Tài liệu Báo cáo khoa học: Specific targeting of a DNA-alkylating reagent to mitochondria Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide doc

Specific targeting of a DNA-alkylating reagent to mitochondria Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro- 5 H -pyrrolo[2,1- c ][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide Andrew M. James 1 , Frances H. Blaikie 2 , Robin A. J. Smith 2 , Robert N. Lightowlers 3 , Paul M. Smith 3 and Michael P. Murphy 1 1 MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Cambridge, UK; 2 Department of Chemistry, University of Otago, Dunedin, New Zealand; 3 Department of Neurology, Medical School, University of Newcastle upon Tyne, UK The selective manipulation of the expression and replica- tion of mitochondrial DNA (mtDNA) within mammalian cells has proven difficult. In progressing towards this goal we synthesized a novel mitochondria-targeted DNA- alkylating reagent. The active alkylating moiety [(11aS)-8- hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c] [1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates guanine bases in DNA (with a preference for AGA tri- plets), preventing its expression and replication. To target this compound to mitochondria it was covalently coupled to the lipophilic triphenylphosphonium (TPP) cation to form a derivative referred to as mitoDC-81. Incorporation of this lipophilic cation led to the rapid uptake of mitoDC-81 by mitochondria, driven by the large mem- brane potential across the inner membrane. This com- pound efficiently alkylated isolated supercoiled, relaxed- circular or linear plasmid DNA and isolated mtDNA. However mitoDC-81 did not alkylate mtDNA within isolated mitochondria or cells, even though it accessed the mitochondrial matrix at concentrations up to 100-fold higher than those required to alkylate isolated DNA. This surprising finding suggests that mtDNA within intact mitochondria may not be accessible to this class of alky- lating reagent. This inability to alkylate mtDNA in situ has significant implications for the design of therapies for mtDNA diseases and for studies on the packaging, expression and turnover of mtDNA in general. Keywords: membrane potential; mitochondria; mitochond- rial DNA; targeting. Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptides and the RNA machinery for their transcrip- tion and translation [1–3]. As these polypeptides are all components of oxidative phosphorylation complexes, mtDNA mutations can severely disrupt mitochondrial function, leading to a number of human diseases for which there are no effective therapies [2–8]. Possibilities for treatment, such as the replacement of the defective gene by gene therapy, are being explored; however, gene therapy for mtDNA diseases is even more challenging than for nuclear gene defects because of the problem of delivering DNA to mitochondria, the difficulty of generating stable insertion or expression of exogenous DNA within mam- malian mitochondria, and the large number of mitochon- dria and mtDNA molecules per cell [9,10]. Even if this approach is effective, it will not be practical for the majority of mtDNA diseases that are caused by mtDNA deletions, or mutations in RNA genes [9,11] until we extend our knowledge of potential RNA import processes in mamma- lian mitochondria [12]. Because of these challenges an alternative ÔantigenomicÕ strategy has been developed as a potential therapy for mtDNA diseases [13–16]. This approach does not introduce a functioning copy of the defective gene; instead it utilizes the following mtDNA properties: mtDNA is present in patients at high copy number as a mixture of both normal and mutated molecules; mtDNA diseases are only pheno- typically expressed above a threshold proportion of mutated mtDNA; and mtDNA is continually degraded and resyn- thesized. Consequently, if the proportion of mutated mtDNA molecules in a patient can be decreased below this threshold the disease phenotype may be suppressed. This could be done by selectively enhancing the degradation, or inhibiting the replication, of mutated mtDNA molecules without affecting wild-type mtDNA [17]. The potential of this approach has been demonstrated for the mtDNA disease neuropathy, ataxia and retinitis pigmentosa Correspondence to M. P. Murphy, MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Hills Road, Cambridge CB2 2XY, UK. Fax: + 44 1223 252905, Tel.: + 44 1223 252900, E-mail: mpm@mrc-dunn.cam.ac.uk, http://www.mrc-dunn.cam.ac.uk Abbreviations: DC-81, (11aS)-8-hydroxy-7-methoxy-1,2,3,11a- tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one; DMEM, Dulbecco’s modified Eagle medium; FCCP, carbonylcyanide-p- trifluoromethoxy-phenylhydrazone; IBTP, 4-iodobutyltriphenyl- phosphonium iodide; mitoDC-81, [4-((11aS)-7-methoxy-1,2,3,11a- tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]tri- phenylphosphonium iodide; mtDNA, mitochondrial DNA; NARP, neuropathy, ataxia and retinitis pigmentosa; PNA, peptide nucleic acid; TPMP, methyl triphenylphosphonium; TPP, triphenyl- phosphonium. (Received 27 February 2003, revised 15 April 2003, accepted 12 May 2003) Eur. J. Biochem. 270, 2827–2836 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03660.x (NARP), by targeting a restriction enzyme to mitochondria that cleaves at a unique site associated with the NARP point mutation [18]. While these experiments show that this approach works in vitro, the targeting of specific restriction enzymes is difficult to achieve in patients and most mtDNA diseases are not associated with a unique restriction site. Therefore other reagents that selectively inhibit the replica- tion of mutated mtDNA molecules are being developed [16]. Among the most promising are peptide nucleic acids (PNA), which are stable nucleic acid analogues that bind tightly to complementary DNA sequences [19], selectively inhibiting the replication of mutated mtDNA sequences without disrupting wild-type sequences that differ by only a single base pair [20]. Furthermore PNAs can be delivered to the mitochondrial matrix, by attachment of a mitochondrial protein import sequence [21], or conjugation to a lipophilic cation [22]. However, even though both approaches appeared to lead to the accumulation of a PNA within the mitochondria of cultured cells, neither affected the proportion of mutated mtDNA in heteroplasmic myoclonic epilepsy with ragged-red fibres (MERRF) cells [23]. Accepting that the PNA has been successfully transpor- ted to the site of mtDNA replication, these negative results suggestthatdeliveryofaPNAthatbindstoaparticular sequence to mitochondria is not sufficient, perhaps because the PNA does not form a complex with the target mtDNA sequence that is durable enough to inhibit mtDNA repli- cation or expression. One approach to increase the duration of PNA binding to DNA is to conjugate it to a DNA- alkylating reagent so that the PNA becomes covalently bound to its target sequence. To do this a DNA alkylating reagent that reacts relatively slowly with DNA is required to ensure that the alkylation occurs following binding to the specific sequence by the PNA. As a first step towards this goal we set out to develop a mitochondria-targeted DNA- alkylating reagent to determine whether it was possible to alkylate mtDNA within intact mitochondria and cells. In addition to facilitating the development of antigenomic therapies, such a reagent might also be useful in investi- gating the rate of turnover of mtDNA and in the derivation of mtDNA-free (q°) clones of the large number of cell lines where the classical treatment with ethidium bromide is ineffective [24]. Therefore, our immediate objective was to synthesize a mitochondria-targeted DNA-alkylating reagent that would covalently bind to mtDNA, leading to inhibition of replication and expression (Fig. 1). For the DNA alkylating reagent we chose the antibiotic (11aS)-8- hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1- c][1,4]benzodiazepin-5-one; (DC-81) [1,25]. This DNA- alkylating reagent binds to the minor groove of double- stranded DNA and then alkylates guanines at position N- 2 [26], favouring guanines flanked by purines [27–29]. The covalent attachment of DC-81-like compounds to DNA causes premature termination of transcription in vitro [29]. To target DC-81 to mitochondria we attached it to a lipophilic triphenylphosphonium (TPP) cation. These cations are accumulated into the mitochondrial matrix several-hundred fold, driven by the large mitochondrial membrane potential, and addition of a TPP-moiety has been used to drive the selective uptake of a wide variety of molecules into mitochondria [23,30–33]. Therefore attach- ing the DNA alkylating reagent DC-81 to TPP should give rise to a high local concentration in the vicinity of mtDNA with the resultant binding and selective alkyla- tion leading to a depletion of mtDNA in intact cells (Fig. 1). Here we report the synthesis and characterization of a novel mitochondria-targeted alkylating reagent and show that it alkylates DNA in vitro and is taken up by mitochondria. However, in spite of its substantial import, it did not alkylate mtDNA in isolated mitochondria or cells. This unexpected finding has significant implications for the development of antigenomic therapies for mtDNA diseases. Fig. 1. Selective uptake of mitoDC-81 by mitochondria and subsequent alkylation of mtDNA. The uptake of mitoDC-81 into cells driven by theplasmamembranepotential(Dw p ) followed by the further accu- mulation of mitoDC-81 into mitochondria driven by the mitoch- ondrial membrane potential (Dw m ) is illustrated. The Nernst equation indicates a 10-fold increase in accumulation for every 61.5 mV of membrane potential. This leads to a millimolar concentration of mitoDC-81 within mitochondria on incubation of cells with high nanomolar to micromolar concentrations of mitoDC-81. This high local concentration of mitoDC-81 within mitochondria could then lead to the alkylation and inactivation of mtDNA, as indicated in the figure. 2828 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Materials and methods Synthesis of [4-((11a S )-7-methoxy-1,2,3,11a-tetrahydro- 5 H -pyrrolo[2,1- c ][1,4]benzodiazepin-5-on-8-oxy)- butyl]triphenylphosphonium iodide (mitoDC-81) 2 Sodium hydride (4.8 mg, 0.12 mmol, 60% suspension in oil) was added to a dry Schlenk tube containing a magnetic stirrer and held under an argon atmosphere. The sodium hydride was washed three times with pentane, then dried in vacuo (0.1 mm Hg). Dimethylformamide (0.1 mL) was then added and the suspension was stirred for 10 min at room temperature. A solution of DC-81 1 [34] (24.6 mg, 0.1 mmol) in dimethylformamide (0.2 mL) and tetra- hydrofuran (0.2 mL) was added dropwise to the reaction vessel and stirred for 3.75 h, after which time the reaction mixture was a yellow/brown suspension (Scheme 1). A solution of (4-iodobutyl)triphenylphosphonium iodide (44.9 mg, 0.1 mmol) [35] in dimethylformamide (0.2 mL) was then added dropwise to the ice-cooled reaction mixture, which was subsequently allowed to warm to room tem- perature overnight and stirred for a further 2 days at room temperature to give a light brown suspension. Distilled water (2 mL) was then carefully added and the mixture was partitioned with dichloromethane (5 · 2mL). The com- bined organic layers were dried (MgSO 4 ) and evaporated to dryness in vacuo. The residual oil was dissolved in minimal dichloromethane, precipitated with excess ether and the solvent layer decanted. The precipitate was then redissolved in minimal dichloromethane, precipitated with excess ether and the solvent layer decanted. This precipitation process was repeated nine times. The residue was dried under reduced pressure for 3 h yielding 2 as a mustard coloured solid (12 mg, 0.014 mmol, 14%). 1 HNMRd 7.6–7.9 (m (16H, P + –ArH and H-11),7.46(1H,s,H-6), 6.49 (1H, s, H-9),4.1–4.2(2H,m,Ar-O-CH 2 ), 3.71 (3H, s, O-CH 3 ), 3.4– 4.0(5H,m,P + –CH 2 , H-11a and H-3), 1.75–2.4 (8H, m, O-CH 2 -CH 2 ,P + –CH 2 -CH 2 , H-1andH-2) p.p.m., 31 PNMR d 25.65 p.p.m. ESMS found (M + ) 563.2469 calculated for C 35 H 36 O 3 N 2 P(M + ) 563.2458. 1 H(300MHz)and 31 P(121MHz)NMRspectrawere recorded on a Varian Unity 300 spectrometer in chloro- form-d 1 standardized against the solvent peak or external 85% phosphoric acid, respectively. The high resolution mass spectrum (ESMS) was obtained by G. Willett, School of Chemistry, University of New South Wales, Sydney, Australia. Isolated DNA incubations Circular plasmid DNA (5 lg; derived from pYES-PA2, 6.8 kb), HindIII digested k DNA (5 lg),oraladderof linearmarkerDNA(5lg of a 1-kb ladder, Invitrogen) were incubated for 6 h at 37 °Cin50lL20m M Tris/HCl pH 8.0, 1 m M EDTA (TE) supplemented with 500 l M mitoDC-81, 500 l M methyl triphenylphosphonium (TPMP) or ethanol carrier. DNA was then precipitated with 1 vol. 0.6 M NaOAc, 20 m M EDTA followed by 2 vols cold ethanol. After centrifugation (13000 g for 30 min at 4 °C) the pellet was resuspended in 50 lL10m M Tris/HCl pH 8.0, 0.1 m M EDTA and 1 lgwasrunat60 Vona0.6% agarose/ethidium bromide gel. The DNA was electrotrans- ferred to a positive nylon membrane (Hybond N+, Amersham Pharmacia Biotech), treated with 0.4 M NaOH, and fixed by UV irradiation. The membrane was then blocked overnight with 1% (w/v) milk powder, 0.05% (v/v) Tween-20 in TBS (TBST) before incubation for 1 h with a 1 : 3000 dilution of rabbit anti-TPP serum in 0.1% (w/v) milk powder/TBST. The membrane was washed three times with TBST then incubated for 30 min with a 1 : 5000 dilution of anti-rabbit I g G conjugated to horseradish peroxidase (Sigma). The membrane was washed five times with TBST and antibody binding to TPP moieties was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). MtDNA was prepared from isolated rat liver mitochon- dria (40 mg protein) using a plasmid spin miniprep kit (Qiagen) at a ratio of 5 mg mitochondrial protein per column. The initial alkaline lysis was shortened to  30 s due to the presence of alkali-labile ribonucleotides in mtDNA [36]. The eluted mtDNA was pooled, precipitated with NaOAc/ethanol, and the pellet resuspended in 15 lL TE. When separated by agarose gel electrophoresis, isolated mtDNA appeared as three species with apparent sizes of 9, 16 and  40 kb compared to linear markers. These corres- pond to supercoiled, linear and relaxed-circular mtDNA forms, respectively. Upon digestion with the single-cutter ClaI, these species resolved into the linear form, migrating at 16 kb. Southern blotting showed that supercoiled and relaxed-circular DNA transferred very poorly compared with linear DNA (data not shown), hence mtDNA was routinely cut with ClaI prior to electrophoresis. MtDNA was incubated for 6 h at 37 °C, digested with ClaI and electrophoresed at 14 V on a 0.4% agarose/ethidium bromide gel. The DNA was then electrotransferred to a positive nylon membrane and TPP moieties were detected using anti-TPP serum as above. Mitochondrial incubations Rat liver mitochondria were prepared by homogenization followed by differential centrifugation [37]. Protein concen- tration was determined using the biuret assay with BSA as standard [38]. The uptake of mitoDC-81 by mitochondria was measured at 30 °C using a mitoDC-81-sensitive ion- Scheme 1. [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1- c][1,4]benzodiazepin-5-on-8-oxy)butyl]triphenylphosphonium iodide (2; mitoDC-81). Ó FEBS 2003 Mitochondrial DNA alkylation (Eur. J. Biochem. 270) 2829 selective electrode suspended in a stirred chamber open to the atmosphere [39,40]. To calibrate the electrode, five stepwise additions of 1 l M mitoDC-81 were made to 3 mL 250 m M sucrose, 5 m M Tris/HCl pH 7.4, 1 m M EGTA, containing isolated rat liver mitochondria (1 mg pro- teinÆmL )1 ). The mitochondria were energized with 10 m M succinate and the membrane potential was dissipated with 1 l M carbonylcyanide-p-trifluoromethoxy-phenyl- hydrazone (FCCP). To measure alkylation of mtDNA, mitochondria (1 mgÆmL )1 protein) were suspended in two 100 mL conical flasks each containing 10 mL 250 m M sucrose, 5 m M Tris/HCl pH 7.4, 1 m M EGTA, 1 m M EDTA, 10 m M succinate supplemented with 5 l M mitoDC-81. The flasks were incubated for 1 h at 30 °Cin a shaking water bath, after which time the mitochondria were pelleted by centrifugation (10 000 g for 10 min) and washed with TE. The mtDNA was isolated as above, resuspended in 15 lL TE, cut with ClaI and separated at 14 V through a 0.4% agarose/ethidium bromide gel. The DNA was electrotransferred to a positive nylon membrane and TPP moieties were detected using anti-TPP serum as above. Respiration rate measurements were carried out in the stirred and thermostatted chamber of a Clark-type oxygen electrode (Rank Brothers, Bottisham, UK). Cell incubations Human 143B osteosarcoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum, glucose (4.5 gÆL )1 ), streptomycin sulfate (100 mgÆL )1 ), penicillin G (100 000 UÆL )1 ), uridine (50 mgÆL )1 ) and pyruvate (100 mgÆL )1 ) unless otherwise stated. Cells were grown in plastic flasks or on glass coverslips at 37 °C in a humidified atmosphere of 5% CO 2 /95% air until confluent. For confocal microscopy, cells were grown overnight on glass coverslips then treated for 6 h with 500 n M (4-iodo- butyl)triphenylphosphonium (IBTP), 500 n M TPMP, or 500 n M mitoDC-81. Coverslips were washed three times in NaCl/P i and then fixed with 4% (w/v) paraformaldehyde for 30 min at room temperature. They were washed four more times, and permeabilized and blocked with 10% (v/v) foetal bovine serum, 0.1% (v/v) Triton X-100, NaCl/P i for 10 min at 4 °C before being shaken overnight at 4 °Cwitha 1 : 500 dilution of rabbit anti-TPP serum and a 1 : 100 dilution of an anti-(cytochrome oxidase subunit 1) mAb (Molecular Probes) in 10% (v/v) foetal bovine serum, 0.05% (v/v) Tween 20, NaCl/P i . They were washed three more times and incubated for 30 min at 4 °C with a 1 : 200 dilution of anti-mouse IgG AlexaFluor 546 (Molecular Probes) and a 1 : 500 dilution of anti-rabbit IgG-Oregon Green in 10% (v/v) foetal bovine serum, 0.05% (v/v) Tween 20. After washing a final five times with NaCl/P i ,the coverslips were mounted on slides and visualized using a Nikon Eclipse E800 confocal microscope and a Nikon · 60 (numerical aperture 1.4) oil immersion Plan Apochromat objective. Oregon green was illuminated with the 488 nm line of an argon laser and fluorescence detected using a HQ515/30 filter. The argon laser was then turned off and Alexafluor 546 was illuminated with a 543-nm neon laser and fluorescence detected using a 570LP filter. The green and red channels were acquired using Biorad Lasersharp 2000 and subsequently merged to indicate colocalization. The laser intensity, iris and gain settings were identical for all images shown. To assess whether mitoDC-81 could completely deplete mtDNA and thereby create q° cells, 143B cells were grown in DMEM/foetal bovine serum supplemented with 50 mgÆL )1 uridine and passaged twice per week in the presence of either 500 n M mitoDC-81 or 500 n M TPMP for a period of 17 days [24]. Cells were cloned by dilution and after a further 28 days their rate of oxygen consumption respiring on succinate was measured after digitonin-perme- abilization [41]. Results Synthesis of mitoDC-81 The compound of interest, mitoDC-81 2, was prepared by reaction of the phenoxide anion derived from 1 with IBTP (Scheme 1). This approach was found to be more effective than creating a bromoalkyl side chain on 1 followed by reaction with triphenylphosphine. The product 2 (Scheme 1) was isolated and characterized as described for previously synthesized complex triphenylphosphonium salts [30,31]. Accumulation of mitoDC-81 by isolated mitochondria An ion-selective electrode sensitive to mitoDC-81 was constructed in order to determine whether mitoDC-81 was accumulated by energized mitochondria (Fig. 2). Mito- chondria were added to the incubation chamber and the response of the electrode was then calibrated by sequential additions of 1 l M mitoDC-81, which showed the expected logarithmic response of the electrode to cation concentra- Fig. 2. Uptake of mitoDC-81 by energized mitochondria. An ion- selective electrode was used to measure the uptake of mitoDC-81 into rat liver mitochondria. To calibrate the electrode five stepwise addi- tions of 1 l M mitoDC-81 were made to a suspension of mitochondria in a stirred incubation chamber. The addition of the respiratory sub- strate succinate generated a membrane potential, which caused mitoDC-81 to be sequestered inside the mitochondria, and this accu- mulation was reversed by addition of the uncoupler FCCP. 2830 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003 tion (Fig. 2). When mitochondria were energized by addi- tion of the respiratory substrate succinate, the concentration of mitoDC-81 in the incubation chamber decreased to about 2.5 l M , due to sequestration of mitoDC-81 within the mitochondrial matrix. This corresponds to uptake of about 2.5 nmol mitoDC-81Æmg mitochondrial protein )1 .Asthe mitochondrial volume is about 0.5–0.9 lLÆmg protein )1 under these conditions [42–44] this corresponds to an intramitochondrial mitoDC-81 concentration of 2.5–5 m M , although the steady-state free matrix concentration is likely to be about 60–80% lower than this due to binding to the matrix-facing surface of the inner membrane [45]. When the membrane potential was dissipated by addition of the uncoupler FCCP, mitoDC-81 was rapidly released from the mitochondria (Fig. 2). Therefore mitoDC-81 is accumu- lated about a thousand-fold within energized mitochondria, driven by the membrane potential. Alkylation of isolated DNA by mitoDC-81 To determine if the conjugation of a lipophilic cation to DC-81 disrupted its ability to alkylate DNA, we determined whether mitoDC-81 could alkylate DNA in vitro.Todothis we incubated 500 l M mitoDC-81 with linearized DNA molecules of different sizes (i.e. a standard DNA ÔladderÕ used as a molecular weight marker). After incubation the DNA was separated by electrophoresis, electrotransferred to a nylon membrane and probed for mitoDC-81 covalently bound to the DNA by immunoblotting using anti-TPP serum [35] (Fig. 3A,B). This showed considerable alkylation of the DNA ladder by mitoDC-81 (Fig. 3B, lane 1). Analysis of the gel prior to electrotransfer showed that the extent of alkylation was proportional to the amount of DNA present and was independent of the size of the DNA fragment (Fig. 3A, lane 1). As mtDNA in vivo is negatively supercoiled it was also important to compare the ability of mitoDC-81 to alkylate linear, relaxed-circular, and super- coiled DNA. Therefore in parallel experiments we investi- gated the ability of 500 l M mitoDC-81 to alkylate a 6.8-kb plasmid that was present in all three conformations (Fig. 3A,B). Analysis of the agarose gel prior to electro- transfer showed three forms, supercoiled, linear and relaxed-circular plasmid DNA, in order of increasing apparent molecular weight (Fig. 3A). Comparison of the ethidium fluorescence (Fig. 3A) and the amount of DNA alkylation (Fig. 3B) showed that all three plasmid species were alkylated by mitoDC-81. Incubation with TPMP, which lacks the DNA-alkylating moiety but is recognized by the anti-TPP serum [35], showed that the DC-81 moiety was essential for DNA-labelling (Fig. 3B, lane 2). The concen- tration of mitoDC-81 used and the time taken for DNA alkylation are in agreement with the conditions used to react DC-81 related compounds and isolated DNA [46]. To confirm that mitoDC-81 could alkylate mtDNA, we isolated mtDNA from rat liver mitochondria and incubated it with 500 l M mitoDC-81 (isolated mtDNA; Fig. 3C,D). This showed extensive alkylation of the isolated mtDNA by mitoDC-81. The higher molecular weight band in the isolated mtDNA is relaxed-circular mtDNA. Its absence from the immunoblot was due to poor transfer from the gel to the membrane, as was confirmed by Southern blotting. The presence of relaxed-circular mtDNA after digestion with ClaI may indicate alkylation of the restriction site as untreated mtDNA, or mtDNA isolated from mitoDC-81- treated mitochondria, was always fully linearized by ClaI under identical conditions. MtDNA that had been ran- domly broken during isolation or incubation with mitoDC- 81 was also extensively alkylated and migrated as a smear when subsequently cut with ClaI( 4–8 kb; data not shown). We conclude that conjugation of the lipophilic cation to DC-81 does not disrupt its ability to alkylate DNA and high micromolar concentrations of mitoDC-81 are sufficient to alkylate linear, circular and supercoiled DNA. As these concentrations of mitoDC-81 are easily achieved within the mitochondrial matrix on incubation with mitoDC-81 (Fig. 2), mitoDC-81 should also alkylate mtDNA within mitochondria and cells. MitoDC-81 does not alkylate mtDNA in isolated mitochondria Having ascertained that mitoDC-81 was sequestered by isolated mitochondria and that it could covalently modify Fig. 3. MitoDC-81 alkylates linear, relaxed-circular and supercoiled DNA in vitro. A 1-kb ladder of linear DNA (1 lgÆmL )1 ) or plasmid DNA (1 lgÆmL )1 ; 6.8 kb) was incubated for 6 h at 37 °Cwith500 l M mitoDC-81, 500 l M TPMP or ethanol carrier (A and B). MtDNA was isolated from rat liver mitochondria (40 mg protein) and incubated with 500 l M mitoDC-81 for 6 h and then cut with ClaI (C and D). DNA was separated by electrophoresis on an agarose gel and the ethidium bromide fluorescence recorded to quantify DNA (A and C). The DNA was then electrotransferred to a nylon membrane and the presence of TPP moieties covalently attached to the DNA was visu- alized by immunoblotting using anti-TPP serum (B and D). Ó FEBS 2003 Mitochondrial DNA alkylation (Eur. J. Biochem. 270) 2831 linear, relaxed-circular and supercoiled DNA in vitro,we next tested whether it could alkylate mtDNA in situ within intact, isolated mitochondria. Incubations of 6 h with a concentration of 500 l M mitoDC-81 led to extensive alkylation of isolated DNA (Fig. 3), but such long incuba- tion times and high concentrations of mitoDC-81 cannot be used with isolated mitochondria due to uncoupling and loss of mitochondrial integrity. Isolated mitochondria can beincubatedwithamaximumof 5 l M mitoDC-81 without uncoupling, but as this concentration leads to an intramitochondrial concentration of 3–5 m M (Fig. 2), the concentration of mitoDC-81 should not be a limiting factor. Respiration rate measurements indicated that isolated mitochondria incubated at 30 °C for 1 h could still be stimulated with uncoupler, but longer incubations led to the loss of mitochondrial coupling (data not shown). Therefore it was important to determine if a 1-h incubation was sufficient to lead to detectable DNA labelling by mitoDC-81. To do this, isolated plasmid DNA was incubated with various concentrations of mitoDC-81 for 1 h (Fig. 4A,B). Extensive alkylation of the DNA was noted after 1 h incubation at concentrations as low as 50 l M mitoDC-81 (Fig. 4A,B). That the concentration of mtDNA within mitochondria is also unlikely to be limiting under these conditions is supported by the following rough calculations. There are about 18.7 · 10 9 mtDNA mole- culesÆmg protein )1 in rat liver mitochondria [47], giving about 3.05 · 10 14 bpÆmg protein )1 , or 335 ng DNAÆmg protein )1 . Under these conditions the mitochondrial matrix volume is about 0.5–0.9 lLÆmg protein )1 [42–44], so the effective mtDNA concentration within the matrix is 372–670 lg DNAÆmL )1 , significantly higher than the concentration of plasmid DNA (100 lgÆmL )1 )usedinFig.4AandB. Consequently, a 1-h incubation of isolated mitochondria with 5 l M mitoDC-81 should lead to detectable alkylation of mtDNA within mitochondria. However, when energized mitochondria were incubated with mitoDC-81 for 1 h and the mtDNA isolated, there was no DNA alkylation (Fig. 4C,D). In summary, loading mitochondria with sufficient mitoDC-81 to alkylate isolated mtDNA does not detectably label mtDNA within mitochondria. MitoDC-81 does not alkylate mtDNA within intact cells It was unexpected that mitoDC-81 did not alkylate mtDNA within isolated mitochondria, even though a concentration of mitoDC-81 50–100-fold higher than that required to alkylate isolated plasmid DNA had accumulated. One possibility is that a longer exposure to mitoDC-81 than was possible with isolated mitochondria could lead to mtDNA labelling. Substantially longer incubation times are possible with cultured cells, which can be incubated with mitoDC-81 indefinitely without disrupting their mitochondrial mem- brane potential. Therefore we next investigated whether mitoDC-81 alkylated mtDNA within intact cells. Cells couldbeincubatedwithupto500n M mitoDC-81 indefin- itely, although higher concentrations (1–5 l M )weretoxic over 2–24 h. Using 500 n M mitoDC-81 will generate an ample intramitochondrial concentration of mitoDC-81 because mitoDC-81 will be accumulated within the cyto- plasm driven by the plasma membrane potential and then be further accumulated within the mitochondria due to the mitochondrial membrane potential [11]. From the known plasma and mitochondrial membrane potentials and the cell and mitochondrial volumes of 143B cells [48] we estimate an intramitochondrial concentration of  450 l M mitoDC-81 for 143B cells incubated with 500 n M mitoDC-81. To see if long-term incubation with mitoDC-81 did result in alkylation of mtDNA, cells were incubated with 500 n M mitoDC-81, IBTP, or TPMP for 24 h and probed using antiserum against TPP and confocal microscopy to visualize any mitoDC-81 bound to mtDNA in situ. IBTP was used as a positive control as it is taken up by mitochondria in cells in Fig. 4. MitoDC-81 does not alkylate mtDNA in situ within isolated mitochondria. In (A) and (B) the concentration dependence of alkyla- tion of plasmid DNA by mitoDC-81 over a 1-h incubation was determined. Plasmid DNA (100 lgÆmL )1 ; 6.8 kb) was incubated for 1hat30°C with 0–1000 l M mitoDC-81. In (C) and (D) rat liver mitochondria(1mgproteinÆmL )1 ) were incubated for 1 h at 30 °C with 5 l M mitoDC-81 after which time mtDNA was isolated and cut with ClaI. All DNA samples were separated on an agarose/ethidium bromide gel and the fluorescence recorded (A and C). The DNA was then transferred to a nylon membrane and TPP moieties covalently attached to the DNA were visualized using anti-TPP serum (B and D). The mitochondrial incubation with mitoDC-81 was repeated twice as described here with identical results. In addition, several similar incu- bations of mitochondria with mitoDC-81 but using a variety of dif- ferent methods to prepare it for electrophoresis, also showed no evidence for DNA alkylation by mitoDC-81. A 1-kb linear DNA ladder (1 lg) and HindIII-digested k DNA (1 lg), which had been incubated for 6 h with 500 l M mitoDC-81, were included as positive controls and molecular mass markers. 2832 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the same way as mitoDC-81, and once there it binds slowly but irreversibly to protein thiols, enabling it to be detected by anti-TPP serum [35]. If mitoDC-81 binds to mtDNA it will also be retained within mitochondria after fixation and should be visible by immunocytochemistry in the same way as IBTP. To see if this was the case, cells were incubated with 500 n M IBTP,TPMPormitoDC-81for24h,then fixed and dual-labelled with antibodies to the mitochondrial enzyme cytochrome oxidase (red) and to TPP (green; Fig. 5). The images were then merged, so yellow indicates colocalization of the TPP and cytochrome oxidase, while red indicates that only the cytochrome oxidase was detected and that there was no TPP immunoreactivity. This experi- ment showed that IBTP colocalized with cytochrome oxidase due to its accumulation by mitochondria and subsequent irreversible reaction with protein thiols (Fig. 5A). Although TPMP will also accumulate within mitochondria, it did not generate any TPP-labelling as it is not covalently bound inside the matrix and is lost on fixation (Fig. 5B). Confocal microscopy of cells incubated with mitoDC-81 (Fig. 5C) gave a pattern of fluorescence that resembled that of TPMP, rather than IBTP. This suggests that mitoDC-81 is not covalently bound to mtDNA within mitochondria. Incubations from 2 h to 3 days with mitoDC-81 concentrations ranging from 100 n M to 5 l M gave similar negative results (data not shown). It is possible that the amount of mitoDC-81-modified mtDNA present is below the detection limit for immuno- cytochemistry. However this seems unlikely because TPP covalently bound to mitochondrial macromolecules was very easy to detect by confocal microscopy after incubation of cells for 30 min with 100 n M IBTP (data not shown). In addition, mitochondrial respiratory complexes such as cytochrome oxidase, present at  200 pmolÆmg protein )1 [49] are easily detected by these procedures, as are other mitochondrial antigens such as those involved in apoptosis (e.g. Smac [50]), which are probably present at a fraction of the content of respiratory complexes. Consequently the limit for the detection of mitochondrial antigens using confocal microscopy is likely to be in the low pmolÆmg protein )1 range. There are  18.7 · 10 9 mtDNA moleculesÆmg mito- chondrial protein )1 [47] or about 6.1 · 10 14 basesÆmg protein )1 , 19.3% of which are guanines corresponding to 1.18 · 10 14 guanine residuesÆmg protein )1 or about 200 pmol GÆmg protein )1 . Therefore we would predict that mitoDC-81alkylationofafewpercentoftheavailable mtDNA guanine residues should have been detectable by confocal microscopy after 24 h. Even so, we cannot entirely exclude the possibility that mitoDC-81 did alkylate a small proportion of the guanine residues in mtDNA, but that the amount alkylated was below the threshold for detection, or that alkylated mtDNA is rapidly degraded or repaired. In summary, we found no evidence for alkylation of mtDNA by mitoDC-81 within cells. Long-term incubation with mitoDC81 did not impair cellular respiration For mitoDC-81 to be of potential use in preventing mtDNA replication it might require only a few molecules of mitoDC-81 bound per mtDNA molecule. Thus, even though we could not directly detect alkylation of mtDNA by mitoDC-81, it remained possible that mitoDC-81 was bound to mtDNA, but at concentrations below the detection limit of immunoblotting or confocal microscopy. If that was the case then long-term incubation of cells with mitoDC-81 should disrupt mtDNA replication, depleting mtDNA and ultimately leading to the production of cells that entirely lacked mtDNA (q°). Therefore we cultured 143B cells with mitoDC-81 for long time periods and attempted to isolate q° clones. 143B osteosarcoma cells were chosen because it is easy to deplete these cells of mtDNA and q° clones derived from 143B cells are robust [51]. For these experiments the culture medium was also supplemented with uridine and pyruvate, as these are required for the growth and survival of any q° cells that arise [51–53]. After 2.5 weeks of culture ( 15 cell divisions) with mitoDC-81, clones were isolated and allowed to recover in the absence of mitoDC-81 before mitochondrial oxygen consumption was measured. None of the individual clones (n ¼ 24) that were analysed had impaired mitochondrial respiration. This lack of detection of q° clones was not due to recovery and expansion of a residual population of nonalkylated mtDNA on removal Fig. 5. MitoDC-81 does not bind to mtDNA in cultured cells. 143B cells were cultured at 37 °C for 24 h in the presence of 500 n M IBTP (A), 500 n M TPMP (B) or 500 n M mitoDC-81 (C). After fixation mito- chondria (red) and TPP moieties (green) were visualized using an anti- (cytochrome oxidase) mAb and anti-TPP serum, respectively. Ó FEBS 2003 Mitochondrial DNA alkylation (Eur. J. Biochem. 270) 2833 of mitoDC-81 as a bulk culture of 143B cells grown in 500 n M mitoDC-81 for 6 weeks had normal rates of mitochondrial respiration (data not shown). Concentra- tions of mitoDC-81 >500 n M were too toxic for long-term culture and 500 n M mitoDC-81 slowed cell growth slightly relative to 500 n M TPMP or no additions (data not shown), indicating that the DC-81 moiety of mitoDC-81 was affecting the cells. Interestingly, this growth inhibition was not related to effects on mtDNA as 500 n M mitoDC- 81 completely prevented the growth of, although did not necessarily kill, a previously established 143B-derived q° cell line (data not shown). This effect was specific to mitoDC-81 and was not due to nonspecific disruption by the lipophilic cation as q° cells cultured with 500 n M TPMP grew at the same rate as control incubations (data not shown). One possible interpretation is that the higher mitochondrial membrane potential of 143B cells causes their mitochondria to sequester mitoDC-81 preventing the toxic reactions in the cytosol or nucleus that disrupt growth in q° cells. However, as the q° cells still survived for several days in the presence of mitoDC-81 this would not have led to the elimination of any q° cells that arose during incubation with mitoDC-81. In summary, we found no evidence for the depletion of mtDNA on long-term incubation of cells with mitoDC-81. Discussion We have synthesized a mitochondria-targeted compound that has the ability to alkylate isolated DNA. This molecule, mitoDC-81 is taken up by energized mitochondria and accumulates within the mitochondrial matrix  1000-fold driven by the membrane potential. Consequently incubation of energized mitochondria with micromolar concentrations of mitoDC-81 leads to millimolar concentrations within the mitochondrial matrix. MitoDC-81 alkylated isolated mtDNA, and reacted with supercoiled, relaxed-circular or linear plasmid DNA. Extensive binding was detectable after a 1-h incubation of isolated DNA with 50 l M mitoDC-81. However, incubation of mitochondria with mitoDC-81 for a similar duration under conditions where the concentration within the mitochondrial matrix was estimated to be 50– 100-fold greater did not lead to detectable binding of mitoDC-81 to mtDNA. This was extended to cells where incubation with mitoDC-81 also failed to yield detectable alkylation of mtDNA by confocal microscopy, even though the mitochondrial uptake of mitoDC-81 was predicted to be ample to alkylate isolated mtDNA, and despite the fact that with cells it was possible to incubate for far longer periods (24 h), than was possible with isolated mitochondria. Finally, alkylation of mtDNA by mitoDC-81 would be expected to disrupt mitochondrial biogenesis and lead to depletion of mtDNA, but there was no generation of q° or respiratory-deficient cells on long-term incubation with mitoDC-81. Therefore we found no evidence that mitoDC- 81 alkylates mtDNA in mitochondria or cells. The reasons for the lack of alkylation of mtDNA within mitochondria by mitoDC-81 are unclear. The local concentrations of mitoDC-81 and DNA, and the duration of the experiments were ample to alkylate isolated DNA. One possibility is that mitoDC-81 does alkylate mtDNA within mitochondria, but this modified DNA is then rapidly degraded. This seems unlikely, as the amount of mtDNA isolated from treated and untreated mitochon- dria was similar. In addition, such a scenario would have been expected to readily generate q° clones, which were not found. Alternatively, upon alkylation the mtDNA, or the mitochondria themselves, may have become difficult to isolate. However, the similar yields of mtDNA from isolated mitochondria treated with mitoDC-81 again make this explanation unlikely. Furthermore, the lack of labelling of mtDNA by mitoDC-81 in intact cells, as explored by immunocytochemistry, makes it unlikely that mtDNA was alkylated in situ, but was then selectively lost in subsequent manipulations during which unmodified mtDNA was retained. Alternatively, mitoDC-81 may react with RNA, nucleotides, nucleosides or other biolo- gical molecules. Such reactions could inactivate a large proportion of the mitoDC-81 taken up by mitochondria, thus preventing its reaction with mtDNA. However, this would require that these other mitoDC-81 targets are lost upon fixation, as the adducts were not seen by confocal microscopy. It is possible that the binding of mitoDC81 to membranes and proteins within mitochondria prevents its reactivity with mtDNA, however, this binding is reversible, leading to a dynamic equlibrium between bound and free compound. The protein thiol reagent IBTP reacts extensively with matrix proteins, even though it is of similar hydrophobicity to mitoDC81 and will therefore be membrane-bound to about the same extent [35]. Consequently it is unlikely that the binding of mitoDC81 to mitochondrial components eliminates its reactivity with mtDNA. We also cannot exclude the possibility that mitoDC-81 does alkylate mtDNA, but that the altered bases are rapidly excised by very rapid and effective endogenous repair processes that are active in isolated mitochondria. While artefactual explanations for the lack of mitoDC81 binding to mtDNA in situ cannot be entirely eliminated, the most likely interpret- ation of the absence of mtDNA alkylation is that even though the mitoDC-81 was accumulated by mitochondria at high concentrations it could not react with mtDNA. Possible reasons for this could be that the mtDNA is not accessible to this alkylating reagent due to its interaction with the inner membrane or the abundance of nucleoid proteins. The latter have been reported under certain conditions to entirely wrap mtDNA and cause a marked increase in nuclease resistance in vitro [54]. Similar targeting of PNAs to mitochondria also failed to show inhibition of mtDNA replication in intact cells [21,23], suggesting that access of the PNA to mtDNA in the matrix was also limited. In summary, even though an active DNA alkylating reagent could be delivered to mitochondria there was no evidence for its reaction with mtDNA in situ.These unexpected findings suggest that the accessibility of mtDNA to some alkylating reagents may be constrained. It may be that more reactive alkylating reagents could be used to modify mtDNA, but nonspecific reactions with mtDNA or modification to nuclear DNA could limit this approach. A better understanding of the selective alky- lation of mtDNA in situ is required in order to develop therapeutic strategies to deplete mutated mtDNA mole- cules selectively. 2834 A. M. James et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Acknowledgement We thank P. Howard (School of Pharmacy, University of London) for thegiftofDC81. References 1. 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Biochem. 270) Ó FEBS 2003 . the synthesis and characterization of a novel mitochondria- targeted alkylating reagent and show that it alkylates DNA in vitro and is taken up by mitochondria. . Specific targeting of a DNA-alkylating reagent to mitochondria Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,1 1a- tetrahydro- 5 H -pyrrolo[2,1- c ][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium