Nuria Garrido

Acisclo Pérez-Martos

Mercedes Faro

José Manuel Lou- Bonafonte

Patricio Fernández-Silva

Manuel José López-Pérez

Julio Montoya

José Antonio Enríquez
email: enriquez@unizar.es.

Cisplatin mediated impairment of mitochondrial DNA metabolism inversely correlates with glutathione levels

Keywords: cisplatin, mitochondria, mtDNA metabolism, glutathione, anticancer drug toxicity

Cisplatin accumulates in mitochondria being this organelle a major target for this drug in cancer cells. Thus, alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro. In order to clarify the potential effect of cisplatin in mitochondrial DNA maintenance and expression, we have analyzed rat liver mitochondrial DNA and RNA synthesis as well as their stability under the influence of in vivo treatment or in vitro exposure to cisplatin. We show that cisplatin causes a direct and significant impairment of mtDNA and mtRNA synthesis and decreased steady state levels of mtRNAs in isolated mitochondria. Furthermore, in vivo treatment of the animals with cisplatin exerted a protective effect from the impairment on mitochondrial RNA metabolism caused by in vitro exposure to the drug, by means of increased mitochondrial GSH levels after in vivo cisplatin treatment.

INTRODUCTION

Cisplatin is one of the most commonly used chemotherapeutic agents, with demonstrated activity against a diverse spectrum of malignancies including testicular cancer, ovarian carcinoma, and head/neck tumors [START_REF] Rosencweig | Cisdiamminedichloroplatinum (II). A new anticancer drug[END_REF][START_REF] Vokes | Favorable long-term survival following induction chemotherapy with cisplatin, fluorouracil, and leucovorin and concomitant chemoradiotherapy for locally advanced head and neck cancer[END_REF]. However, the therapeutic effectiveness of the drug is limited by the severity of its side effects and the potential progression of tumor cells to a cisplatin-resistant state [START_REF] Hoff | Toxic effects of cisdiamminedichloroplatinum (II) in man[END_REF].

In blood, where the chloride concentration is relatively high, cisplatin exists mainly in the dichloro neutral form. Inside the cell, the relatively low chloride concentration favors the replacement of one or both chloride moieties by H 2 O, resulting in a positively charged molecule that reacts with nucleophilic sites to crosslink DNA, RNA, and protein [START_REF] Kartalou | Mechanisms of resistance to cisplatin[END_REF]. Cisplatin cytotoxicity correlates with DNA adduct formation [START_REF] Lindauer | Cellular distribution and cellular reactivity of platinum (II) complexes[END_REF]. Some of the Pt-DNA adducts can be removed by the nucleotide excision repair (NER) mechanism, but others cannot [START_REF] Zamble | Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug[END_REF]. Pt lesions on DNA impair template function blocking DNA replication, induce a G 2 cell cycle arrest, inhibit RNA transcription and finally promote cell death through apoptosis [START_REF] Eastman | Cisplatin: Chemistry and Biochemistry of a Leading Anticancer Drug[END_REF]. The pathway from cisplatin-induced DNA damage to irreversible apoptotic commitment has not been yet fully defined [START_REF] Rich | Defying death after DNA damage[END_REF].

Enhanced ability to repair DNA lesions through up-regulation of components of the NER pathway has been proposed to contribute to cisplatin resistance in a number of resistant tumors [START_REF] Dabholkar | Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy[END_REF]. Selection for cisplatin resistance in some models has been associated with development of mismatch repair deficiency [START_REF] Fink | In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair[END_REF]. Failure to appropriately recognize cisplatin adducts in cells with defects in mismatch repair is hypothesized to lead to replication bypass of lesions, resulting in frequent incorporation of incorrect nucleotides and failure to trigger cell death.

Intracellular cisplatin inactivation by glutathione (GSH) has also been proposed as a mechanism of cisplatin resistance [START_REF] Godwin | High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis[END_REF]. GSH can either quench DNA-platinum mono-adducts before their conversion to cytotoxic DNA crosslinks [START_REF] Eastman | Cross-linking of glutathione to DNA by cancer chemotherapeutic platinum coordination complexes[END_REF] or form a complex (or complexes) with cisplatin, thereby reducing the amount of intracellular drug available for interaction with DNA [START_REF] Ishikawa | Glutathione-associated cisdiamminedichloroplatinum (II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance[END_REF].

Although there is evidence that the mechanism of tumor killing involves the formation of DNA crosslinks, the sensitivity of cells to platinum drugs does not always correlate with the formation of DNA adducts [START_REF] Strandberg | The significance of DNA cross-linking to cis-diamminedichloroplatinum (II)-induced cytotoxicity in sensitive and resistant lines of murine leukemia L1210 cells[END_REF]. Moreover, the acute side effects of cisplatin are not completely understood indicating that other mechanisms may be involved in the toxicity of the drug.

Mitochondria are subcellular organelles involved in critical metabolic pathways including cellular energy metabolism. Besides the mitochondrial role on cellular ATP production, these organelles are known to be effectors of major apoptotic pathways [15]. A direct inhibition of the respiratory chain rapidly depletes cellular ATP and promotes non-apoptotic (necrotic) cell death [16]. Because of their critical role in cell survival, mitochondria are targets for cellular toxins and chemotherapeutic agents [START_REF] Szewczyk | Mitochondria as a pharmacological target[END_REF].

It has been shown that cisplatin accumulates in mitochondria [START_REF] Sharma | Cis-platinum: subcellular distribution and binding to cytosolic ligands[END_REF]. The electrochemical gradient resulting in a negative charge within mitochondria may play a role in the accumulation of positively charged cisplatin in this organelle [19]. Indeed, mitochondria are thought to be a major target for cisplatin in cancer cells [20] and alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents [START_REF] Harper | Characterization of a novel metabolic strategy used by drug-resistant tumor cells[END_REF]. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro [22,23]. Mitochondria contain their own DNA that is transcribed and translated to synthesize 13 proteins of the mitochondrial electron transport chain [START_REF] Fernandez-Silva | Replication and transcription of mammalian mitochondrial DNA[END_REF]. It has been proposed that cisplatin may also induce mtDNA damage [25,26]. However it remains to be demonstrated if the damage of mtDNA is a primary or a secondary consequence of the cisplatin toxicity.

In this report, we have analyzed the influence of the in vivo treatment and the in vitro addition of cisplatin on mtDNA and mtRNA synthesis and stability. We show that cisplatin caused a direct and significant impairment of the synthesis of mtDNA and the synthesis and steady state of mtRNAs. Furthermore, we provide evidence indicating that GSH levels dramatically influence mitochondrial DNA metabolism. Thus, the in vivo treatment of the animals with cisplatin increases GSH levels, both in cytoplasm and in mitochondrial compartments with a parallel protection of mtDNA metabolism to cisplatin exposure. On the contrary, experimentally induced depletion of mitochondrial GSH impairs mtDNA expression.

These studies provide the basis for the understanding of the effects of cisplatin on mitochondrial activity and the mechanisms of cellular toxicity and resistance caused by this drug.

EXPERIMENTAL PROCEDURES

Chemicals-Cisplatin, glutathione (GSH), meta-phosphoric acid (MPA), Nethylmaleimide (NEM), sodium borohydride (NaBH 4 ), 1-octanol, N-ethylmorpholine, monobromobimane (mBBr), acetonitrile (HPLC-grade), ammonium nitrate and ammonium formate were purchased from Sigma Chemical Company. Cisplatin was dissolved in NaCl 0.9% (1 mg/ml) within 1 hour before use. Concentrated solutions of MPA (10%) and NEM (0.1 M) were prepared fresh immediately prior to use. The column and the guard column for high pressure liquid chromatography were obtained from Waters Corporation. Animals-Wistar rats weighing 200 to 250 gr. were fed laboratory chow and water ad libitum and used for the experiments. In cisplatin in vivo studies, groups of 3 rats were injected intraperitoneally (i.p.) with a simple dose of cisplatin: 10 or 20 mg/kg of body weight. The animals were killed by decapitation 5, 24 or 48 hours after the administration. Controls were injected with saline alone, and one control animal was killed with each one of the experimental groups. Isolation of rat liver mitochondria-The liver was rapidly removed chilled in homogenization medium and mitochondrial fractions were purified as described elsewhere [27]. The final mitochondrial pellet was resuspended in the corresponding buffer for DNA or RNA synthesis or for GSH determination. Sample preparation for GSH determination-The sample preparation for GSH analysis was performed according to Rebrink et al. [START_REF] Rebrink | Effects of age and caloric restriction on glutathione redox state in mice[END_REF] with little modification. Total GSH determination was carried out on crude homogenates just after homogenization of the liver as mentioned above. 200 µl aliquots of the crude homogenates were mixed with 200 µl of ice-cold 10% (w/v) meta-phosphoric acid (MPA) for deproteinization. After 30 min of incubation on ice, samples were centrifuged at 14000 g for 20 min at 4°C. Supernatants were used immediately for GSH determination or stored at -80°C for up to 4 months.

For measurement of mitochondrial GSH content, 200 µl aliquots of fresh mitochondrial preparations containing 2 mg protein/ml were washed once with a 10fold excess of buffer and pelleted by centrifugation. The mitochondrial pellets were suspended in 200 µl of 5% (w/v) MPA, incubated 30 min on ice and centrifuged at 18000 g for 20 min at 4°C. Supernatants were used immediately for GSH determination or stored at -80°C until analysis.

Protein concentration in homogenates and mitochondrial fractions was determined by the Bradford method [29].

Derivatization of GSH and quantitation by HPLC-Derivatization procedure was performed essentially as described by Pastore et al. [START_REF] Pastore | Fully automated assay for total homocysteine, cysteine, cysteinglycine, glutathione, cysteamine, and 2-mercaptopropionylglicine in plasma and urine[END_REF]. Briefly, 10 µl of 4 M NaBH 4 (dissolved in 333 µl/ml dimethyl sulfoxide and 66 mM NaOH), 5 µl of a solution containing 2 mM EDTA and 2 mM DTT, 5 µl of 1-octanol, and 5 µl of 2 M HCl were mixed and spun down in a derivatization vial. 10 µl of homogenate or mitochondrial supernatant (in 5% w/v MPA) was added, mixed, and spun down. After 1 min incubation, 25 µl of N-ethylmorpholine buffer (2 M N-ethylmorpholine, pH 8.0) and 10 µl of 25 mM bromobimane (in 1:1 acetonitrile:H 2 O by volume) were added to the derivatization vial. After 1 min of incubation the reaction was stopped with 4.2 µl of glacial acetic acid. All operations were carried out at room temperature.

GSH was analyzed with a Waters HPLC system, equipped with two 515 HPLC pumps. 10 µl of derivatized sample were injected using a Waters 717 autosampler into a 150 x 3.9 mm Nova-Pack C-18 column (4 µm particle size), equilibrated with 5% buffer A (acetonitrile)-95% buffer B (30 mM ammonium nitrate and 40 mM ammonium formate buffer, pH 3.6). Samples were eluted from the column with a 20 min gradient of buffer A (0-7.50 min, 5% buffer A-95% buffer B; 7.50-10.50 min, 100% buffer A; 10.50-16.00 min, 100% buffer A; 16.00-18.00 min, 5% buffer A-95% buffer B; 18.00-20.00 min, 5% buffer A-95% buffer B) at a flow rate of 1.5 ml/min. The column was run at 33°C. Under these conditions, sample analysis was completed in 20 min and GSH was the only thiol eluting peak with a retention time of 7.90 min. Calibration standards for GSH were prepared in duplicate by diluting of 2 mM stock solution of GSH in 5% (w/v) MPA. For the detection of derivatized GSH a Waters 474 scanning fluorescence detector operating at an excitation wavelength of 390 nm and an emission wavelength of 478 nm was used. The Empower Login program for Windows 2000 (Waters) was used for controlling HPLC system and analyzing GSH results. Each sample was injected twice and the average of peak areas was used for calculations of the GSH concentrations.

Synthesis of DNA and RNA in isolated rat liver mitochondria-Mitochondrial DNA and RNA synthesis assays were performed as described elsewhere [27]. Briefly, reactions were performed in 0.5 ml at mitochondrial concentration of 2 mg/ml in both cases. For DNA synthesis, 50 mM dATP, dGTP, dCTP and 20 µC [α-32 P] dTTP (2000 µC/mmol) were added to the incubation tube and incubation was carried out at 37ºC for 3 hours on a rotating wheel [27].

For RNA synthesis, 20 µC [α-32 P] UTP (800 µC/mmol) was added to the medium. The incubation was maintained at 37ºC for 60 min on a rotary shaker (12 rpm) [START_REF] Enriquez | In organello RNA synthesis system from mammalian liver and brain[END_REF].

After DNA or RNA synthesis mitochondria were centrifuged at 14000 g for 1 min and washed twice with 0.5 ml of 10% glycerol, 0.15 mM MgCl 2 , 10 mM Tris-HCl, pH 6.8.

Mitochondrial nucleic acids extraction-Mitochondrial lysis and nucleic acid extraction

were carried out as described before [27]. Briefly, the mitochondrial pellet was resuspended in 500 µl of pronase buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA-Na), lysed with 2% SDS in the presence of 100 µg the pronase (autodigested to remove nucleases) and incubated for 15 min at 37ºC. Total mitochondrial nucleic acids were extracted twice with an equal volume of a phenol/chloroform/isoamylic alcohol mixture (25:25:1) at room temperature. After ethanol precipitation and centrifugation, the pelleted nucleic acids were dissolved in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA.

Mitochondrial DNA was analyzed directly or after digestion with DNase RNase free and the restriction enzyme BamHI by vertical 1% agarose slab gel electrophoresis [27]. Mitochondrial RNAs were analyzed by methylmercury hydroxide-agarose gels as previously described [32].

After the run, the gels were first stained with ethidium bromide, photographed under UV light, dried and exposed for autoradiography either at -70ºC with a DuPont screen intensifier or at room temperature as needed.

The amount of total mitochondrial DNA or RNA was quantified by densitometry of the gel bands after electrophoresis, stain with ethidium bromide and photography of the gel with an LKB Ultroscan XL laser densitometer and Gel Scan XL software.

The Amount of DNA or RNA synthesized by isolated mitochondria was quantified by densitometry of the autoradiograms with an LKB Ultroscan XL laser densitometer and Gel Scan XL software. Images were acquired with an Epson Perfection 1250 scanner and Adobe Photoshop CS 8.0.1 software. O 2 consumption-Mitochondrial O 2 consumption was measured polarographically at 37°C in a closed 1.0 ml reaction chamber fitted with a Clark O 2 electrode (Yellow Springs Instrument, Yellow Springs OH). One milligram of mitochondrial protein was added to 0.9 ml incubation medium to initiate the study of mitochondrial respiration. The incubation medium was composed of 25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 0.05 mM EDTA, 5 mM MgCl 2 ,10 mM H 3 PO 4 and 10 mM Tris-HCl, pH 7.4, also containing 1 mg/ml fatty acid-free bovine serum albumin (BSA). Respiration started with the addition of Complex I substrates 10 mM glutamate and 2.5 mM malate. After 1-3 min, 0.2 mM ADP was added to initiate state 3 respiration. After ADP was fully converted to ATP, state 4 respiration was measured. 0.2 µM KCN was added to inhibit Complex I activity. Mitochondrial respiration rates (state 3 and state 4) were expressed as nanoatom O 2 •min -1 •mg mitochondrial protein -1 or nanoatom O 2 •min -1 •mtDNA -1 (a.u.). The respiratory control ratio (RCR) is indicative of mitochondrial coupling, and was expressed as the state 3/ state 4 ratio.

The P/O ratio (moles of synthesized ATP per atom of oxygen transformed to water during oxidative phosphorilation) was also calculated as a measurement of mitochondrial coupling. PCR amplification of mtDNA-Mitochondria and mitochondrial DNA were isolated as described before and incubated with and without cisplatin as described in the figures. The oligonucleotide primers used in this study, as indicated below, were prepared by Gibco BRL. Various segments of mtDNA were amplified using the following oligonucleotides:

H-OH-5´-CCTGTGGAACCTTTTAGTTAAG-3´ L-OH-5´-TTTGGCATTGAAGTTTCAGGTG-3´ H-IL-5´-CCCCAAAAACATTAAAGCAAGA-3´ L-IL-5´-GAATCCATCTAAGCATTTTCAG-3´ H-ND-5´-GATTAGTGTTAGGATATTA-3´ L-16S-5´-CCCAGTTACGAAAGGACAAGAG-3´ L-Cys-5´-CCCAAGTTATTAGGGTGGCA-3´ H-Cys-5´-GCGTATTCGACGTTAAAGCC-3´
All amplification reactions were carried out in 0.2 ml PCR tubes in a final volume of 50 µl. Each reaction consisted on 0.5 µg of mtDNA, 2.5 µM of each primer, 200 µM dNTP, 2 mM MgCl 2 , 2.5 units Taq DNA polymerase (Epicenter Technologies, Madison, WI), and the supplied reaction buffer. For the amplification of mtDNA, samples underwent an initial denaturation at 94°C for 10 min, followed by 40 cycles of template denaturation at 94°C for 1.30 min, primer-template annealing at 55°C for 1.30 min and primer extension at 72°C for 1.30 min. At the end of the 30 cycles, extension was allowed to continue for an additional 7 min. PCR amplification was performed on a programmable thermal cycler (Biometra, Trio Termocycler). The amplified products were separated on horizontal 1% agarose gel electrophoresis with 1% ethidium bromide. After the run, the gels were photographed under UV light. Amounts of amplified DNA were quantified by densitometry with an LKB Ultroscan XL laser densitometer and Gel Scan XL software. Statistical analysis-Results were shown as the mean plus the standard deviation. The statistical analysis was carried out by Mann Whitney test. p<0.05 was considered statistically significant.

RESULTS

Cisplatin impairs mitochondrial respiration.

Mitochondria isolated from rat liver were incubated at 37°C in the absence or in the presence of different cisplatin concentrations for 1, 2 or 3 hours and subjected to oxygen consumption measurement. State 3 and state 4 respiration were measured and the respiratory control ratio (RCR) and the P/O ratio were calculated (Fig. 1A). Cisplatin caused a dose and time dependent decrease in the RCR (Fig. 1A, upper panel) and the P/O ratio was less sensitive to cisplatin, but it also showed a dose and time dependent increase (Fig. 1A, lower panel) indicating an impairment of the OXPHOS system function.

Cisplatin inhibits mitochondrial DNA synthesis.

Since cisplatin can form adducts with DNA, we investigated if mitochondrial DNA was functionally affected by the presence of the drug. To evaluate this possibility, the direct effect of cisplatin on mitochondrial DNA replication in isolated organelles was evaluated. Rat liver mitochondria were incubated for 3 hours at 37ºC with [α-32 P] TTP in the presence of cisplatin concentrations varying from 0 to 0.7 mM. After incubation, mitochondrial nucleic acids were isolated and examined by electrophoresis. As shown in Fig. 1B, cisplatin significantly inhibited mtDNA replication in organello. The inhibition was dose-dependent with a range of cisplatin concentrations between 0 and 0.7 mM. At concentrations higher than 0.2 mM, cisplatin inhibited mtDNA synthesis within 1 hour. The inhibition affected all the different mtDNA conformations [27]. Cisplatin did not have any effect on radioisotope capture by the isolated mitochondria (TTP uptake, Fig. 1B).

Inhibition of mtDNA replication could be either due to impairment of the OXPHOS capacity induced by cisplatin or to a direct modification of the mtDNA template, as it has been postulated for nuclear DNA. In order to evaluate which one of the two mechanisms is determining the observed effect, we used a PCR-based assay [START_REF] Govan | Fine-mapping of DNA damage and repair in specific genomic segments[END_REF] in which many DNA lesions would block the Taq DNA polymerase progression and result thereby in a specific decrease in amplification of the damaged DNA template. Mitochondria isolated from rat liver were incubated for 1 hour at 37°C in the presence of varying concentrations of cisplatin (0 to 1.4 mM). After the incubation, nucleic acids were extracted and used as template for PCR amplification. Also, 1.4 mM cisplatin was added to one of the DNA control samples just before the PCR amplification. PCR products were then analyzed on agarose gels. As shown in Fig. 1C, left panel, the amount of amplified mtDNA decreased in a dose-dependent manner after incubation with cisplatin, while it was not affected by the presence of cisplatin exclusively during the PCR amplification reaction (C 1 ). Various mtDNA regions were affected by cisplatin at different extent, a fact that could be explained by the length of the amplification fragment (Fig. 1C, right panel). The region amplified by H-Cys/L-Cys (1), the largest one, showed the higher synthesis inhibition, whereas H-OH/L-OH (4), the shorter one, showed the less amplification inhibition.

These results indicated that cisplatin produced a direct and non-specific structural alteration on mtDNA, which caused an inhibition of mtDNA amplification by PCR and, very likely, contributed to the inhibition of mitochondrial DNA synthesis in organello.

Cisplatin inhibits mitochondrial DNA transcription.

The next question to be answered was whether cisplatin also interfered with mtDNA transcription. Again, mitochondria isolated from rat liver were incubated for 1 hour at 37ºC with [α-32 P] UTP in the presence of cisplatin concentrations varying from 0 to 1.4 mM. After incubation, mitochondrial nucleic acids were isolated and examined by electrophoresis.

Analogous to the effect on replication, cisplatin caused a dramatic inhibition of mtDNA transcription in organello (Fig. 2A). The inhibition was already detectable at concentrations as low as 0.17 mM and was complete at 0.7 mM. The densitometric analysis of the RNA electrophoretic patterns normalized by the mtDNA amount loaded in each lane, showed that the inhibition affected to all mtRNA species: ribosomal and messenger RNAs. Cisplatin did not have any effect on radioisotope capture by the isolated mitochondria (UTP uptake, Fig. 2A, left panel). In addition, the effect of 0.17 mM cisplatin was time-dependent, observing increased inhibition from 1 to 3 hours (Fig. 2A, right panel).

Cisplatin decreases mitochondrial RNA stability.

Incubation of the organelles in the presence of cisplatin did not affect mtDNA steady state (Fig. 2B, upper band), but the levels of the in vivo mtRNAs were decreased in a dose and time-dependent manner (Fig. 2B, lower bands) as determined from the stained gels (see Material and Methods). We observed a general decrease in the amount of the mtRNAs that was particularly evident for the 12S and 16S ribosomal RNAs. Thus, we determined whether a decrease in mitochondrial RNA stability (Fig. 2B) caused by cisplatin could explain the reduction on the amount of newly synthesized RNA in isolated mitochondria (Fig. 2A). Comparison of the effect of cisplatin on mtRNA stability with the effect on RNA synthesis revealed that the decreased RNA levels could not be explained only because of the reduction of the stability (not shown).

The results described above demonstrated that cisplatin causes a direct damage of mitochondrial biogenesis by inhibiting mitochondrial DNA synthesis and transcription and by destabilizing the mitochondrial RNA, effect that is already evident after a short time exposure.

Effect of in vivo cisplatin-treatment on mitochondrial RNA metabolism

In order to evaluate the in vivo relevance of the effects observed in vitro, we treated rats with cisplatin. The drug was injected intraperitoneally once at doses of either 10 or 20 mg per kg of body weight. For each dose, the animals were sacrificed at 5, 24 or 48 hours after the administration (see Materials and Methods).

Liver mitochondria were isolated from animals treated in vivo with cisplatin and incubated in organello for 1 hour with [α-32 P] UTP. After incubation, mitochondrial 7 nucleic acids were isolated and examined by electrophoresis. In vivo treatment with cisplatin caused various effects on mtRNA synthesis by isolated mitochondria depending on the dose and the time after the treatment (Fig. 3A and3B). Thus, 10 mg/kg cisplatin increased in organello RNA synthesis rate, whereas in vivo treatment with 20 mg/kg cisplatin slightly decreased it. These effects were already evident after 5 hours of cisplatin adeministration, raising to a maximum after 24 hours and returning to control levels after 48 hours (Fig. 3D, upper panel).

The effect of in vivo cisplatin treatment on mtRNA steady state levels was next examined. An increase on mitochondrial ribosomal RNA steady state level was found for both doses assayed (Fig. 3C). The change was moderate for the 20 mg/kg dose, but it reached a 3-fold increase for the 10 mg/kg dose. This effect was already evident after 5 hours of cisplatin administration, reaching a maximum after 24 hours and returning to control levels after 48 hours (Fig. 3D, lower panel).

Effect of in vitro addition of cisplatin on mtRNA metabolism in mitochondria isolated from in vivo cisplatin-treated animals

The addition of cisplatin to the incubation medium was found to directly inhibit mitochondrial RNA synthesis in organello, but the in vivo treatment of the animals with the drug increased mitochondrial transcription. In an attempt to clarify what is the effect of cisplatin on mitochondrial DNA metabolism and on mitochondrial activity, organelles from animals treated in vivo with 10 mg/kg cisplatin for 24 h were incubated with doses of cisplatin varying from 0 to 1.3 mM for 1 hour. As expected, in organello RNA synthesis was inhibited by cisplatin both in mitochondria isolated from in vivo treated and non-treated animals, but surprisingly, this inhibition was less pronounced on the mitochondria isolated from in vivo cisplatin-treated animals (Fig. 4A, upper panel). In vitro addition of cisplatin to the incubation medium decreased mtRNA steady state levels both in mitochondria isolated from control and from in vivo cisplatin-treated animals, but the decrease was far more dramatic in the former case (Fig. 4A, lower panel, and Fig. 4B).

The above results pointed out the existence of in vivo mechanisms that reduce the damage of cisplatin to mtRNA metabolism.

Effect of in vivo cisplatin treatment on GSH concentration

To test this hypothesis, GSH metabolism was analyzed. GSH concentration was measured in total liver homogenates and in mitochondrial preparations from controls and from animals treated in vivo with cisplatin with a 10 mg/kg dose 5, 24 or 48 hours prior to sacrifice. As shown in Table 1, after 24 hours of in vivo cisplatin treatment, the GSH levels increased both in total homogenate as in the mitochondrial fraction. The increase was significant in both cases when a non-parametric test was applied (Mann Whitney test, p=0.0495 and p=0.0495, respectively). After 5 and 48 hours of in vivo cisplatin treatment the GSH levels were slightly higher in both total homogenate and mitochondrial fraction, but this increase was not significant in any case. Mitochondria isolated from control and in vivo treated animals with 10 mg/kg cisplatin for 24 h were next treated with 0.2 mM of N-ethylmaleimide (NEM) for 15 minutes at room temperature. NEM modifies thiols groups, rendering them incapable of binding cisplatin. The HPLC analyses showed that >75% of mitochondrial GSH in control samples and >70% of mitochondrial GSH in in vivo cisplatin-treated samples was modified by NEM (GSH concentration was 2.28±0,1 and 2.71±0,5 nmoles/mg mitochondrial protein for control and cisplatin in vivo treated mitochondria after NEM treatment respectively).

Next, the effect of mitochondrial GSH concentration on mitochondrial RNA synthesis in organello and mitochondrial RNA stability was analysed (Fig. 5). As described before (Fig 4A ), in organello RNA synthesis was inhibited by the in vitro addition of cisplatin both to mitochondria isolated from in vivo treated or non-treated animals, being the inhibition higher on mitochondria isolated from in vivo cisplatin non-treated animals (Fig. 5A and5B). Very interestingly, when equivalent samples of mitochondria were used but pre-incubated with NEM (+NEM), in organello RNA synthesis was completely abolished even in the absence of cisplatin. Moreover, mitochondrial depletion of GSH impaired mtRNA stability and reverted the protective effect on mtRNA stability induced by in vivo treatment with cisplatin (Fig. 5C and5D).

DISCUSSION

The results presented in this report, together with previous studies, allow us to propose that cisplatin accumulates in the mitochondria, binds to mtDNA, and most likely also to mtRNA, inducing their physical modification. This modification would be responsible for the inhibition of mtDNA and mtRNA synthesis and the decrease of mtRNA stability. Since mtDNA encodes for polypeptides that are components of the mitochondrial respiratory chain complexes, cisplatin direct effect on mitochondrial DNA metabolism could be the cause of the long-term decline of the mitochondrial respiration activity and ATP synthesis.

We show here that cisplatin causes a decrease of the mitochondrial oxygen consumption in isolated mitochondria and an increase of mitochondrial uncoupling between oxygen consumption and ATP synthesis. This result is in good agreement with previous studies describing that cisplatin induces decline in mitochondrial respiratory activity in vivo [START_REF] Gordon | Mitochondrial alterations in cisplatininduced acute renal failure[END_REF] and inhibition of the mitochondrial respiratory chain complexes in vitro [23,[START_REF] Kruidering | The use of porcine proximal tubular cells for studying nephrotoxicity in vitro: The role of oxidative stress in cisplatin-induced cell death[END_REF]. However, this inhibition was observed at doses and/or exposure times higher than those needed to inhibit mtDNA synthesis and expression. This would agree with recent studies showing that cisplatin caused little or no immediate effect on mitochondrial function in various cancer cell lines, tumor cells, and beef heart submitochondrial particles as measured by oxygen consumption [START_REF] Souid | Immediate effects of anticancer drugs on mitochondrial oxygen consumption[END_REF] but caused a long term inhibition of mitochondrial oxygen consumption which could be an indirect effect of the drug on mitochondrial activity [20].

By contrast, mtDNA synthesis appeared to be greatly inhibited at low cisplatin concentrations. It is well known that cisplatin binds to mtDNA. In studies with human malignant melanoma cells, cisplatin binding to mtDNA was 50 fold greater than to chromosomal DNA [START_REF] Giurgiovich | Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure[END_REF]. Cisplatin is able to induce intra-strand and inter-strand adduct formation on mtDNA [38], and mtDNA crosslink has been described in animals treated in vivo [START_REF] Giurgiovich | Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure[END_REF] and in in vitro treated cancer cells [39]., However, little is known about the consequences of these lesions on mitochondrial activity. DNA adducts can interfere with DNA synthesis. We have shown that cisplatin caused an impairment of mtDNA when used as template for PCR amplification. It has previously been demonstrated that cisplatin-DNA adducts do block Taq-polymerase and PCR essays have been used to analyze the effects of cisplatin on genomic DNA as well as on mitochondrial DNA [START_REF] Daoud | Polymerase chain reaction analysis of cisplatin-induced mitochondrial DNA damage in human ovarian carcinoma cells[END_REF]. Therefore, the formation of DNA adducts seems to be the more plausible explanation for the strong inhibition of mtDNA replication induced by cisplatin.

DNA adducts can interfere with DNA expression as well. We have shown that cisplatin directly inhibits mitochondrial DNA expression. Previous reports revealed that cisplatin-DNA adducts block elongation during in vitro RNA synthesis by procariotic and eucariotic RNA polymerases [41,[START_REF] Corda | RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cisdiamminedichloroplatinum (II)[END_REF]. Additionally, a 2 to 3-fold decrease in transcriptional activity was observed when platinum-modified reporter genes were 9 transfected into human or hamster cells [START_REF] Mello | DNA adducts of cisdiamminedichloroplatinum (II) and its trans isomer inhibit RNA polymerase II differentially in vivo[END_REF]. Cisplatin was also shown to substantially reduce transcription from the mouse mammary tumor virus promoter stably incorporated into mouse cells [START_REF] Mymryk | Cisplatin inhibits chromatin remodeling, transcription factor binding, and transcription from the mouse mammary tumor virus promoter in vivo[END_REF].

The decrease on mtRNAs steady-state levels was an interesting and unexpected observation. To our knowledge, such an effect had never been described before in cisplatin treatments. The decrease on mtRNAs newly synthesized by isolated mitochondria could have been due either to modifications in mtDNA transcriptional activity or to alterations of the mtRNA stability. We must stress that cisplatin induced both inhibition of mtRNA synthesis and decreased mtRNA stability, being both effects independent. One explanation would be that cisplatin interacts directly with mitochondrial RNAs and cause their structural modification inducing their earlier degradation. In any case, this hypothesis requires further investigation.

Unexpectedly in vivo treatment with cisplatin (10 mg/kg) promoted an increase of mtRNA synthesis in organello. Similarly, the stability of mtRNAs was increased in mitochondria isolated from animals treated in vivo with the drug. These results were apparently contradictory with the results we found in mitochondria incubated in vitro in presence of the drug. A scenario that conciliates both the in vitro and the in vivo results can be envisioned. After the in vivo treatment of the animals with cisplatin, the drug inhibits directly mtDNA synthesis and expression and decreases mtRNA stability. As a consequence, mitochondrial respiration is impaired and the cellular ATP levels are decreased. The cell reacts triggering compensatory mechanisms to maintain cellular ATP levels. Those would be the induction of genes involved in: (i) mitochondrial biogenesis, (ii) repair of mtDNA damage, (iii) cellular protection mechanisms such as decreased cellular uptake, (iv) increased cellular efflux of cisplatin, or (v) inactivation of the drug by sulfhydryl groups. These in vivo putative compensatory/defense mechanisms could be maintained, at least in part, in the mitochondria after their isolation and would explain the resistance to a direct alteration of mtDNA metabolism by cisplatin in mitochondria isolated from in vivo treated-animals.

In support of increased mitochondrial biogenesis, it has been reported that in vivo cisplatin treatment caused an elevation of mtDNA levels in rat liver [45]. The transcript level of mitochondrial 12S rRNA was increased by cisplatin treatment in two different cultured cell lines [46]. Cisplatin may increase mitochondrial transcription by altering activities of nuclear-encoded transcription factors in response to mtDNA damage, oxidative stress or other metabolic signals. It has been shown in cell culture that incubation with cisplatin resulted in up-regulation of Tfam, a mitochondrial transcription factor [47], which could explain the increase in mtRNA synthesis. However, the finding that mitochondria isolated from animals treated in vivo with the drug are more refractory, compared to control mitochondria, to the inhibition of transcription, reinforces the idea that a protection mechanism is induced by cisplatin in vivo treatment.

In this respect, we analyzed the levels of glutathion (GSH) as a potential protective pathway. GSH is critical in maintaining the protein sulphydryl groups in a reduced state and in the protection against oxidative stress through detoxification of reactive oxygen species [START_REF] Anderson | Glutathione: an overview of biosynthesis and modulation[END_REF]. Glutathion can similarly detoxify many exogenous toxins, including cisplatin, through the formation of glutathion adducts [49]. Pt ions entering the cell may preferentially bind to GSH and metallothionein, both present in millimolar concentrations in the cytoplasm. Formation of these complexes limits the amount of drug available for DNA binding and a positive correlation between GSH levels and resistance to cisplatin has been reported [START_REF] Godwin | High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis[END_REF].

We found that in vivo treatment with cisplatin (10 mg/kg) induced an increase in the cytoplasmic and mitochondrial GSH concentration as it had been described in previous studies [START_REF] Godwin | High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis[END_REF]. Modification of mitochondrial GSH with N-ethylmaleimide, NEM, caused a dramatic impairment of mitochondrial RNA synthesis in organello. The influence of sulphydryl groups on nuclear DNA platination by cisplatin have been demonstrated by an 8-fold increase in the amount of Pt-DNA adducts when cellular thiols were blocked by NEM [50]. A similar effect could work for mitochondria incubated in vitro with NEM. Mitochondrial GSH depletion may leave mtDNA unprotected for cisplatin damage.

It should be noted that all the in vivo effects we have described here were observed when we used the 10 mg/kg dose. Treatment of the animals with a higher dose, 20 mg/kg, did not increase mtRNA synthesis nor stability. It is possible that the raising of the cisplatin dose, overwhelmed the compensatory mechanisms and the cells were no longer able to overcome the toxicity. The fact that the animals treated with 40 mg/kg dose died before they were sacrificed may show a strong alteration of cell viability.

The in vitro and in vivo cisplatin concentrations used in our studies are clinically relevant [START_REF] Kruidering | The use of porcine proximal tubular cells for studying nephrotoxicity in vitro: The role of oxidative stress in cisplatin-induced cell death[END_REF][START_REF] Giurgiovich | Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure[END_REF][START_REF] Zhang | Role of mitochondria in cisplatininduced oxidative damage exhibited by rat renal cortical slices[END_REF]. Cisplatin-induced nephrotoxicity and neurotoxicity are the most severe and dose-limiting side effects for the therapeutic use of cisplatin, but dosedependent cisplatin-induced hepatotoxicity can alter the clinical situation in patients. It is known that cisplatin uptake by human liver is significant [START_REF] Zicca | Reduction of cisplatin hepatotoxicity by procainamide hydrochloride in rats[END_REF]. Hepatotoxicity was reported in studies where similar doses of cisplatin to those used in our study were administrated to rats [START_REF] Zicca | Reduction of cisplatin hepatotoxicity by procainamide hydrochloride in rats[END_REF][START_REF] Pratibha | Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats[END_REF] and to humans [54].

In summary, the results presented in this report are the first biochemical evidence of a direct effect of cisplatin on mtDNA metabolism. These results together with the evidence of the protective effect exerted by GSH are fundamental for a better understanding of the cisplatin mechanism of action on tumor cells and of its toxic side effects on non-tumor cells. -Effect of cisplatin on mitochondrial RNA synthesis and stability (A) Autoradiogram after electrophoresis on methylmercury hydroxide-agarose gels of newly mtRNAs synthesized in organello by isolated rat liver mitochondria incubated in the presence of 0, 0.17, 0.35, 0.7, 1 and 1.4 mM cisplatin (left), and incubated without cisplatin (-) or with 0.17 mM cisplatin (+) during 1, 2 or 3 hours (right). Each lane shows mtRNAs synthesized by equivalent amounts of mitochondria. UTP uptake: percent of radioactivity trapped by mitochondria but not incorporated into mtRNA. (B) Ethidium bromide staining after electrophoresis on methylmercury hydroxideagarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria incubated with 0, 0.17, 0.35, 0.7, 1 and 1.4 mM cisplatin (left), and without cisplatin (-) or with 0.17 mM cisplatin (+) during 1, 2 or 3 hours (right). Each lane shows nucleic acids isolated from an equivalent amount of mitochondria. (C) Ethidium bromide staining after electrophoresis on methylmercury hydroxideagarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria of three different animals (1, 2 and 3) treated in vivo without (Control) or with 10 mg/kg cisplatin for 24 hours (Treated). Each lane shows nucleic acids isolated from an equivalent amount of mitochondria. Animals were treated in vivo without (Control) or with cisplatin 10 mg/kg for 5, 24 or 48 hours. Liver was extracted from the animals and GSH levels were determined by HPLC from total homogenates or mitochondrial fractions and after treatment of mitochondria with N-ethylmaleimide for 15 min at RT (Data for 3 different experiments ± SD). * Significant differences respect to control p<0.05.

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20071615 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071615 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20071615 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071615 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20071615 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071615 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 .

 1 Figure 1.-Influence of cisplatin on mitochondrial oxidative phosphorylation and mtDNA replication (A) Effect of cisplatin on mitochondrial oxidative phosphorylation. RCR and P/O ratios in mitochondria isolated from rat liver and incubated at 37ºC for 1, 2 or 3 hours with different cisplatin concentrations: 0, 0.17, 0.35 and 0.7 mM cisplatin respectively. Data are from three different experiments and expressed as mean ± SD. (B) Effect of cisplatin on mitochondrial DNA synthesis. Autoradiogram after electrophoresis through agarose slab gel of newly synthesized mtDNA by isolated rat liver mitochondria incubated in the presence of 0, 0.007, 0.035, 0.07, 0.35 and 0.7 mM cisplatin. Each lane shows mtDNA synthesized by an equivalent amount of mitochondria. TTP uptake: percent of radioactivity trapped by mitochondria but not incorporated into mtDNA. (C) Left: PCR amplification of mtDNA isolated from rat liver mitochondria after incubation for 1 hour at 37°C with 0, 0.17, 0.35, 0.7, 1 and 1.4 mM cisplatin. C 1 : mtDNA control sample were cisplatin 1.4 mM was added just before PCR amplification. Right: PCR amplification of mtDNA isolated from rat liver mitochondria and then incubated in vitro for 1 hour with the indicated cisplatin concentration. C 2 : mtDNA control sample where cisplatin 1.5 nM was added just before PCR amplification.

Figure 2 .

 2 Figure 2. -Effect of cisplatin on mitochondrial RNA synthesis and stability(A) Autoradiogram after electrophoresis on methylmercury hydroxide-agarose gels of newly mtRNAs synthesized in organello by isolated rat liver mitochondria incubated in the presence of 0, 0.17, 0.35, 0.7, 1 and 1.4 mM cisplatin (left), and incubated without cisplatin (-) or with 0.17 mM cisplatin (+) during 1, 2 or 3 hours (right). Each lane shows mtRNAs synthesized by equivalent amounts of mitochondria. UTP uptake: percent of radioactivity trapped by mitochondria but not incorporated into mtRNA. (B) Ethidium bromide staining after electrophoresis on methylmercury hydroxideagarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria incubated with 0, 0.17, 0.35, 0.7, 1 and 1.4 mM cisplatin (left), and without cisplatin (-) or with 0.17 mM cisplatin (+) during 1, 2 or 3 hours (right). Each lane shows nucleic acids isolated from an equivalent amount of mitochondria.

Figure 3 .

 3 Figure 3. -Effect of the in vivo cisplatin treatment on mitochondrial RNA synthesis and stability (A and B) Autoradiograms after electrophoresis on methylmercury hydroxide-agarose gels of newly synthesized mtRNAs by rat liver mitochondria isolated from animals treated in vivo without C, or with cisplatin T, at doses of 10 mg/kg (A) or 20 mg/kg (B) for 5, 24 or 48 hours. Each lane shows mtRNAs synthesized by an equivalent amount of mitochondria.(C) Ethidium bromide staining after electrophoresis on methylmercury hydroxideagarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria of three different animals (1, 2 and 3) treated in vivo without (Control) or with 10 mg/kg cisplatin for 24 hours (Treated). Each lane shows nucleic acids isolated from an equivalent amount of mitochondria.

Table 1 . -GSH concentration in total homogenate and in the mitochondrial fraction

 1

		Control	Cisplatin	Control	Cisplatin	Control	Cisplatin
		5hours	5hours	24hours	24hours	48hours	48hours
	nmolGSH/	25,40±1,2 26,75±1,3 25,50±0,5 31,50±0,8* 24,50±1,5 25,05±0,9
	mg protein
	nmolGSH/mg	10,50±0,7 10,85±0,8 10,75±0,6 15,25±0,7* 10,25±0,5 10,75±0,8
	mitochondrial
	protein
		Control	Cisplatin	Control	Cisplatin	Control	Cisplatin
		5hours	5hours	24hours	24hours	48hours	48hours
		+NEM	+NEM	+NEM	+NEM	+NEM	+ NEM
	nmolGSH/mg	2,42±0,4	3,36±0,5	2,28±0,1	2,71±0,5 1,78±0,1 2,53±0,4
	mitochondrial
	protein

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 22 Apr 2008 as manuscript BJ20071615 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071615 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Cersosimo, R. J. (1993) Hepatotoxicity associated with cisplatin chemotherapy. Ann. Pharmacother. 27, 438-441 FOOTNOTES * We would like to thank Santiago Morales for his technical assistance. Our work was supported by the Spanish Ministry of Education (SAF2006-00428), the Health Institute Carlos III (REDEMETH-G03/054 and REDCIEN C03/06-Grupo RC-N34-3), EU EUMITOCOMBAT-LSHM-CT-2004-503116), DGA research group of Excellence grant (B55 to NG, APM, PFS and JAE) and DGA Consolidated research group grant (B33 MLP and JM). 1 The abbreviations used are: mtDNA, mitochondrial DNA; H, mtDNA heavy strand; L, mtDNA light strand; OH, heavy strand replication origin; IL, light strand transcription origin; ND1, ND2, ND3, ND4, ND4L, ND5 and ND6 are subunits 1 to 6 and 4L of the NADH Dehydrogenase; Cytb, Cytochrome b; COI, COII and COIII are Cytochrome c oxidase subunits 1 to 3; A6, ATP synthase F0 subunit 6; A8, ATP synthase F0 subunit 8; rRNA, ribosomal RNA; mRNA, messenger RNA; tRNA, transfer RNA; 12S and 16S are the mitochondrial rRNAs; pre-RNA is for the precursor of mitochondrial RNAs; 7S mRNA synthesized from the light strand; Pt, platinum; NER, nucleotide excision repair; PCR, polymerase chain reaction; RCR, respiratory control ratio; GSH, gluthatione; sodium borohydride (NaBH 4 ); monobromobimane (mBBr); meta-phosphoric acid (MPA); N-ethylmaleimide (NEM). ) and animals treated in vivo with 10 mg/kg 24 hours (closed circles) and then incubated with 0, 0.4 and 1.3 mM cisplatin, and ratio between the amount of RNA synthesized by rat liver mitochondria isolated from animals treated in vivo without or with 10 mg/kg cisplatin for 24 hours (squares). The radioactivity incorporated on mtRNA has been normalized by the amount of mtDNA in each sample. Data represent % synthesis above the control (100). Lower panel: densitometric quantification from the ethidium bromide stained gels of 16S mitochondrial rRNA isolated from rat liver mitochondria from control animals (open circles) and animals treated in vivo with 10 mg/kg cisplatin for 24 hours (closed circles) and then incubated with 0, 0.4 and 1.3 mM cisplatin, and ratio between the amount of in vivo 16S mitochondrial ribosomal RNA isolated from rat liver mitochondria of animals treated in vivo without or with 10 mg/kg cisplatin for 24 hours (squares). Data represent % synthesis above the control (100). (B) Ethidium bromide staining after electrophoresis on methylmercury hydroxideagarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria of animals treated in vivo without (Control animal) or with 10 mg/kg cisplatin for 24 hours (Treated animal) and incubated in vitro with 0, 0.4 and 1.3 mM cisplatin. Each line shows nucleic acids isolated from an equivalent amount of mitochondria. A andB) Autoradiograms after electrophoresis on methylmercury hydroxide-agarose gels of newly synthesized mtRNAs by rat liver mitochondria isolated from animals treated in vivo without (Control animal) or with 10 mg/kg for 24 hours (In vivo cisplatin-treated animal), incubated in vitro without (-NEM) or with NEM 0.2 mM (+NEM) 15 min at room temperature and then incubated with 0, 0.4 and 1.3 mM cisplatin 1 hour at 37°C. Each lane shows mtRNAs synthesized by an equivalent amount of mitochondria. (C and D) Ethidium bromide staining after electrophoresis on methylmercury hydroxide-agarose gels of mtDNA and mtRNAs isolated from rat liver mitochondria of animals treated in vivo without (Control animal) or with 10 mg/kg cisplatin for 24 hours (in vivo cisplatin-treated animal), and incubated in vitro without (-NEM) or with NEM 0.2 mM (+NEM) 15 min at room temperature and then incubated with 0, 0.4 and 1.3 mM cisplatin for 1 hour at 37°C. Each lane shows nucleic acids isolated from an equivalent amount of mitochondria.


Helen M Cooper

Johannes N Spelbrink
email: hans.spelbrink@uta.fi

The human Sirt3 protein deacetylase is exclusively mitochondrial

Keywords: Sirtuin, deacetylase, mitochondrial, Leptomycin B, import, immunofluorescence HsSirt3, human Sirt3, HAT, histone acetyl transferase, HDAC, histone deacetylase, AceCS2, mitochondrial acetyl CoA synthetase (2), HEK293, human embryonal kidney 293, U2OS, U2 osteosarcoma, CRM1, chromosome region

It has recently been suggested that perhaps as many as 20% of all mitochondrial proteins are regulated through lysine acetylation while Sirt3 has been implicated as an important mitochondrial protein deacetylase. It is therefore of crucial importance that the mitochondrial localization of potential protein deacetylases is unambiguously established.

While mouse Sirt3 was recently shown to be mitochondrial, human Sirt3 (HsSirt3) was reported to be both nuclear and mitochondrial and to relocate from the nucleus to the mitochondrion upon cellular stress. We show here using various HsSirt3 expression constructs and a combination of immunofluorescence and careful subcellular fractionation that in contrast to the earlier report human Sirt3 is exclusively mitochondrial. We discuss possible experimental explanations for these discrepancies. In addition, we suggest on the basis of the analysis of public genome databases that the fulllength mouse Sirt3 protein is a 37 kDa mitochondrial precursor protein contrary to the previously suggested 29 kDa protein.

Introduction

An important aspect of the regulation of chromatin remodelling and consequently geneexpression involves post-translational modifications of histones, abundant nuclear DNAbinding proteins with regulatory and packaging functions [START_REF] Strahl | The language of covalent histone modifications[END_REF][START_REF] Marmorstein | Protein modules that manipulate histone tails for chromatin regulation[END_REF]. These modifications include phosphorylation, methylation and acetylation. Acetylation of lysine residues involves histone acetyl transferases (HATs) such as P300/CBP, while deacetylation involves histone deacetylases, or HDACs. HDACs can be divided in three distinct families based on their domain architecture, and originally based on similarity with yeast HDACs [START_REF] Marmorstein | Structure of histone deacetylases: insights into substrate recognition and catalysis[END_REF]. Class I and II HDACs share similar catalytic domains, whereas the Class III 'Sir' or Sirtuin family of deacetylases are catalytically distinct and depend on NAD+ as a cofactor. Although the names suggests HDACs are specific for histones, this is not the case. Indeed, many proteins have been identified as targets of HATs and HDACs, many of them transcription factors, but also for example the cytoplasmic protein tubulin, a major structural component of the microtubular network [START_REF] Kouzarides | Acetylation: a regulatory modification to rival phosphorylation?[END_REF]. In addition, in recent years acetylation/deacetylation has been show to play an important role in protein stability [START_REF] Freiman | Regulating the regulators: lysine modifications make their mark[END_REF].

The deacetylase Sirt3 as well as Sirt4 and 5 were recently identified as mitochondrial members of the Sir family of deacetylases [START_REF] Schwer | The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase[END_REF][START_REF] Onyango | SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria[END_REF][START_REF] Michishita | Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins[END_REF], which suggested the possibility of acetylated mitochondrial protein substrates. A recent proteomics approach to identify lysine-acetylated proteins indeed has suggested that as many as 20% of all mitochondrial proteins are regulated by lysine acetylation [START_REF] Kim | 1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T Substrate and Functional Diversity of Lysine Acetylation Revealed by a Proteomics Survey[END_REF]. The first bona fide human Sirt3 (HsSirt3) substrate to be identified was the mitochondrial acetyl CoA synthetase or AceCS2 [START_REF] Schwer | Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2[END_REF][START_REF] Hallows | Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases[END_REF] and further work has now shown that Sirt3 in mouse is a global mitochondrial deacetylase involved in the regulation of steady-state acetylation levels of many proteins [START_REF] Lombard | Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation[END_REF]. Whereas Sirt3 is a deacetylase, Sirt4 has been show to be an ADPribosylase functioning in the regulation of Insulin secretion [START_REF] Haigis | SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells[END_REF][START_REF] Ahuja | Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase[END_REF]. The identification of Sirt3 and Sirt4 substrates and the involvement of Sirt1 in e.g. metabolic syndrome has suggested roles of several Sirt proteins in the regulation of whole organism metabolism in response to food availability [15][START_REF] Haigis | Mammalian sirtuins--emerging roles in physiology, aging, and calorie restriction[END_REF][START_REF] Lagouge | Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha[END_REF][START_REF] Gerhart-Hines | Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha[END_REF]. Finally, both Sirt3 and 4 but not Sirt 5 have been implicated in nutrient sensing via mitochondrial NAD + levels [START_REF] Yang | Nutrient-Sensitive Mitochondrial NAD(+) Levels Dictate Cell Survival[END_REF].

In order to understand the consequences of mitochondrial protein acetylation and deacetylation it is of crucial importance to unambiguously establish the mitochondrial localization of HDACs. The human Sirt3 has an additional 142 amino acids at its Nterminus. Whereas the mouse protein is exclusively mitochondrial [START_REF] Lombard | Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation[END_REF], it was recently suggested that the N-terminal extension of human Sirt3 mediates an initial localization to the nucleus [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF] and mitochondrial relocation occurs upon stress, induced by its own overexpression, by UV damage or etoposide. A second family II HDAC, HDCA7 has been shown to be localized in the inner mitochondrial membrane and nucleus and relocate to the cytoplasm upon apoptosis inducing stress [START_REF] Bakin | Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes[END_REF]. In this paper we re-address the issue of HsSirt3 nuclear localization by using similar approaches [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF] and show contrary to the original publication that human Sirt3 is exclusively mitochondrial similar to the mouse protein.

Materials and Methods

Reagents and antibodies

Cloning of expression constructs

Full length HsSirt3 was originally obtained by RT-PCR using highly purified HeLa

Stable inducible cell-lines, transient transfections and siRNA transfections

Stable inducible cell lines expressing HsSirt3-FLAG and POLG2-HA were generated and maintained as previously described [22]. To induce expression the indicated amount of doxycycline (Sigma) was added to the growth medium from an ethanol stock, and cells were processed for further analyses following the required induction. U2OS and HeLa cells were maintained in DMEM medium (Cambrex Bioscience) with 2 mM L-glutamine (Cambrex Bioscience) and 10% FCS (Euroclone). For immunofluorescence cells were transfected using TransIT ® -LT1 transfection reagent (Mirus, Madison, USA) according to the manufacturer's instructions and 1 µg plasmid per 6-well plate well. Leptomycin B was added as required at 5 ng/ml 2-3 hours prior to processing for immunofluorescence or Western blot analysis. For the RNAi, cells were transfected with a total of 420 pmol of siRNAs/10 cm plate, either a combination of 3 HsSirt3 specific siRNAs or a single nontargeting siRNA, using Lipofectamine 2000 reagent at 30 µl/420 pmol of RNA according to the manufacturers' instructions. Cells were grown for 48hrs after transfection.

Immunofluorescence

For immunofluorescent detection cells were grown on coverslips in 6 well plates.

Following transfection for 1-2 days and Leptomycin B treatment cells were fixed using Red CMXRos treatment was done prior to fixation essentially as described [24]. Slides were mounted using ProLong ® Gold antifade with DAPI (Invitrogen). Image acquisition using confocal microscopy was carried out as described [START_REF] Garrido | Composition and dynamics of human mitochondrial nucleoids[END_REF], using an Andor iXon DV885 EMCCD camera and the Andor iQ software (Andor, Belfast N. Ireland). Images were further processed using Photoshop CS2. xg for 30 min. Alternatively, the low-speed pellet obtained from hypotonic lysis was further purified on a 25%/30%/35% Optiprep gradient to obtain purer nuclear fractions free of whole cell and mitochondrial contaminations according the manufacturers protocol, and then extracted by high salt as above. Western blot analysis by Enhanced

Subcellular fractionation and Western blot analysis

Chemiluminescence was done essentially as described [START_REF] Spelbrink | In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells[END_REF]. Western blot analysis used Prestained Broad-Range markers from Biorad. Peroxidase-coupled secondary anti-mouse or rabbit was obtained from Vector Laboratories. In some instances the Supersignal ® West Femto Maximum kit (Pierce) was used for detection according to the manufacturer's protocol. Detection and quantification used a Biorad Chemi Doc XRS system.

Results and Discussion

As Unfortunately commercial antibodies against HsSirt3 worked very poorly in immunofluorescence unless the protein was overexpressed, suggesting that endogenous HsSirt3 expression levels were relatively low in the cells examined. Finally, we transfected cells to express an HsSirt3 variant that lacked the first 142 amino acids previously suggested to be important for initial nuclear localization where it was suggested to be processed prior to mitochondrial re-localization [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF]. Our hypothesis was that if indeed the full-length protein is processed in the nucleus to yield a protein that is mitochondrial-import competent, deletion of the first 142 amino-acids should result in a protein that is mainly mitochondrial. In addition, it has been suggested that mouse Sirt3 starts at the equivalent of human methionine 143 [START_REF] Yang | Cloning and Characterization of Two Mouse Genes with Homology to the Yeast Sir2 Gene[END_REF][START_REF] Shi | SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes[END_REF], but is nevertheless a mitochondrial protein [START_REF] Lombard | Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation[END_REF] (see also below). The results (Fig 1D ) show however that the human variant lacking amino acids 1-142 is present in the cytoplasm and at high expression levels also in the nucleus. LMB treatment of these cells did not alter this distribution (not shown). Co-staining with Mitotracker® CMXRos did not show any evidence of mitochondrial targeting of truncated HsSirt3 (not shown).

To further corroborate the immunofluorescence experiments we relied on Western blot analysis and careful subcellular fractionation. Although the use of a hypotonic lysis method to obtain mitochondrial fractions from cultured cells is common practice, it is not appropriate to use the resulting nuclear pellet without further purification, as was done in the earlier demonstration of nuclear localization of HsSirt3 [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF]. The problem is that the nuclear fraction resulting from hypotonic lysis typically contains 20-30 % unbroken cells and has a considerable mitochondrial contamination that is not always clear in a subsequent high salt extraction because many mitochondrial contaminants, especially membrane proteins, will be in the unused pellet following centrifugation of the high salt extract. To illustrate this problem we repeated essentially the same procedure as previously published [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF], and furthermore tried to remove the nuclear mitochondrial contamination from the nuclear pellet by an additional NP40 detergent extraction which is a detergent commonly used to prepare crude nuclear fractions from whole cells. The Apart from cells overexpressing HsSirt3-FLAG we used a cell line engineered to express an HA tagged version of the accessory subunit of mitochondrial DNA polymerase gamma POLG2 (P2HA), which is a well established mitochondrial protein(see e.g. [START_REF] Kaguni | DNA polymerase gamma, the mitochondrial replicase[END_REF]).

Although with immunofluorescence this protein is clearly mitochondrial, the use of the crude isolation method shows also this protein to be present in all fraction including the high-salt 'nuclear' extract.

To obtain a clean nuclear fraction we subjected the low-speed nuclear pellet fractions of hypotonic lysates to a further purification step using an iodixanol gradient fractionation. Because nuclei are of considerably higher density than mitochondria and whole cells, this procedure yields a very pure nuclear fraction essentially free from mitochondrial contaminants. The resulting fraction was again subjected to a high salt extraction and the various fractions were subjected to Western blot analysis. Using inducible HEK293 FlpIn™ TREx™ cells this furthermore allowed us to use very short induction times similar to the immunofluorescence approach previously used [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF] which suggested that short induction showed apparent nuclear HsSirt3 localization. Both the tagged overexpressed HsSirt3 as well as the endogenous protein were detected with commercial Sirt3 antibodies. The results (Fig. 2B) unambiguously demonstrate that both HsSirt3-FLAG and endogenous HsSirt3 are mitochondrially localized with no evidence of protein residing in the nucleus. Similar results were also obtained with regular or for 1-1,5 hours showed the presence of the 44 kDa precursor HsSirt3-FLAG protein.

This was detected with the FLAG antibody but more importantly also with one of the commercial HsSirt3 antibodies showing this antibody is capable of detecting also the endogenous precursor protein. The precursor form of HsSirt3-FLAG was clearly detectable in the mitochondrial lysates but was not observed on any occasion in clean nuclear extracts. A potential precursor form of the endogenous protein was not observed on any occasion in the nuclear fraction. In fact, as most inducible systems, the cell culture system used in this study is slightly leaky resulting in steady-state HsSirt3-FLAG in noninduced cells that is comparable with endogenous HsSirt3 steady-state levels (see below).

Non-induced cells that continuously express HsSirt3-FLAG at very low levels did not show the HsSirt3-FLAG precursor form in their mitochondrial (or nuclear) extracts, but only upon a short 1-1,5 hour induction the precursor form became visible in the mitochondrial lysates but not in the nuclear lysates and without a visible increase in the processed form. These data show that the precursor is generally shortlived (not visible in the 'leaky' non-induced cells but visible after short induction) and mitochondrially targeted where it is further processed to yield the mature protein as previously suggested [START_REF] Schwer | The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase[END_REF].

To demonstrate that the band tentatively assigned to the endogenous HsSirt3 is indeed HsSirt3, we treated cells with HsSirt3 siRNA. Figure 2C shows that in cells overexpressing HsSirt3-FLAG, siRNA treatment for 48 hours had the desired effect of anti-acetyl lysine antibody, as previously described [START_REF] Schwer | Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2[END_REF] (not shown). Treatment of siRNA and subcellular fractionation followed by Western blot analysis shows that indeed the assigned endogenous mitochondrial HsSirt3 band is HsSirt3.

Combined the above results unequivocally demonstrate the mitochondrial localization of HsSirt3 and suggest that processing of the full length protein very likely takes place in the mitochondrion. There are various explanations for the discrepancy of our results with the previous results [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF]. First, as we have shown here, the subcellular fractionation previously used to obtain a nuclear fraction was flawed in that any crude nuclear fraction derived from hypotonic lysis will still contain both whole cells and mitochondria. Furthermore the use of an antibody marker to detect a mitochondrial membrane protein is not appropriate to exclude contamination from the nuclear high-salt extract. Second, the previous paper heavily relied on an antibody that was specific for the N-terminus of HsSirt3 but the specificity of that antibody for HsSirt3 was not adequately demonstrated and various appropriate immunofluorescence controls were lacking. For example, an HsSirt3 siRNA experiment to demonstrate a reduction or complete loss of the nuclear fluorescent signal assumed to be from the precursor form of HsSirt3 was not shown. Finally, some of the images that appear to suggest nuclear retention of signal upon LMB treatment could be explained by clustering of a mitochondrial signal around and on top of the nucleus.

One of the premises to suggest that human Sirt3 behaves differently has been the absence of an N-terminal extension in the predicted mouse Sirt3 protein [START_REF] Scher | SirT3 is a nuclear NAD+dependent histone deacetylase that translocates to the mitochondria upon cellular stress[END_REF][START_REF] Yang | Cloning and Characterization of Two Mouse Genes with Homology to the Yeast Sir2 Gene[END_REF][START_REF] Shi | SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes[END_REF], i.e. and its localization [START_REF] Yang | Cloning and Characterization of Two Mouse Genes with Homology to the Yeast Sir2 Gene[END_REF][START_REF] Shi | SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes[END_REF]. In addition both the human and mouse proteins when starting at M143 have a very poor mitochondrial targeting prediction. For these reasons we searched the mouse genome initially using the mouse cDNA sequence coding for the 29 kDa Sirt3. This analysis using the 'ab initio RNA' database yielded several predicted mRNAs from the Celera mouse genome (sequence provided in suppl Figure 1) that when translated would give a larger protein more compatible with the full length human protein. Using this prediction we looked for confirmation in the EST databases by using the 5' end of the cDNA upto and including the equivalent of the human M143 codon.

This confirmed that the predicted mRNAs that would code for a larger mouse Sirt3 protein do exist in vivo (as also indicated in Suppl Figure 1). It should be noted here that the larger protein variant was originally also predicted by Yang et al. [START_REF] Yang | Cloning and Characterization of Two Mouse Genes with Homology to the Yeast Sir2 Gene[END_REF] but was discarded on the basis of it being rare and on a poor alignment with the predicted fulllength human protein. For reasons unknown, this alignment did not take into consideration the possibility of large gaps. In stead it was proposed that the major Sirt3 mRNA in mouse would be created by an 8 nucleotide extension to the mRNA derived from the 5' end of exon 2. ESTs that agree with this can indeed be found in abundance, When we searched for protein homologues using our prediction for the mouse full length protein the best hits were with various predicted mammalian Sirt3 proteins such as those from horse, pig, and cow. Sequence alignment of these proteins showed an excellent alignment (Fig 3), with all proteins having a predicted N-terminal stretch of 75-77 amino-acids prior to the equivalent of human M143. In addition, all proteins had an excellent prediction for mitochondrial localization. Of the various proteins in the alignment the mouse protein is the only one to have a second methionine 14 amino acids downstream of the first methionine that we assign here as M1. Further experiments are required to test whether M1 or the downstream methionine (M15) is preferentially used.

In conclusion, based on experimental data we show here the unambiguous and exclusive localization of human Sirt3 in mitochondria. In addition, based on the reanalysis of the mouse Sirt3 gene and transcripts we suggest that mouse Sirt3 has an Nterminal mitochondrial targeting sequence similar to that predicted for other mammals, but shorter than the human protein. This is supported by the observation that a truncated human protein that starts at the previously predicted mouse N-terminus is not mitochondrially targeted despite a high degree of similarity with the mouse protein from this residue onwards. Nevertheless, the endogenous mouse protein has been demonstrated

unequivocally to be mitochondrial [START_REF] Lombard | Mammalian Sir2 Homolog SIRT3 Regulates Global Mitochondrial Lysine Acetylation[END_REF]. Our results greatly simplify various ambiguous published results relating to the function of the Sirt3 protein and should help in clarifying its potential roles in metabolism. First, based on our data there is no support for a nuclear function of human Sirt3. Second, the effects of mouse Sirt3 overexpression on for nuclear/cytoplasmic localization of HDAC7 and thus also seem to contradict this previous report. In addition, when we expressed in various cell lines an HDAC7-FLAG variant of essentially the same sequence as reported [START_REF] Bakin | Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes[END_REF] we see the same nuclear/cytoplasmic pattern as observed with the endogenous protein and the same LMB responsiveness. Nevertheless, since in our opinion it is difficult to fully exclude a mitochondrial localization of at least a minor fraction of HDAC7 we are hesitant to fully exclude a mitochondrial role for this protein.

Biochemical

  Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T

  mRNA, and cloned in pcDNA3.1(-) /Myc-His A (Invitrogen) using Not I and BamHI. Truncated (Δ1-142) HsSirt3-Myc-His and HsSirt3-FLAG were obtained by PCR amplification from the original full-length construct and cloned either in pcDNA3.1(-) Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T /Myc-His A or pcDNA5/FRT/TO (for FLAG) (Invitrogen). The FLAG epitope was introduced at the C-terminus by PCR and followed immediately by a stop-codon (all primer sequences can be obtained upon request). All constructs were fully verified by sequencing.

Biochemical

  Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T either 3.3% paraformaldehyde (PFA) in cell culture medium for 25 min or in methanol for 5 min at -20ºC [23]. This was followed by three washes in PBS and lysis for 10 min with 0.5% Triton X100 in PBS/10% fetal calf serum (FCS) after PFA fixation. No lysis step was performed after methanol fixation. Primary and secondary antibodies were incubated at recommended concentrations in PBS/10%FCS for 1 hr-o/n. Mitotracker ®

  Cells were isolated by centrifugation and washed once with ice cold PBS. For hypotonic lysis the cell pellet was resuspended by gentle pipetting in 2-3 volumes of ice-cold homogenization buffer (4 mM Tris-HCl, pH7.8, 2.5 mM NaCl, 0.5 mM MgCl2, 0.1 mM PMSF), kept on ice for 6 min, then homogenized in a glass homogenizer with 20-25 strokes of a tight-fitting pestle. Disruption of the cells was monitored by microscopy. One-ninth volume of 2M sucrose, 10 mMTris-HCl, 1 mM EDTA pH 7.8 was added following lysis and nuclei and cell debris were pelleted by centrifugation at 1,200xg max for 3 min at 4 °C. Mitochondria from the post-nuclear supernatants were recovered by centrifugation at 12,000xg max for 3 min at 4 °C. Mitochondrial pellets were washed once with 1 ml of ice-cold sucrose wash solution (0.25 M Sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH, pH 7.8) and the mitochondrial pellet lysed for 15 min on Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T ice in 50 mM TrisHCl pH7.5, 300 mM NaCl, 1 mMEDTA, 1% Triton X100. An equal volume of 2x Laemmli sample buffer was added and the sample was denatured at 95°C for 5 min prior to SDS-PAGE. Nuclear pellets following hypotonic lysis were either extracted first by 0.5% NP40 in 50 mMTrisHCl pH7.5, 150 mM NaCl, again pelleted and extracted with 20 mM Tris-HCl at pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1 mM PMSF, 0.5 mM DTT [26] for 20 min and centrifuged at 20.000

  others we observe a clear mitochondrial localization of HsSirt3 upon transient or stable overexpression in various human cell lines, including HeLa, U2OS, and HEK293 FlpIn™ TREx™ a stable inducible cell line engineered to express HsSirt3-FLAG. It has been suggested that overexpression per se is enough to induce HsSirt3 translocation from Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T the nucleus to the mitochondria [20] but that preventing nuclear export by inhibition of CRM1 by leptomycin B would retain the protein in the nucleus. We chose to similarly test leptomycin B (LMB) in combination with immunofluorescence in U2OS cells following transient transfection of various HsSirt3 expression plasmids because these cells spread well and have a large surface area of their cytoplasm. In addition, an advantage of using transient transfection is that a large variation can be observed in expression levels with cells expressing the recombinant protein at high levels but also cells in which the protein is expressed at levels just above the detection limit. Thus cells not treated with LMB showed a clear mitochondrial localization of HsSirt3 irrespective of the level of expression or the epitope tag, being either the FLAG or myc epitope (Fig1, panels A1-3, and not shown). Treatment of these cells with LMB did not show any evidence of a nuclear fluorescent signal suggestive of nuclear retention of HsSirt3 (Fig1, panels B1 and B2). We used a commercial antibody against HDAC7 in co-staining to demonstrate the efficacy of the LMB treatment showing a clear redistribution of HDAC7 from mostly cytoplasmic to mostly nuclear, as previously observed [28]. Similar results were observed using HeLa cells although results were sometimes more difficult to interpret due to clustering of mitochondria in the nuclear periphery (Fig1, panel C).

  Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T

  Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T results in Figure 2A show that it is essentially impossible to avoid a mitochondrial contamination of a crude nuclear fractionation, even following an additional NP40 step.

HEK293 cells not engineered

  to overexpress any protein (not shown). Enrichment of typical nuclear proteins in the iodixanol nuclear fraction was demonstrated by reprobing Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T membranes for HDAC1 (Fig. 2B) and e.g. nucleoporin (not shown) showing that the high salt extraction had worked. Neither the short induction nor LMB treatment (not shown) had any effect on the above results. Strong overexpression for a longer period (2-3 days)

Biochemical

  Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T knocking down HsSirt3-FLAG expression, albeit modestly. Furthermore, the same treatment in regular HEK293 cells increased acetylation of AceCS2 as detected by an

Biochemical

  Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T the mouse protein has been predicted to start at the methionine equivalent to human M143. Since the human and mouse protein are very highly conserved from M143 onward and we have not seen any evidence of mitochondrial localization of a truncated human variant starting at M143 (Fig 1D) we questioned the original mouse protein prediction

  but curiously the intron-exon boundary resulting from the 8 nucleotide extension of exon 2 of mouse Sirt3 is less a consensus boundary than the one without this extension. Since the Sirt3 genomic region is gene dense we suggest that the abundant ESTs with the 8 Biochemical Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T nucleotide extension result from (pre)-mRNA of the gene adjacent to mouse Sirt3, coding for a 26S proteasomal subunit and transcribed in the opposite orientation.

Biochemical

  Journal Immediate Publication. Published on 23 Jan 2008 as manuscript BJ20071624 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071624 S t a g e 2 ( a ) P O S T -P R I N T example PGC1α in brown adipocytes [30] is likely an artefact caused by the overexpression of a truncated mouse Sirt3 that is targeted not to mitochondria but to the cytoplasm. Although Shi et al. did show apparent co-localization with the mitochondrial dye Mitotracker® this appears to be an experimental artefact, possibly caused by severe overexpression, since the Mitotracker® staining does not actually show a typical mitochondrial network as normally seen with mouse 3T3 cells that were used in that study. Finally, as an additional cautionary note, the HDAC7 antibody used in this study was identical to the antibody used by Bakin et al [21], who suggested a mitochondrial localization of HDAC7. Our immunofluorescence results in U2OS cells (Fig 1, panels B1-2) and similar results obtained using C4-2 cells (not shown) showed

BiochemicalFigure 1 :Figure 2 :

 12 Figure 1: Overexpressed HsSirt3 is mitochondrial irrespective of expression level or

Figure 3 : 2 .

 32 Figure 3: The predicted mouse Sirt3 is highly similar to the predicted proteins from

Fig 3 .

 3 Fig 3. Cooper & Spelbrink

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