG DNA sequence specificity of 9-aminoacridine carboxamide Pt complexes.

In this study, the DNA sequence specificity of four DNA-targeted 9-aminoacridine carboxamide Pt complexes was compared with cisplatin, using two speci...
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