DNA Condensation by Chiral α-Methylated Polyamine Analogues and

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DNA Condensation by Chiral r-Methylated Polyamine Analogues and Protection of Cellular DNA from Oxidative Damage Irina Nayvelt,† Mervi T. Hyvo¨nen,‡ Leena Alhonen,‡ Ipsit Pandya,† Thresia Thomas,† Alex R. Khomutov,| Jouko Vepsa¨la¨inen,§ Rajesh Patel,† Tuomo A. Keina¨nen,‡ and T. J. Thomas*,† Departments of Medicine, Environmental & Community Medicine and Pathology & Laboratory Medicine and the Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903, Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio, Finland, Laboratory of Chemistry, Department of Biosciences, Biocenter Kuopio, University of Kuopio, Finland, and Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russia Received August 21, 2009; Revised Manuscript Received October 21, 2009

Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of R,R′-dimethylated spermine [(R,R)-Me2Spm, (S,S)-Me2Spm, (R,S)-Me2Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me2Spm displayed more efficient condensing ability than spermine, with significantly lower EC50 (concentration for 50% compaction) values (p e 0.01). However, spermine exerted slightly more duplex stabilization than Me2Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me2Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me2Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.

Introduction The natural polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), are ubiquitous cellular components, with exquisite mechanisms for maintaining cellular homeostasis.1 These low-molecular-weight aliphatic amines are water-soluble and fully protonated at physiological pH. Polyamines act as polycations in their interactions with cellular macromolecules such as DNA, RNA, and anionic phospholipids. However, spatial positioning and distribution of charge within polyamine molecules impart unique functional characteristics in their ability to interact with biological macromolecules. High levels of polyamines in cancer cells are well known to promote increased cell proliferation.2,3 Polyamine depletion leads to growth inhibition, apoptosis, or both. Compounds that affect polyamine homeostasis are being investigated as therapeutic agents for diseases involving disorders of polyamine metabolism.4 Several polyamine analogues down-regulate polyamine levels, inhibit cellular uptake, and activate catabolism. A large percentage of studies on polyamine analogues over the last three decades focused on potential cancer chemotherapeutic properties of compounds with alkyl-substituted terminal amino * To whom correspondence should be addressed. Tel: (732) 235-8460. Fax: (732) 235-8473. E-mail: [email protected]. † University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. ‡ A. I. Virtanen Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio. § Laboratory of Chemistry, Department of Biosciences, Biocenter Kuopio, University of Kuopio. | Russian Academy of Sciences.

groups, which include down-regulation of natural polyamine uptake and inability to support cellular functions.5,6 Recently, polyamine analogues with methyl groups on R-carbon atoms were synthesized and characterized.7,8 These analogues are chiral molecules and are more resistant than natural polyamines to degradation by catabolic enzymes. It has been reported that R-methylated polyamines analogues are able to support cellular functions. A recent study demonstrated that all R-methylspermidine (MeSpd) and R,R′-dimethylspermine (Me2Spm) stereoisomers could support the growth of DU145 cells during short-term treatment with polyamine biosynthesis inhibitor, R-difluoromethylornithine (DFMO).9 In vivo, the stereoisomers restored delayed hepatic regeneration occurring after partial hepatectomy of transgenic rats with activated polyamine catabolism.9 Furthermore, administration of MeSpd was able to prevent the development of severe acute pancreatitis caused by pancreatic polyamine depletion, indicating that this analogue was successfully internalized by pancreatic cells and that it could substitute natural polyamines in vivo.10 Another recent study demonstrated that the stereoisomers differed in their ability to regulate the expression of the key metabolic enzymes of polyamine homeostasis.11 Therefore, it seems feasible to explore the intricacies of polyamine functions using R-methylated polyamines and to examine their applications in DNA packaging and in diseases involving excessive polyamine degradation. Natural and synthetic polyamines have been shown to affect the stability of double and triple stranded DNA, protect DNA from oxidative stress, ionizing radiation, and endonuclease digestion, and promote DNA packaging to nanoparticles.12-19 Under physiologi-

10.1021/bm900958c CCC: $40.75  2010 American Chemical Society Published on Web 11/17/2009

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cal ionic and pH conditions, positively charged polyamines interact electrostatically with negatively charged DNA and induce DNA compaction when 89-90% of the charges have been neutralized.20,21 Polyamines facilitate the coil-globule transition by lowering the free energy threshold, whereas the DNA globule remains in the B-form conformation.22 DNA condensation by multivalent ions is believed to occur when the ions have at least three positive charges, and ions with higher valence are able to promote DNA compaction at lower concentrations. Spectroscopic evidence suggests that N-alkylated polyamine analogues bind guanine bases and backbone phosphate groups as major targets in DNA, whereas Spd and Spm bind to major and minor grooves as well as to phosphate groups. To examine the impact of polyamine structure on DNA condensation, we compared the abilities of Spm and the stereoisomers of Me2Spm to condense and stabilize calf thymus (CT) DNA. We found significant differences in the DNA compacting abilities of these compounds, indicating that polyamine spatial geometry plays a part in the condensation process. In addition, we tested and compared the abilities of Spd, Spm, MeSpd, and Me2Spm to stabilize DNA against various stress-inducing agents in vitro and in a cell-based model system.

Experimental Section Materials. DNA. CT DNA was purchased from Worthington Biochemical Corporation (Lakewood, NJ) or Sigma Chemical (St. Louis, MO). For the condensation and melting temperature experiments, DNA (Worthington) was dissolved in 1 mM Na cacodylate buffer (1 mM Na cacodylate, 0.5 mM EDTA, pH 7.4) and dialyzed against the same buffer for 3 h. The average molecular weight of the DNA, determined by multiangle laser light scattering and Zimm plot, was 6 × 106. DNA solution’s absorbance ratio (A260/A280) was measured at 1.9, indicating that the DNA was free from protein contamination. For DNA damage experiments, CT DNA was dissolved in sterile double distilled water (ddH2O). Plasmid pGT-N28 was obtained from New England Biolabs (Ipswich, MA) and dissolved in sterile ddH2O. Reagents. Spermine tetrahydrochloride and spermidine trihydrochloride were purchased from Sigma Chemical. The stereoisomers of MeSpd and Me2Spm (Figure 1) and racemic (Rac) compounds were synthesized as previously reported.23 DFMO was obtained from ILEX Oncology (San Antonio, TX). Dulbecco’s modified Eagle’s medium (DMEM), gentamicin, and fetal bovine serum (FBS) were obtained from Sigma Chemical. Coomassie Brilliant Blue and bovine serum albumin were purchased from Bio-Rad (Hercules, CA). Methods. Cell Culture. The human prostate carcinoma cell line DU145 was obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in humidified atmosphere of +37 °C and 10% CO2 in DMEM supplemented with 10% heat-inactivated FBS and 50 µg/mL gentamicin. Total Intensity Light Scattering. Static light scattering experiments were performed using a Fluoromax-2 spectrofluorometer (Jobin YvonSpex Instruments S. A., Edison, NJ).24,25 The excitation and emission monochromators were both set to a wavelength of 305 nm with 5 nm band pass. The scattered light intensity was collected at a 90° angle with respect to the incident beam. Small quantities of spermine or isomers were added to the CT DNA/buffer solution (0.5 µg/mL DNA) in 2 mL borosilicate glass tubes to achieve the desired concentration of the condensing agent. The solutions were vortexed gently for 5 s and allowed to equilibrate for 30 min at room temperature. They were then centrifuged in a Beckman GS 6KR centrifuge for 10 min at 500g to avoid light scattering from aggregated particles. Centrifugation at 500g did not induce phase separation or a reduction in DNA concentration in solution. Therefore, the light scattering measurements are representative of the DNA concentrations used. Dynamic Laser Light Scattering. Dynamic light scattering experiments were performed using DynaPro model MSX equipment (Protein Solutions, Charlottesville, VA).24,25 Spm or analogue solutions were

Figure 1. Chemical structures of R-methylated polyamines used in this study.

added to DNA solutions (0.5 µg/mL) to achieve the desired condensing agent concentrations. The samples were mixed and allowed to attain equilibrium for 30 min at 22 °C. Samples were centrifuged for 10 min at 500g and 4 °C to remove aggregate particles. We measured hydrodynamic radii by transferring 45 µL of sample solution to a standard quartz cuvette, and the scattered light was detected at a 90° angle to the incident beam. All measurements were performed in the same cuvette to avoid variations introduced by minor differences between cuvettes. The cuvette was washed with ddH2O and vacuumdried before each measurement. A laser beam from a 2W laser (800 nm wavelength) was passed through a quartz cell holding the sample, and the scattered light was detected at a 90° angle with respect to the incident beam. The scattered light was analyzed with an autocorrelator to generate the first-order autocorrelation function. The following equation describes the autocorrelation function, g(l)(τ), for monodisperse particles that are much smaller than the incident beam

g(l)(τ) ) exp[-Dq2(τ)] In this equation, τ is the decay time and q () 4πn/[λo sin(θ/2)]) is the scattering vector, which is a function of the incident beam wavelength, λo, the scattering angle, θ, the refractive index of the solvent, n, and the diffusion coefficient, D. The hydrodynamic radius (Rh) is calculated from the diffusion coefficient using the Stokes-Einstein equation

Rh ) kT/6πηD where T is the absolute temperature, η is solvent viscosity, and k is the Boltzmann constant. Data were analyzed by a Dynamics version 6 software package obtained from Protein Solutions. Microscopy. Structural morphology of condensates was analyzed using a JEOL 1200EX electron microscope. We prepared samples by adding appropriate amounts of spermine/isomer solutions to DNA/buffer solutions (0.5 or 2.5 µg/mL). The solutions were vortexed lightly and allowed to equilibrate for 30 min. Formvar-coated copper grids were

Chiral R-Methylated Polyamine Analogues glow-discharged for 60 s. Samples were placed on the grids for 30 min, and excess solution was blotted using a filter paper. The grids were stained with either 1% uranyl acetate or 1% phosphotungstenic acid solution (pH ∼7.0) for 1 min and allowed to air-dry. Melting Temperature (Tm) Measurements. Tm experiments were performed using a Beckman DU640 spectrophotometer. Tm cell block contained six cells, one of which was filled with buffer (used as a blank). Other cells were filled with DNA solution alone or in combination with an appropriate concentration of polyamines. Melting temperature was obtained by increasing the temperature of the sample at a rate of 0.5 °C/min, within a range of 50-95 °C, and monitoring the absorbance (A) every 30 s. Tm values were taken as the temperature at which half of the complex was dissociated. Computer-generated first derivative of the melting curve, dA/dT (where A is absorbance and T is temperature) was also used for determining Tm. Tm measurements obtained by both methods did not deviate by more than 1 °C. DNA Damage in Vitro. Protection of DNA from reactive oxygen species (ROS)-induced strand breakage was measured by Cu(II)/H2O2 oxygen-radical-generating system (Fenton-type).26 Plasmid pGT-N28 (0.2 µg) was preincubated with polyamines/analogues for 10 min and then incubated in a solution containing 30 µM H2O2 and 10 µM CuSO4 in phosphate-buffered saline (PBS) (pH 7.4) for 1 h at 37 °C (total reaction volume 30 µL). After incubation, 6 µL of 5X gel-loading solution was added, and samples were electrophoresed on a 0.9% agarose gel containing ethidium bromide. Gel was destained in sterile ddH2O for 15 min, and bands were visualized using a Typhoon 9400 scanner (GE Healthcare). Relative band intensities were analyzed using Quantity One software. The aforementioned assay was also used for the detection of DNA damage induced by UV-C irradiation and DNase I treatment. For UV-C irradiation, a 10 µL mixture of plasmid DNA and polyamines was pipetted into an open dish and incubated for 10 min. The dish was irradiated (254 nm) with RPN 2500 cross-linker (GE Healthcare) for a time sufficient to supply 12 300 J/m2. Then, 2 µL of 5× gel loading buffer was added, and samples were analyzed by gel electrophoresis. For DNase I treatment, plasmid DNA was preincubated with polyamines/analogues for 10 min and then incubated with 0.1 U of DNase I (Fermentas International, Burlington, Ontario, Canada) for 5 min at 37 °C in a buffer containing 10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, and 0.1 mM CaCl2 (total reaction volume 30 µL). We added 6 µL of 5× gel loading buffer, and samples were analyzed by gel electrophoresis. DNA Damage in DU145 Cells. Cells were plated (2 × 106/10 cm plate) and grown overnight. The cells were pretreated for 72 h with medium supplemented with 5 mM DFMO (to deplete natural polyamines and increase analogue uptake), 1 mM aminoguanidine (to inhibit degradation of natural polyamines by serum amine oxidases in the medium), and 100 µM tested polyamine/analogue. For UV-C irradiation, cells were washed with PBS, and the plates were immediately irradiated for a time sufficient to supply 1000 J/m2. The cells were harvested immediately after irradiation. The extent of DNA damage (presence of alkali-labile sites) after UV-C irradiation was determined by Fast Micromethod.27 After the addition of NaOH to obtain a pH 12.4, the kinetics of alkaline unwinding were monitored by fluorometry with a Victor2 multilabel counter (PerkinElmer, Waltham, MA). For oxidative stress or drug-induced damage, analogue pretreated cells were incubated for 2 h with 500 µM H2O2 or 24 h with 10 µM etoposide, respectively. The extent of DNA fragmentation was measured by terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling (TUNEL) assay according to manufacturer’s instructions (in situ cell death kit, Roche Applied Science, IN) and analyzed by flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ). The number of aldehyde-reactive sites in DNA was measured with a commercial kit, DNA damage quantification kit (BioVision, Mountain View, CA). Determination of Polyamine Concentrations. Cells were harvested by trypsinization and washed with PBS, and the cell pellets were stored at -70 °C. Cells were counted electronically after harvesting by Coulter

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Figure 2. Typical plots of relative intensity of scattered light at 90° against concentrations of Spm and Me2Spm analogues. The CT DNA solution had a concentration of 0.5 µg/mL. The experiment was performed in 1 mM Na cacodylate buffer (pH 7.4) and repeated at least three times.

Counter model Z1 (Beckman Coulter, Fullerton, CA). Pellets were lysed in lysis buffer (20 mM Tris pH 7.4, 1 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol) and incubated on ice for 20 min. A portion of samples was taken for polyamine concentration measurement and mixed in a 9:1 ratio with 50% sulphosalicylic acid with 100 µM diaminoheptane as internal standard. Intracellular polyamine concentrations were determined by high-pressure liquid chromatography (HPLC), as previously described.28 Statistical Analysis. For DNA damage assays, analysis of variance (ANOVA) with Tukey’s posthoc test was used for multiple comparisons. Tests were performed using GraphPad Prism 4.03 software (GraphPad Software). For total intensity light scattering, dynamic light scattering, and melting temperature experiments, t test comparisons of polyamines/analogues were performed using SigmaStat 3.5 software (Systat Software).

Results Condensation of CT DNA. To determine the influence of the chirality of R-methyl substituents on DNA condensation, we studied condensation of CT DNA by Me2Spm stereoisomers and racemic mixture [25% (R,R); 25% (S,S); 25% (R,S); 25% (S,R)] and Spm. In Figure 2, the total intensity of scattered light is graphed against the concentration of each analogue. At a critical concentration of the condensing reagent, we observed a sharp rise in scattered light intensity. At higher concentrations, the scattered light intensity reached a plateau, which corresponded to complete condensation of CT DNA at this concentration range. We compared the DNA condensing ability of each compound by calculating their EC50 values (Table 1), which correspond to compound concentrations at which half of the DNA was in the compacted form. The condensing efficiency of spermine and Me2Spm stereoisomers appeared as follows: (R,R)-Me2Spm > (R,S)-Me2Spm > Rac-Me2Spm > (S,S)-Me2Spm > Spm. The EC50 values of (R,R)-, (R,S)-, and Rac-Me2Spm were significantly different from Spm, and (S,S)Me2Spm was a significantly weaker condensing agent than (R,R)- or (R,S)-diastereomers. Superior DNA compacting abilities of methylated analogues compared with spermine led to a question of whether they also induce faster DNA condensation. Previous studies using stoppedflow titration and fluorescence lifetime correlation spectroscopy showed that DNA condensation is a very fast process that occurred in a matter of milliseconds (intramolecular) or seconds (intermolecular).29,30 Unfortunately, the limitations of our laboratory equipment do not allow us to make measurements on the milliseconds to seconds time scale. We, however, found that DNA condensation was complete within 1 to 2 min after mixing in the case of spermine and stereoisomers.

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Table 1. EC50 Values of Spm and Me2Spm Stereoisomers and the Hydrodynamic Radii (Rh) of Formed Condensates compd

EC50 (µM)a mean ( SD

hydrodynamic radii (nm)b mean ( SD

Spm (S,S)-Me2Spm (R,R)-Me2Spm (R,S)-Me2Spm Rac-Me2Spm

4.17 ( 0.06 (a,b,c) 3.97 ( 0.12 (a,b) 3.40 ( 0.10 (d,e,f) 3.55 ( 0.06 (d,e,f) 3.94 ( 0.12 (a,b,g)

40.4 ( 1.0 (a,b) 48.4 ( 1.2 (c,d,e,f) 45.1 ( 2.5 (g,h,i) 39.6 ( 1.1 (a,b,j) 42.6 ( 2.4 (a,k)

a Measurements were performed in 1 mM Na cacodylate buffer (pH 7.4) with CT DNA concentration of 0.5 µg/mL. ( indicates SD from at least four separate experiments. (a) p e 0.05 versus Spm; (b) p e 0.01 versus Spm; (c) p e 0.01 (S,S); (d) p e 0.01 (R,R); (e) p e 0.01 (R,S); (f) p e 0.01 Rac; (g) e 0.05 Rac. b Measurements were performed in 1 mM Na cacodylate buffer (pH 7.4) with DNA concentrations of 0.5 µg/mL and condensing agent concentration of 10 µM. ( indicates SD from 4-6 separate experiments. (a) p e 0.01 versus spermine; (b) p e 0.001 versus Spm; (c) p e 0.01 versus (S,S); (d) p e 0.05 versus (S,S); (e) p e 0.001 versus (R,R); (f) p e 0.05 versus (R,R); (g) p e 0.001 versus (R,S); (h) p e 0.01 versus (R,S); (i) p e 0.05 versus (R,S); (j) p e 0.01 versus Rac; (k) p e 0.05 versus Rac.

Hydrodynamic Radius of Condensates. Next, we determined the size of DNA nanoparticles produced by Spm and Me2Spm stereoisomers using the compound concentrations found in the plateau regions of static light scattering curves (DNA in the fully condensed form). Hydrodynamic radii (Rh) of the condensates ranged from 39.6 ( 1.1 [(R,S)-Me2Spm] to 48.4 ( 1.2 nm [(S,S)-Me2Spm] and did not follow the same pattern as the EC50 values (Table 1). Particle sizes increased in the following order: (R,S)-Me2Spm < Spm < Rac-Me2Spm < (R,R)-Me2Spm < (S,S)-Me2Spm (Table 1). Rh values of nanoparticles prepared from (S,S)- and (R,R)-Me2Spm differed significantly from those produced by Spm, and (S,S)-Me2Spm produced significantly larger particles than (R,R)- or (R,S)Me2Spm. We also examined nanoparticle formation in the presence of Spd and MeSpd stereoisomers. In contrast with Spm/Me2Spm system, discrete condensates were formed at very high Spd/ MeSpd concentrations (400-500 µM). Particle sizes ranged from 75 ( 4.9 to 85.2 ( 3 nm for Spd and (R)-MeSpd, respectively. Electron Microscopy. We next analyzed the shapes and sizes of nanoparticles produced in the course of CT DNA condensation by Spm and Me2Spm analogues. Representative electron micrographs of condensate structures are shown in Figure 3. In the absence of condensing agents, DNA remained in an aggregated, random polymeric state (panel A). Upon the addition of polyamines, spheroids and toroids were formed (panels B-F). Whereas Spm and the stereoisomers generally produced circular structures, (S,S)-Me2Spm also formed spindle-shaped particles (panel C (b,c)). Particle size varied but was similar overall to our dynamic light scattering measurements. (It should be noted that larger structures shown in Figure 3 do not represent the majority of nanoparticle population but were selected because of better negative staining). Tm of Polyamine-DNA Complex. In the next set of experiments, we compared DNA stabilizing abilities of Spm and Me2Spm stereoisomers by determining their Tm values. We obtained Tm data by monitoring the UV absorbance of DNA at 260 nm as the temperature varied from 50 to 95 °C. At a critical temperature, a sharp rise in UV absorbance was observed in the melting profile of DNA, corresponding to DNA strand separation/melting (melting profile not shown). Each compound induced DNA stabilization in a concentration-dependent manner, as evidenced by the increasing Tm (65-90 °C) as the compound concentrations increased from 0 to 100 µM (Table 2). However,

Figure 3. Representative electron micrographs of CT DNA alone (panel A), and CT DNA condensed in the presence of Spm (panel B), (S,S)-Me2Spm (panel C), (R,R)-Me2Spm (panel D), (R,S)-Me2Spm (panel E), and Rac-Me2Spm (panel F). Scale bar is 100 nm.

the most efficient DNA condensing agents did not provide the highest degree of duplex stabilization. Spm, which showed the lowest condensing ability, had the maximal DNA stabilizing ability. (R,R)-Me2Spm, the most potent DNA compacting isomer in our investigation, also provided a high degree of DNA stabilization by Tm measurements. These results indicate that DNA condensation and stabilization may occur through divergent mechanisms. In addition, we examined the effect of Spd and MeSpd stereoisomers on the melting temperature of DNA. These compounds also conferred duplex DNA stability in a concentration-dependent manner, with Tm rising from 62 to 81 °C as the polyamine concentration varied from 0 to 100 µM (data not shown). However, there was no significant difference between Spd and MeSpd isomers, pointing to a similar degree of stabilization by all compounds. To examine the effect of methylated polyamines on DNA conformation, we conducted a circular dichroism (CD) investigation with (R,R)-Me2Spm, the most effective condensing agent used in this study. The addition of the analogue (e10 µM) did not change the conformation of DNA from the B-DNA conformation (results not shown). This result is consistent with a previous finding that polyamines do not alter the B-conformation of DNA in a significant manner.31 Protection against DNA Damage in Vitro. Because Me2Spm stereoisomers demonstrated differential ability to condense and stabilize DNA, we examined whether the stereoisomers of MeSpd and Me2Spm could stabilize and protect DNA in vitro from various damaging agents, such as UV-C irradiation, oxidative stress, and endonucleases. To measure DNA damage, we used the plasmid strand break assay, visualized by agarose gel electrophoresis. A single-strand break in intact, supercoiled plasmid DNA results in the formation of open circular DNA, whereas a double-strand break leads to the formation of linear DNA. The ability of polyamine analogues to protect DNA from oxidative stress was tested using a ROS-generating system, where hydroxyl radicals are produced by Fenton-type reaction from H2O2 catalyzed by copper(II) ions. Figure 4A shows DNA bands from an ethidium bromide stained gel. The majority of DNA in the untreated sample was in the fast-moving supercoiled state. A single nick in the double-stranded plasmid DNA

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Table 2. Effects of Spm and Me2Spm analogues on the melting temperature (Tm) of DNA Tm (°C)a conc., µM 0 1 10 25 100

Spm mean ( SD 66.1 71.6 83.5 85.7 91.5

( ( ( ( (

1.4 0.8 0.1 0.2 0.3

(i) (d,l) (f,j,l) (d,g,i,k)

(S,S)-Me2Spm mean ( SD 66.1 72.0 81.1 84.6 89.7

( ( ( ( (

1.4 0.7 0.4 (a,g,i) 0.6 (c) 0.3 (a)

(R,S)-Me2Spm mean ( SD 66.1 74.9 82.8 84.1 90.1

( ( ( ( (

1.4 0.1 0.2 0.7 0.3

(b,l) (e) (c) (b)

(R,R)-Me2Spm mean ( SD 66.1 72.3 82.8 85.0 90.1

( ( ( ( (

1.4 1.1 0.4 (e) 0.7 (m) 0.3 (b)

Rac-Me2Spm mean ( SD 66.1 71.6 81.8 83.5 89.6

( ( ( ( (

1.4 0.9 0.7 0.5 0.3

(i) (b) (b,h) (a)

a Tm measurements were performed in at least three separate experiments, with 6.25 µg/mL of CT DNA in 1 mM Na cacodylate buffer. (a) p e 0.001 versus Spm; (b) p e 0.01 versus Spm; (c) p e 0.05 versus Spm; (d) p e 0.001 versus (S,S); (e) p e 0.01 versus (S,S); (f) p e 0.05 versus (S,S); (g) p e 0.01 versus (R,R); (h) p e 0.05 versus (R,R); (i) p e 0.01 versus (R,S); (j) p e 0.05 versus (R,S); (k) p e 0.001 versus Rac; (l) p e 0.01 versus Rac; (m) p e 0.05 versus Rac.

Figure 4. (A) Induction of strand breakage by oxidative stress and (B) protection by natural polyamines in vitro. Supercoiled plasmid DNA (0.2 µg) was preincubated with polyamines for 10 min and then treated with 30 µM H2O2 and 10 µM CuSO4 at +37 °C for 60 min. The relative amounts of linear (ds break), open circular (ss break), and supercoiled (intact) forms were analyzed by agarose gel electrophoresis. Data are expressed as means ( SD (n ) 8).

produced the slow-moving circular form. Nicking both strands of the duplex DNA produced the linear form that migrates at intermediate speed during electrophoresis. Natural polyamines were first tested at physiological concentration range (0.1 to 10 mM). Results presented in Figure 4B show the effect of different concentrations of Spd and Spm on the retention of intact supercoiled DNA after treatment with reagents causing oxidative damage. Physiologically compatible concentrations of 1 mM Spm retained 30% of the DNA in the intact supercoiled state. Concentrations of 1 mM Spm/Me2Spm or 10 mM Spd/MeSpd were used in further investigation of the ability of these compounds to protect DNA from oxidative damage. Figure 5A shows the effect of the compounds on the protection of DNA from oxidative damage in comparison with Spd or Spm. (R)-MeSpd and (R,R)-Me2Spm provided better defense against oxidative stress than Spd or Spm, respectively. However, (S)-MeSpd, (S,S)-Me2Spm, and (R,S)-Me2Spm offered a similar degree of protection as Spd or Spm, respectively. In addition, natural polyamines and the analogues were excellent defenders of plasmid DNA integrity against DNase I-mediated cleavage (Figure 5B). Whereas control plasmid was totally digested in 5 min, all tested compounds markedly inhibited DNA digestion. However, no significant differences were detected between the stereoisomers. Polyamines/analogues did not demonstrate a protective effect against UV-C irradiationinduced DNA damage in vitro, as assessed by plasmid strand

Figure 5. Ability of natural polyamines and MeSpd and Me2Spm stereoisomers to protect DNA in vitro from strand breakage induced by (A) reactive oxygen species and (B) DNase I digestion. Supercoiled plasmid DNA (0.2 µg) was preincubated with polyamines and their methylated analogs for 10 min and then treated with 30 µM H2O2 and 10 µM CuSO4 at +37 °C for 60 min or DNase I (0.1 U) at +37 °C for 5 min. The relative amounts of linear (ds break), open circular (ss break), and supercoiled (intact) forms were analyzed by agarose gel electrophoresis. The relative amount of intact plasmid is shown. Data are expressed as means ( SD (n ) 8). *** represents p < 0.001 as compared with control (Co) sample.

break assay or alkaline unwinding assay of DNA (Fast Micromethod) (data not shown). Protection of DU145 Cells against DNA Damage. In the next series of experiments, we tested the ability of the compounds to provide protection against DNA damage in DU145 cells. Cells were pretreated for 72 h with 100 µM polyamine/analogue, 5 mM DFMO (to deplete natural polyamines and enhance extracellular polyamine uptake), and 1 mM aminoguanidine (to prevent the degradation of natural polyamines by serum amine oxidases) and then exposed to physical and chemical DNA-damaging agents. The intracellular polyamine

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Table 3. Polyamine Concentrations in DU145 Cells Pretreated with 5 mM DFMO and 100 µM Polyamines/Analogues for 72 ha Put

Spd

Spm

treatment control DFMO DFMO + DFMO + DFMO + DFMO + DFMO + DFMO + DFMO + b

Spd (R)-MeSpd (S)-MeSpd Spm (R,R)-Me2Spm (S,S)-Me2Spm (R,S)-Me2Spm

MeSpd

Me2Spm

total analogue

(pmol/µg DNA) 23 ( 7 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

248 ( 18 N.D. 557 ( 35 N.D N.D. 138 ( 4 N.D. N.D. N.D.

293 ( 19 176 ( 17 152 ( 8 50 ( 2 58 ( 4 330 ( 14 26 ( 1 27 ( 1 25 ( 5

633 ( 31b 808 ( 19b N.D. 27 ( 6 N.D.

161 ( 4 99 ( 4

794 907

522 ( 19 490 ( 11 551 ( 132

522 517 551

a All plates contained 1 mM aminoguanidine. Values are means ( SD, n ) 3. N.D., not detectable. Experiment was repeated three times with similar results. MeSpd is metabolized to MeSpm.

Figure 6. Protection of DU145 cells from etoposide-induced apoptosis by natural polyamines and the stereoisomers of MeSpd and Me2Spm. Cells were pretreated for 72 h with 100 µM polyamines and their methylated analogs in the presence of DFMO (5 mM) to enhance polyamine uptake and to replace natural polyamines with analogs. Aminoguanidine (1 mM) was included in all plates to prevent the oxidation of polyamines by serum amine oxidases. DNA damage was induced by incubating cells with 10 µM etoposide for 24 h. The cells were analyzed for DNA damage by (A) TUNEL assay and (B) aldehyde-reactive probe (ARP)-labeling. Data are expressed as means ( SD (n ) 3). * represents p < 0.05; *** represents p < 0.001 as compared with DFMO + etoposide-treated sample.

levels after 72 h of preincubation are presented in Table 3. The analogues efficiently replaced natural polyamines, and their accumulated levels were in the same concentration range as those of the natural counterparts in control cells. We first tested etoposide, a topoisomerase II inhibitor, which causes DNA double-strand breaks and apoptosis. TUNEL analysis (indicating the amount of fragmented DNA) showed that 24 h of treatment with 10 µM etoposide produced a marked increase in the proportion of TUNEL-positive cells (Figure 6A). In contrast, pretreatment with polyamines or analogues significantly reduced the amount of apoptotic cells compared with the DFMO-treated control group. Of the natural polyamines, Spm was more effective than Spd in protecting cells against apoptosis. Among the analogues, (R)-MeSpd and (R,R)-Me2Spm conferred the best protection against apoptosis induced by etoposide. DNA damage determined by the quantification of aldehyde-reactive sites in DNA produced similar results (Figure 6B), although the extent of overall protection from the drug, as assessed by the latter method, was less than the protection against apoptosis, as measured by the TUNEL assay. We then exposed DU145 cells to 500 µM H2O2 for 2 h and measured the DNA damage as the amount of aldehyde-reactive sites in DNA. As indicated in Figure 7, all tested compounds protected cells from DNA damage, and Spm was superior to Spd in its defensive ability. Among the methylated analogues, (R)-MeSpd and (S,S)-Me2Spm were significantly more efficacious DNA-protective agents than the other stereoisomers. We also measured direct DNA damage caused by UV-C irradiation. Here the presence of alkali-labile sites was determined immediately after irradiation without time for DNA repair because

Figure 7. Effect of the natural polyamines and the stereoisomers of MeSpd and Me2Spm on the extent of DNA damage induced by H2O2 in DU145 cells. The cells were pretreated for 72 h with 5 mM DFMO and 100 µM polyamine analogues, after which they were treated with 500 µM H2O2 for 2 h. All plates also contained 1 mM aminoguanidine. The extent of DNA damage was measured using aldehyde-reactive probe (ARP)-labeling. Data are expressed as means ( SD (n ) 3). ** represents p < 0.01; *** represents p < 0.001 as compared with DFMO-treated sample.

we wanted to study the effects of polyamines on direct DNA damage rather than on DNA repair. We found that in DU145 cells, both natural polyamines and analogues, afforded little protection against UV-C irradiation (1000 J/m2), as assessed by alkaline unwinding of DNA (Fast Micromethod) (Figure 8).

Discussion Most studies on polyamine analogues are focused on Nalkylated derivatives in which the amino groups undergo

Chiral R-Methylated Polyamine Analogues

Figure 8. Effect of natural polyamines and the stereoisomers of MeSpd and Me2Spm on the extent of DNA damage induced by UV-C irradiation in DU145 cells. The cells were pretreated for 72 h with 5 mM DFMO and 100 µM polyamine analogues, after which they were irradiated for a time sufficient to supply 1000 J/m2 and collected immediately. All plates contained 1 mM aminoguanidine. The extent of DNA damage was assayed by monitoring alkaline unwinding with fluorometry (Fast Micromethod). Data are expressed as means ( SD (n ) 3). * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001 as compared with DFMO-treated sample.

modification. Many of those analogues have been shown to be cytotoxic against tumor cell lines, and efforts have been directed toward improving their cytotoxicity toward cancer cells while reducing their adverse effects.32 R,R′-dimethyl modification leaves the primary amino groups intact and does not affect the charge distribution of a polyamine moiety.7,8 Me2Spm is resistant to acetylation by spermidine/spermine N1-acetyltransferase (SSAT) and subsequent degradation by acetylpolyamine oxidase. Spermine oxidase is able to metabolize (S,S) but not (R,R) diastereomer in vitro and in vivo. Although metabolically more stable than their natural counterparts, MeSpd and Me2Spm have the ability to substitute for some cellular functions of natural polyamines. However, the interaction of these molecules with biological macromolecules has not been investigated. The present study sheds light on the stereospecificity in the interaction of R-methylated polyamines with DNA. Previous studies with the stereoisomers of R-methylated polyamines indicate that the 3D structure has an impact on the physicochemicalandbiologicalactivityofpolyamineanalogues.11,33 Vijayanathan et al. found that structural characteristics of Spm homologues, such as the number of methylene spacing between the secondary amine groups, play an important role in their DNA condensing ability and the sizes of resulting nanoparticles.34 Saminathan et al. further studied the structural effects of polyamine analogues on DNA aggregation and liquid crystal formation.35 Valasinas et al. found that a cis-decamine was less efficient at inducing DNA aggregation and less cytotoxic than other decamines.36 This finding was attributed to its less stretched conformation and resulting differences in its binding to DNA. The study of salicyl diamines of different conformations indicated that the (R,R)-stereoisomer was the most effective cytotoxic agent in MCF-7 cells.37 The isomer also had the greatest effect on cyclin D1 mRNA down-regulation (down to 40% of control) compared with other isomers that were able to lower mRNA expression to only 68-71% of control. In a study of putrescine analogues, it was found that the distance between amino groups and spatial orientation influenced the ability of an analogue to stimulate antizyme synthesis by inducing ribosomal +1 frameshifting.38 Recently, it was found that among MeSpd and Me2Spm stereoisomers, (S) and (S,S) were more efficient than (R) and (R,R) at inducing antizyme frameshifting.11

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Our present results show that R-methylated polyamine analogues have unique characteristics compared with Spm in interacting with DNA. Whereas the number of positive charges is important in their ability to condense DNA, the spatial arrangement and the positioning of methyl groups in Me2Spm stereoisomers influence DNA compaction efficacy and the size of resulting DNA nanoparticles. We found that (R,R)- and (R,S)Me2Spm, but not (S,S)-Me2Spm, were more efficient DNA condensing agents than Spm. Hydrodynamic radii ranged from 39.6 ( 1.1 to 48.4 ( 1.2 nm, and (S,S)- and (R,R)-Me2Spm produced significantly larger condensates than Spm. Melting temperature measurements demonstrated that Spm conferred greater duplex stabilization than Me2Spm stereoisomers at a concentration range of 10 to 100 µM. Polyamines and their R-methylated analogues protected DNA against damage, both in vitro and in DU145 cells, and the degree of protection depended on the optical properties of the stereoisomer. (R)MeSpd and (R,R)-Me2Spm were particularly effective in protecting DNA from oxidative damage in vitro and in protecting DU145 cells from apoptosis induced by etoposide. However, no difference in protection was observed between stereoisomers in protection against DNase I digestion in vitro, and the most potent stereoisomers protecting against oxidative stress in cells were (R)-MeSpd and (S,S)-Me2Spm. Distinct differences between methylated analogues of spermidine and spermine were evident during nanoparticle formation. Spd stereoisomers condensed DNA at much higher concentrations and produced particles of roughly twice the size of their Spm counterparts. This difference is generally attributed to the larger positive charge and greater binding affinity and condensing efficiency of spermine compared with spermidine. Sizes of nanoparticles produced in this study generally correspond to the sizes of polyamine-DNA condensates previously reported by us and others. Particle size measurements can be influenced by many factors, including the size of the DNA undergoing condensation, starting reactant concentrations, ionic conditions, mixing protocol, and the method of measurement. In a previous study, Thomas and Bloomfield observed T4 DNASpd condensates with hydrodynamic radii of 48 to 49 nm.39 Using both dynamic light scattering and atomic force microscopy, Vijayanathan et al. reported particle formation between PGL-3 plasmid (>5 kb in size) and polyamines/polyamine analogues with radii of 51-96 nm.31 Sizes of nanoparticles decreased with increasing positive charge of the compacting agent. Another study with λ-phage DNA reported 41 nm-sized condensates similar to those observed in our study. The size of λ-DNA nanoparticles depended on polyamine structural specificity, increasing with the length of the methylene spacer between the secondary amino groups. Our melting temperature investigation demonstrates that Spm and Me2Spm stereoisomers stabilize CT DNA in a concentration-dependent manner. These findings are in agreement with other melting studies by Thomas and Bloomfield40 and Saminathan et al.17 The lack of correlation between DNA-condensing and duplex-stabilizing abilities in our study may be attributed to the fact that DNA condensation and stabilization occur through different mechanisms. Terui et al. found that although longer polyamines stabilized DNA more effectively than shorter counterparts, the mechanism of stabilization was similar and independent of polyamine structural features.41 Studies with protamines and polyamines demonstrated their preferential binding to the minor groove of DNA.42 We hypothesize that differences in spatial arrangement of methylated stereoisomers

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can influence their steric interactions with DNA, thereby affecting condensing abilities and particle size. Natural polyamines have been shown to protect DNA against various damaging agents, including oxidative stress, endonuclease-digestion, alkylating agents, and ionizing radiation.12-19 Proposed mechanisms of polyamine protection include physical blocking of DNA against damaging agents, direct scavenging of damaging radicals, binding to and inhibiting proteins, stimulation of DNA compaction that limits target site accessibility, or a combination of these events. The mechanism of polyamine protection against DNase I digestion has not been clearly elucidated. It is possible that polyamines bind to and inhibit DNase I, but they may also act by stimulating DNA compaction and restricting enzyme access to DNA. Polyamineprotein interactions have been reported.43,44 Brune et al. showed that spermine protected DNA against digestion by purified liver endonuclease, and this effect was related to spermine’s ability to modify chromatin structure.45 Moreover, because polyamines have been found to inhibit DNA digestion by many different restriction enzymes with the same efficiency, it is more likely that the protection is mediated via interaction with DNA rather than interaction with the enzyme.46 In our study, polyamine depletion in DU145 cells with DFMO resulted in more pronounced DNA damage compared with control cells, and supplementation of natural polyamines or the analogues counteracted DFMO’s effect, indicating that polyamines have a protective role with respect to DNA. Although (R)MeSpd and (R,R)-Me2Spm were more effective in providing protection against H2O2-induced DNA damage than the other isomers in vitro, (S,S)-Me2Spm was more effective than the other Me2Spm stereoisomers in DU145 cells. This may be attributed to the fact that cells treated with (S,S)-Me2Spm may have already induced their protective mechanisms because (S,S)-Me2Spm is metabolized by spermine oxidase to (S)-MeSpd, generating H2O2 and reactive aldehyde as byproduct. Because the interpretation of cell culture results is obviously very complicated by direct interaction of the analogue as well as byproducts of analogue metabolism, structural specificity insight can be drawn from testing the ability of polyamine analogues to protect DNA against damaging agents in vitro. Our present cell culture and in vitro data indicate that polyamines/analogues provide little or no protection against UV-C irradiation-induced direct DNA damage, whereas polyamines are known to stabilize DNA against ionizing radiation (γ, X-ray).18 The differences might be explained by the different mechanism of action in damaging DNA of UV-C and ionizing radiation. UV-C radiation generally produces dimeric lesions (cyclobutane dimers and (6-4)-photoproducts), which are alkali-labile sites and potential sites for single- and double-strand breaks during DNA repair. In contrast, most of the DNA damage caused by ionizing radiation is mediated by the reactive oxygen radicals, not by dimeric lesions. Proposed protective mechanisms of polyamines against ionizing radiation include direct scavenging of OH radicals and limiting the accessibility of OH radicals to target sites by provoking DNA compaction. Therefore, our present finding that polyamines and analogues protected DNA against hydroxyl radical-induced damage in vitro (Fenton-type reaction) and in DU145 cells is in agreement with results that polyamines protect against ionizing radiation but not against UV-C irradiation.

Conclusions In summary, our study demonstrates that in addition to ion valence, spatial arrangement of substituents plays an important

Nayvelt et al.

role in the ability of polyamine analogues to condense and stabilize DNA. Nanoparticle sizes are also dependent on the structural conformation of the condensing agents. In addition, methylated polyamine analogues provided protection against DNA damage caused by hydroxyl radicals, topoisomeraseinhibitor etoposide, and endonuclease. The degree of protection conferred by various stereoisomers was determined by their structural specificity. These findings might help us to design various applications of chiral polyamine analogues as therapeutic agents in cancer and other diseases as well as in DNA packaging and gene delivery. Acknowledgment. We thank Mrs. Tuula Reponen and Mrs. Anne Karppinen for skillful technical assistance. This work was supported by grants from National Institutes of Health/National Cancer Institute (CA080163-09 and CA42439-20 to T.J.T. and T.T.) and the Academy of Finland (grant numbers 124185 and 128702 to M.T.H., A.R.K. and J.V.), and the program of Molecular and Cell Biology of the Presidium of the Russian Academy of Sciences (A.R.K.).

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