From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only. 2008 111: 1690-1699 Prepublished online November 1, 2007; doi:10.1182/blood-2007-07-102335Redistribution of accumulated cell iron: a modality of chelation withtherapeutic implicationsYang-Sung Sohn, William Breuer, Arnold Munnich and Z. Ioav CabantchikUpdated information and services can be found at:http://bloodjournal.hematologylibrary.org/content/111/3/1690.full.htmlArticles on similar topics can be found in the following Blood collections Red Cells (1174 articles)Information about reproducing this article in parts or in its entirety may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about ordering reprints may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about subscriptions and ASH membership may be found online at:http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlBlood (print ISSN 0006-4971, online ISSN 1528-0020), is published weeklyby the American Society of Hematology, 2021 L St, NW, Suite 900,Washington DC 20036.Copyright 2011 by The American Society of Hematology; all rights reserved.
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1691Table 1. Iron binding parameters of agents used in this study tion onto 96-well plates, or onto microscopic slides attached to perforatedIron ligands (abbreviations) pFe(III)* tissue culture 3-cm plates. Probe loading into cells. For cytosolic loading of CALG, cells inCalcein green (CALG) 20.3† DMEM plus 10mM Na-HEPES (DMEM-HEPES) at 37°C were exposedDeferrioxamine (DFO) 26.6 for 10 minutes to 0.25 M CALG-AM, washed and incubated inDeferiprone (DFP) 19.3 DMEM-HEPES containing 0.5 mM probenecid (to minimize probe leak-Diethylene-triamine-pentaacetic acid (DTPA) 28.2 age). For CALG-Fe(III) (1:1) complexes and for sulforhodamine loadingNitrilotriacetic acid (NTA) 18.1 into endosomes, cells were exposed to 50 M to 100 M complex inTransferrin (Tf) 22.3 DMEM-HEPES for 30 minutes at 37°C and washed extensively with theRhodamine B- (1, 10-phenanthrolin-5-yl) 7.7 (11.5 for Fe(II))‡ same probe-free medium and subsequently with HEPES-buffered saline aminocarbonyl benzyl ester (RPA) (HBS; 20 mM HEPES, 135 mM NaCl; pH 7.4).20,21 RPA was loaded into *pFe(III) values are log M , where M is the concentration of the free metal ion mitochondria as described in earlier studies.21-23when ligand 10 M and Fe(III) 1 M at pH 7.4. Data from Harris.17 Histone-CALG. For loading into the nucleus, CALG was coupled to †Data from Thomas et al.18 core histones from calf thymus (H) 7.2 mg/mL, in 50 mM Na-MES, ‡Values shown are for 1,10-phenanthroline. 100 mM NaCl, pH 5.5, containing 1.25 mM CALG:Cobalt (Co protects CALG metal-binding groups). Coupling was initiated by adding 12 mM EDC, followed by incubation for 1 hour at room temperature and overnightfound to reduce iron accumulation in the dentate nuclei of at 5°C. After exhaustive dialysis, 11 mM DTPA was added (to removeFriedreich ataxia patients,13 indicating its ability to cross the Co(II)) and histone-CALG (H-CALG) was further dialyzed against HBS.blood-brain barrier in humans, which is consistent with previous CALG coupling to H was estimated at 1.1:1 by ﬂuorescence ( excitationﬁndings in animals.14-16 480 nm, emission 520 nm). H-CALG-Fe was prepared by titrating CALG In the present study we assess DFP’s capacity to act as an with FAS. Loading 10 M H-CALG or H-CALG-Fe into cells was done iniron-relocating agent at the cellular level. To trace labile iron DMEM-HEPES containing 100 M chloroquine (for improved FMSmobilized by chelators, we used model cardiac and macrophage targeting to cell nuclei),24 followed by HBS washes.cells in culture and ﬂuorescent metal sensors (FMS) targeted to Fluorescein-DFO-histone. The probe was prepared by coupling ofcellular and extracellular compartments. We show that DFP can N-(ﬂuorescein-5-thiocarbamoyl)-desferrioxamine (Evrogen, Moscow, Rus- sia) to H using ﬂuorescein-DFO (FlDFO)–Fe (1:1) complex generated byrelocate iron accumulated in cell compartments within and across adding 1.1 mM FeSO4 to 1 mM FlDFO in HBS. To 2.64 mL of FlDFO-Fethe cell, and we demonstrate that iron withdrawn from sites of cell complex was added 8.5 mg H, followed by 0.18 mL of 1 M MES (Na form),accumulation can be safely transferred to extracellular transferrin pH 6.5, and 15 mg of EDC. After overnight agitation at 5°C, the mixturefor physiologic reuse. was dialyzed (3.5 kDa cut-off tubing) against 10 mM acetic acid; 1 mM EDTA; 150 mM NaCl, pH 4.5 (to remove bound iron); and ﬁnally HBS. The ﬂuorescein-DFO-histone (H-FlDFO) ratio was 1:1, based on stoichiometric iron-quenching of H-FlDFO ﬂuorescence ( excitationMethods 494 nm, emission 520nm). H-FlDFO, like H-CALG, was loaded into cells in DMEM-HEPES.Materials Rhodamine-labeled apotransferrin. Human apotransferrin (4 mg/mLCalcein green (CALG) 3,3 -bis[N,N-bis(Carboxymethyl) aminomethyl]ﬂuo- in 25 mM Na2CO3, 75 mM NaHCO3; pH 9.8), was incubated at 5°Crescein and its acetomethoxy (AM) precursors CALG-AM, lissamine overnight with 1 mM lissamine rhodamine sulfonyl chloride and the labeledrhodamine sulfonyl chloride, and sulforhodamine were from Molecular protein isolated by gel ﬁltration on Sepharose G25 (Sigma-Aldrich)Probes (Eugene, OR). Human serum holotransferrins and apotransferrins preequilibrated with 150 mM NaCl, 20 mM MES; pH 5.3.(aTf) were from Kamada (Rehovot, Israel). Diethylene-triamine- Transfer of iron from DFP-Fe to apotransferrin. DFP-Fe complexespentaacetic acid (DTPA), nitrilotriacetate (NTA), EDC (1-ethyl-3-(3- were generated by incubating Fe-NTA (50:150 M) with either 150 M ordimethylaminopropyl) carbodiimide), ferric ammonium citrate (FAC), 250 M DFP in HBS for 20 minutes, and completion of complex formationferrous ammonium sulfate (FAS), succinyl acetone, HEPES (N-2- was assessed by absorption at 455 nm. The complexes were mixed withhydroxyethylpiperazine-N -2-ethanesulfonic acid), 4-morpholine ethanesul- 50 M aTf and 25 mM NaHCO3 and incubated for 1.5 hours in a 5% CO2fonic acid (MES), core histone mixture from calf thymus (H), N,N - incubator. Because of the large spectral overlap of DFP-Fe and transferrin-hexamethylene-bis-acetamide, and octyl-glucoside were from Sigma- Fe, we added DTPA (10 mM) to selectively scavenge iron from DFP-Fe.Aldrich (St Louis, MO). Rhodamine isothiocyanate was from Fluka (Buchs, Transferrin that was free of low-molecular components was obtained bySwitzerland). DFP (1,2-dimethyl-3-hydroxypyridin-4-one) was from Apo- either ﬁltering 0.5 mL through 30-kDa cut-off spin ﬁlters (Pall, East Hills,Pharma (Toronto, ON). DFO was from Novartis (Basel, Switzerland). The NY) via centrifugation (2900g for 20 minutes), or by use of a 5-mLred ﬂuorescent mitochondrial metal–sensor rhodamine B-[(1, 10-phenanth- dry-spin–gel ﬁltration-centrifugation at 1100g over precentrifuged Seph-rolin-5-yl aminocarbonyl] benzyl ester (RPA), was a kind gift from U. adex G-50 medium columns (Sigma-Aldrich). The complex-free transferrinRauen and R. Sustmann, University of Duisburg-Essen, Essen, Germany. was diluted in HBS, pH 7.4. Tf-Fe was analyzed by absorbance at 465 nm,A summary of the probes used and their properties is given in Table 1. and aTf by tryptophan ﬂuorescence (280 nm excitation, 306 nm emission; Felix spectroﬂuorimeter station version 2.5; Photon Technology Interna- tional, Lawrenceville, NJ).Methods Iron-loading of cells was done by overnight incubation of cells withComplexes of iron with NTA (Fe-NTA) were generated by mixing FAS and 100 M FAC in culture conditions followed by washing with HBSNTA (1:3 molar ratio) in water and allowing iron to oxidize in ambient containing 100 M DFO, and with HBS alone. To achieve selectiveconditions. The DFP-Fe(III) complexes were generated by mixing Fe-NTA accumulation of iron in mitochondria, cells were treated with 1 mMwith DFP in water; formation of fully substituted DFP-Fe complexes was succinyl acetone for 3 hours at 37°C.conﬁrmed by measuring absorbance at 455 nm.19 Cell hemoglobin synthesis. MEL cells cultured for 2 days in media Cell-culture lines. Rat H9C2 cardiomyocytes, J774 mouse macro- supplemented with 5mM hexamethylene-bis-acetamide were washed andphages, and erythroleukemia (MEL) were grown in 5% CO2 Dulbecco- lysed with octyl-glucoside (1.5%). After 2 minutes centrifugation atmodiﬁed Eagle medium (DMEM) supplemented with 10% fetal calf serum, 12 000g, 100 L of lysate supernatants was transferred to a 96-well4.5 g/L D-glucose, glutamine, and antibiotics (Biological Industries, microplate for estimating hemoglobin (by measuring absorbance atKibbutz Bet Haemek, Israel). Cells were plated 1 day before experimenta- 410 nm). Alternatively, lysates were mixed with 100 L freshly prepared
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.1692 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 Figure 1. Transfer of iron from extracellular DFP-iron complexes to mitochondria. H9C2 cells loaded with the mitochondrial iron-sensor RPA were incubated with or without DFP-Fe (15:5 M) and epiﬂuorescence micro- scopic images were recorded every 5 minutes under settings for rhodamine. Representative ﬁelds of initial cell ﬂuorescence at time 0 (A,C) and after incubation for 1 hour in the presence (B) or absence (D) of 5 M DFP-Fe complex. Magniﬁcation was 600; oil objective was a (Plan Apo) 60 /1.40 NA. (E) Mean ﬂuorescence values in relative units (r.u.) plus or minus SD of 5 cells per ﬁeld calculated for each time-point image and normalized to the initial ﬂuorescence, representing cells incubated without (None) and with 5 M Fe-complex (DFP-Fe or NTA-Fe); the lines denoted as DFP-Fe and NTA-Fe were cor- rected for spontaneous decay given by the control (None). Arrow indicates time the Fe complex was added. (F) Effect of various iron chelates on the ﬂuorescence (f normalized to initial value, f0, in r.u.) of 0.5 M RPA in solution (HBS buffer). The iron chelates all contained 5 M Fe complexed to NTA (1:3 ratio) in the absence (f) and presence ( ) of 100 M ascorbate (Fe:NTA, Fe:NTA ASC) or to DFP, with 100 M ascorbate, at DFP:Fe ratios of 3:1 (F), 5:1 (E), and 7:1 (‚). The values of ﬂuorescence intensity are means of triplicate samples run in parallel, plus or minus SEM.tetramethylbenzidine (0.5 mg/mL in 10% acetic acid) and ﬁnally 8 L of endogenous labile iron and/or to probe photobleaching. On the30% H2O2. Absorption (604 nm) was read after 90 seconds. other hand, addition of preformed DFP-Fe(III) complexes (DFP- Data acquisition and analysis. Epiﬂuorescence imaging (Nikon TE Fe, ratio 3:1) evoked a time-dependent (t ⁄ 15 minutes) quenching 122000 microscope; Melville, NY; and Orca-Era CCD camera; Hamamatsu, of mitochondrial RPA ﬂuorescence (Figure 1E). Consistent withBridgewater, NJ) coupled to a Volocity 4 system (Improvision, Coventry,United Kingdom) was used for image data acquisition and analysis.20 For RPA’s selectivity for Fe(II), no quenching of RPA by Fe-NTAhigh-throughput ﬂuorescence monitoring we used ﬂuorescence plate read- occurred in solution unless ascorbate was added. Transfer of ironing (Tecan-Saﬁre; Neotec, Mannedorf, Austria) essentially as described.20 ¨ from DFP-Fe complexes to RPA in the presence of ascorbate was detected over a wide range of DFP to Fe ratios (1:1 to 7:1). Transfer of iron from extracellular DFP-Fe complexes toResults nuclei. With the view of targeting a ﬂuorescent iron sensor into the cell nucleus, we constructed a conjugate of H with theTo follow DFP-mediated translocation of iron from the medium to high-afﬁnity iron chelator and iron sensor H-FlDFO.11 To minimizecells, within cells, and from cells to the medium, we used uptake via the endocytic pathway, incubation was carried out atﬂuorescent acceptors and donors of iron that undergo quenching or room temperature and in the presence of chloroquine, as describeddequenching upon metal binding or removal. An additional feature in “Histone-CALG.” H-FlDFO within nuclei was rapidlyof these probes was their localization in speciﬁc cell compartments (t ⁄ 12 5 minutes) quenched by DFP-Fe (3:1) added to the extracel-or in the extracellular medium. lular medium (Figure 2E), consistent with the high cell permeabil- ity of DFP-Fe complexes shown in Figure 1. In solution, H-FlDFOTransfer of iron from extracellular DFP-iron complexes to is efﬁciently and rapidly (t ⁄12 1 minute) quenched by DFP-Femitochondria complexes even at a 9:1 DFP:Fe ratio (Figure 2F), as expected fromIngress of ionic iron from the extracellular medium to the DFP’s capacity to shuttle iron to DFO both in vivo and in vitro.11mitochondria of H9C2 cardiomyocytes was followed with the DFP-mediated redistribution of iron between cellular compart-high-afﬁnity Fe(II)–acceptor probe, RPA22,23 (Figure 1). The hydro- ments: from nuclei to mitochondria. DFP’s facilitation of ironphobic cationic rhodamine B is responsible for RPA’s potentiomet- movement between discrete cell compartments was followed inric partitioning in the mitochondria, and phenanthroline is respon- H9C2 cells sequentially labeled in cell nuclei with the iron-donorsible for detection of Fe(II).22 Exposure of cells to Fe(III)-NTA probe H-CALG-Fe(III) and in mitochondria with the Fe(II)-evoked no signiﬁcant changes in RPA ﬂuorescence relative to the acceptor probe RPA. H-CALG-Fe penetrates the plasma membranespontaneous decrease (t ⁄ 60 minutes), probably due to binding of 12 and accumulates initially in the cytosol and subsequently in the
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1693Figure 2. Transfer of iron from extracellular DFP-iron complexes to nuclei.H9C2 cells loaded with the nuclear iron sensor H-FlDFO were incubated with or Figure 3. DFP-mediated relocation of iron between cellular compartments:without 5 M DFP-Fe complex (DFP:Fe ratio 3:1) and epiﬂuorescent microscopic from nuclei to mitochondria. H9C2 cells were loaded with the nuclear iron sensorimages were recorded every 5 minutes under settings for ﬂuorescein. Representative H-CALG that had been precomplexed to iron (H-CALG-Fe), followed by loading withﬁelds of initial cell ﬂuorescence at time 0 (A,C) and after incubation for 1 hour in the the mitochondrial iron sensor RPA. The double-labeled cells were then exposed toabsence (B) or presence (D) of 5 M DFP-Fe complex. (E) Mean ﬂuorescence values 50 M DFP and epiﬂuorescence images were recorded every 5 minutes. Representa-in r.u. plus or minus SD of 5 cells per ﬁeld, calculated for each time-point image and tive ﬁelds of cell ﬂuorescence observed under settings for ﬂuorescein (A,B) andnormalized to the initial ﬂuorescence (f/f0), representing cells incubated without rhodamine (C,D). Images are shown at time 0 (A,C) and after incubation for 1 hour in(None) and with 5 M DFP-Fe complex (DFP-Fe). Arrow indicates time the Fe the presence of 50 M DFP (B,D). (E) Mean ﬂuorescence values plus or minus SD incomplex was added. (F) Effect of DFP-Fe chelates (DFP:Fe at ratios of 1:1, 3:1, 5:1, r.u. of 5 cells per ﬁeld, calculated for each time-point image and normalized to the9:1) on the ﬂuorescence (normalized to initial value, f0, in r.u.) of 0.5 M H-FlDFO in initial ﬂuorescence (f/f0), representing cells incubated with 50 M DFP (circles) andsolution (HBS buffer). The values of ﬂuorescence intensity are means of triplicate with no addition (squares). Fluorescence of H-CALG is indicated by ﬁlled symbolssamples run in parallel, plus or minus SEM. (F and f), and ﬂuorescence of RPA is indicated by open symbols (E and ). The scheme illustrates entry of DFP into the cytosol (C), nuclei (N) containing H-CALG-Fe (iron donor) and mitochondria (M) containing RPA (iron acceptor), and transfer of iron from nuclei to mitochondria. E refers to endosomes.nucleus, particularly in nucleolus-like sites.21 H-CALG-Fe wasused subsaturated with iron so that its accumulation in the nucleicould be followed via changes in the residual ﬂuorescence of the CALG-Fe into endosome/pinosomes and its accessibility to DFP, asunquenched fraction of the probe (revealed by chelation; Figure evidenced by dequenching, in a previous study.20 Moreover, CALG-Fe3A). Loading of RPA into mitochondria was apparent from the coendocytosed with the classical ﬂuid phase markers sulforhodaminecharacteristic perinuclear staining pattern (Figure 3C), from colo- and rhodaminated dextran, both of which showed less than 65% cellcalization with other potentiometric probes and sensitivity to colocalization as obtained with the Volocity program (not shown).protonophores.22,23 Addition of 50 M DFP produced an increase inthe ﬂuorescence of H-CALG-Fe in the nuclei and a concomitant H-FlDFO was detectable in the nuclear area, while CALG-Fe wasdecrease in the ﬂuorescence of RPA in the mitochondria, both of concentrated in punctuate, perinuclear spots, attributable to endosome-which occurred within 5 to 8 minutes and were complete within trapped CALG-Fe (Figure 4). However, after incubation with 50 M20 minutes (Figure 3E). The changes are attributable to dequench- DFP, the ﬂuorescence in the endosomes increased signiﬁcantly, whileing of H-CALG-Fe due to removal of iron by DFP and to that in the nuclei decreased relative to the respective untreated controls.quenching of RPA due to transfer of iron from DFP-Fe. Only minor The kinetics of the changes in the 2 compartments followed similarchanges in ﬂuorescence were observed in untreated control cells. It patterns, although in opposite directions. To ascertain whether the ironis likely that nuclear H-CALG-Fe was not the only source of iron acquired by DFP was exclusively intracellular, the cells were washeddelivered to RPA by DFP, because the addition of DFP to with 0.1 mM DFO to remove all extracellular iron before the addition ofRPA-loaded cells also led to quenching of mitochondrial probe DFP. Furthermore, as the addition of DFP to cells loaded with H-FlDFO(data not shown), although to a much lesser extent than in the alone failed to produce a decrease in its ﬂuorescence (data not shown),presence of H-CALG-Fe. the quenching of H-FlDFO in cells coloaded with CALG-Fe is most DFP-mediated relocation of iron between cellular compart- likely due to DFP-mediated mobilization of iron from endosomes.ments: from endosomes to nuclei. H9C2 cells were labeled However, the possibility that in the course of the experiment somesequentially in the nuclei with the iron-acceptor probe H-FlDFO and in iron might have spontaneously leaked from endosomes to cytosolthe endosomes with the iron-donor probe CALG-Fe (Figure 4). We and rendered it transferable to mitochondria by added DFP cannotshowed the bulk-phase pinocytic uptake of the iron-quenched probe be ignored.
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.1694 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 Figure 4. DFP-mediated relocation of iron between cellular compartments: from endosomes to nuclei. H9C2 cells were loaded with the nuclear iron-sensor H-FlDFO and subsequently exposed to preformed complexes of CALG and iron (CALG-Fe, 1:1 ratio) that were taken up by adsorptive pinocytosis into endo- somes. The cells containing both labels were then exposed to 50 M DFP and epiﬂuorescent images were recorded every 5 minutes under ﬂuorescein set- tings. Representative ﬁelds of cell ﬂuorescence are shown at time 0 (A) and after incubation for 20 minutes (B) in the presence of 50 M DFP. (C) The nuclei (N) and endosomes (E) are indicated by arrows. (D) Mean ﬂuorescence values in r.u. within selected subcellular areas corresponding to endosomes (Di) and nuclei (Dii), using 5 cells per ﬁeld, calculated for each time-point image and normalized to the initial ﬂuorescence (f/f0). Transfer of iron from DFP-Fe complexes to transferrin. We The rationale was that intracellular iron mobilized to the extracellu-followed iron complexation by transferrin using DFP-Fe com- lar medium by DFP would be transferred to the ﬂuorescent Tf,plexes (3:1 and 5:1, 50 M in Fe) and NTA-Fe complexes (3:1, which, upon binding iron, would then bind to cell-surface trans-50 M in Fe) as iron sources and apotransferrin (50 M) as ferrin, and after receptor-mediated endocytosis, would becomeacceptor. To remove from the reaction mixture any traces of iron concentrated in endosomes. To increase the level of mitochondrialassociated with low molecular weight complexes or free ligands, labile iron, the cells were pretreated with the heme-synthesiswe added DTPA after 1 hour of reaction and followed it by size inhibitor succinylacetone, which causes selective accumulation ofﬁltration. Filtration and differential chelation allowed the assess- unused, chelatable iron in the mitochondria.22 After 1 hour ofment of DFP-Fe transfer of iron to aTf both by light absorption of incubation with DFP, the level of cell-bound ﬂuorescent aTf roseTf-Fe complexes at 465 nm (Figure 5) and by tryptophan ﬂuores- by 2.3-fold over controls without DFP (Figure 6A,B). The bindingcence of aTf that is quenched after speciﬁc binding of iron25 was speciﬁc and iron dependent, as it was fully blocked by excess(Figure 5). While the 2 analytical methods monitored binding of holotransferrin (Figure 6C,D) and enhanced by addition of saturat-iron to transferrin, the optical changes are mirror images of each ing concentrations of Fe-NTA (Figure 6E,F), both in the presenceother. Transfer of iron from DFP-Fe to transferrin ensued at both and in the absence of DFP. In control cells not treated with3:1 and 5:1 stoichiometries of DFP:Fe and was of a stable nature: it succinylacetone, DFP failed to enhance ﬂuorescent-Tf bindingpersisted after addition of chelators that differentially dissociate (data not shown), indicating that under normal conditions whereDFP-Fe complexes (but not Fe-transferrin). On the other hand, iron incorporation into heme is not blocked, the concentration ofDFP alone and NTA alone evoked only minor changes that were DFP-mobilized iron is insufﬁcient to be detectable in this system.eliminated by addition of DTPA before the reaction with aTf, DFP-mediated iron delivery for hemoglobin synthesis. Theindicating that the changes were associated with traces of iron potential of DFP-Fe complexes as donors of iron for physiologicpresent in the medium. Essentially similar results were obtained by use was assessed using a model system of cellular iron incorpora-assessing the transfer of iron to apotransferrin using ﬂuorescein- tion, synthesis of hemoglobin (Hb) by differentiating preerythroidlabeled aTf (not shown). However, with lissamine rhodamine- cells. The well-documented induction of Hb synthesis in MEL cellslabeled aTf (R-aTf), the acquisition of iron evoked insigniﬁcant by hexamethylene bisacetamide (HMBA) is highly dependent onchanges in ﬂuorescence. the supply of iron.26 It is repressed in serum-free medium lacking transferrin, but it is signiﬁcantly enhanced by the addition ofDFP-mediated mobilization of iron from H9C2 cells to Fe-NTA irrespective of the presence of Tf (Figure 7). DFP-Fe (3:1)extracellular transferrin: receptor-mediated uptake of supported Hb synthesis to a degree similar to that seen withholotransferrin as an index of iron shuttling Fe-NTA, both in the absence and presence of added Tf. FullyMobilization of cellular iron by DFP and its transfer to extracellular saturated holotransferrin (15 M) was equally effective as Fe-NTAtransferrin are critical features of DFP’s potential function as an and DFP-Fe (data not shown), indicating that various forms of ironiron shuttle. This capability was assessed using H9C2 cells can equally support Hb synthesis in this experimental system. Onincubated with lissamine R-aTf in the presence of DFP (Figure 6). the other hand, DFP without added iron depressed Hb synthesis
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1695Figure 5. Transfer of iron from DFP-Fe complexesto transferrin. DFP-Fe complexes (1:3 and 1:5 ratios;50 M Fe(III)) were mixed with apotransferrin aTf(50 M) and incubated for 1.5 hours in a humidiﬁed 5%CO2 incubator at 37°C. At the conclusion of the reac-tion, 10 mM DTPA was added and the low–molecularweight material was removed by ﬁltration as describedin “Histone-CALG.” The tryptophan (trp) ﬂuorescenceat 280 nm excitation/306 nm emission (top graph) wasobtained from the emission spectra (inset). The high–molecular weight fractions were diluted in HBS, pH 7.4,and the absorbance was read at 465nm (bottom graph).Data are given as means plus or minus SD of3 independent experiments. The labels below the barsindicate the complexes tested and their concentrations.The values above the bars represent the percentagechange in absorbance or ﬂuorescence.below control levels, indicating that the chelator can have an (data not shown), an effect similar to DFO-induced iron depriva-iron-withdrawing effect under conditions of limited iron supply. tion in other hematopoietic cells.27 To demonstrate that transfer ofIn the serum-free medium used in these experiments, DFP iron from DFP-Fe to aTf gave rise to functionally active holotrans-( 100 M) without added iron, as well as DFO (100 M), ferrin, DFP-Fe was allowed to interact with aTf for 1 hour, and thenprevented differentiation and ultimately caused massive cell death the mixture was exhaustively dialyzed (cut-off 12 kDa) against Figure 6. DFP-mediated mobilization of iron from H9C2 cells to extracellular apotransferrin: receptor- mediated uptake of holotransferrin as an index of iron transfer. H9C2 cells were pretreated with succinylac- etone as described in “Methods” to increase mitochon- drial labile iron levels. They were then incubated at 37°C in DMEM-HEPES medium containing 20 M lissamine R-aTf with various additions, and epiﬂuores- cence microscopy images were obtained under rhodamine settings after 60 minutes of incubation. Representative images of cells with no addition (A), 30 M DFP (B), 100 M unlabeled holotransferrin (C), 100 M unlabeled holotransferrin with 30 M DFP (D), 20 M Fe-NTA, 1:3 ratio (E), and 20 M Fe-NTA with 30 M DFP (F). (G) The illustration represents entry of DFP into cells, mobilization of iron from the cytosol (C), nuclei (N) and mitochondria (M), followed by exit of DFP-Fe complexes from the cells (step 1). This is followed by transfer of iron from DFP-Fe to R*-aTf to form R*-Tf-Fe (step 2), which then binds to transferrin receptors on the cell surface (step 3), concen- trates in the endosomes, and is detected by ﬂuorescence microscopy as punctuate ﬂuorescence typical of mi- crovesicles. (H) Mean cell-associated ﬂuorescence values in r.u. of 5 cells per ﬁeld ( SD, from 3 separate experi- ments), calculated from snapshots such as shown in panels A through F, obtained after 1 hour of incubation.
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.1696 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3Figure 7. Stimulation of Hb synthesis in murine erythroleukemia cells. (A) DFP-mediated iron delivery to murine erythroleukemia (MEL) cells synthesizing hemoglobin isschematically depicted. (B) MEL cells suspended in serum-free DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA were supplemented with various ironcomplexes (step 1 in panel A) and cultured for 48 hours. Hemoglobin synthesis (step 2 in panel A) was assessed in terms of hemoglobin content in cell lysates as described in“Cell hemoglobin synthesis” and is expressed relative to the hemoglobin content in control cells with no additions (set as 100%). The additions included 10 M Fe complexed to30 M NTA without (Fe-NTA) and with 15 M human apotransferrin (Fe:NTA aTf); 10 M Fe complexed to 30 M DFP without (DFP-Fe) and with 15 M humanapotransferrin (DFP-Fe aTf); 30 M DFP alone (DFP); 15 M human apotransferrin alone (aTf). Generation of holotransferrin from DFP and apotransferrin (DFP-Fe aTfDial.): 10 M Fe:30 M DFP complex was preincubated with 15 M human for 1 hour and dialyzed. Results shown are averages of 3 separate experiments plus or minus SD;the average Abs604 of control cells was 0.39. (C) DFP-mediated mobilization of iron from J774 (Fe donor; step 1a in panel A) cells to the extracellular medium (step 1b in panelA), followed by entry of DFP-Fe complexes into MEL (Fe acceptor) cells (step 1c in panel A) and intracellular donation of iron for hemoglobin (Hb) synthesis (step 2 in panel A).MEL cells were cultured for 48 hours in DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA without (Con) or with 10 M Fe-NTA (NTA-Fe), or with varioussupernatants of J774 cell lysates that were supplemented with 5 mM HMBA, and lysates were assayed for hemoglobin content. To obtain J774 mouse macrophagesupernatants, J774 cells were cultured overnight without (untreated J774) or with 100 M FAC (Fe-loaded J774), washed with 100 M DFO to remove all extracellular iron, andincubated for 2 hours at 37°C in serum-free DMEM containing 1.25 mg/mL bovine serum albumin, without ( DFP) or with 30 M DFP ( DFP). The cell supernatants werecollected and centrifuged to remove detached cells, and HMBA (5 mM) was added to MEL cells. Shown are values of hemoglobin (Hb; from a representative experiment)obtained in MEL cells exposed for 48 hours to the various conditions.buffer containing 80 mg/L aTf to remove DFP-Fe without gaining support for the proposed use of DFP as an iron shuttle even withoutcontaminant iron from the solution. When added to differentiating the mediation of transferrin, such as might occur in the centralMEL cells, this preparation, generated from DFP-Fe and aTf, fully nervous system.supported Hb synthesis (Figure 7B). Finally, we assessed DFP’s ability to shuttle iron from cell tocell (Figure 7C). In vivo, direct intercellular transfer of iron via DiscussionDFP is not likely, due to the presence of more than 50 M due tothe presence of more than 25 M transferrin in plasma.28 However, The recognition that intracellular iron accumulation is one of theintercellular transfer of iron via DFP could occur in the brain where underlying causes of some clinical syndromes has led to thethe iron-binding capacity of transferrin in cerebrospinal ﬂuid (CSF) suggestion that it might be amenable to treatment with ironis estimated to be in the submicromolar range.29,30 In the experi- chelators.3,33,34 These syndromes include Parkinson disease, neuro-ment illustrated in Figure 7A, J774 mouse macrophages were degeneration with NBIA, and Friedreich ataxia, where iron depos-chosen as the iron-donating cells because of their high capacity for its in speciﬁc areas of the brain have been identiﬁed by histologic oriron accumulation.31 DFP at a concentration of 30 M, achieved in imaging techniques34 and are thought to be a central factor in thevivo with moderate oral doses,1,32 mobilized sufﬁcient iron from development of the diseases.35 Similarly, in anemia of chroniciron-loaded J774 cells, but not from untreated ones, to signiﬁcantly disease, iron is retained within erythrocyte-phagocytosing macro-enhance Hb synthesis in MEL cells (Figure 7C). Spontaneous phages, presumably to prevent access by invading pathogens toefﬂux of iron from iron-loaded J774 cells took place, as evidenced iron from the circulation.7-9,31 While regional iron mobilizationby the increased MEL cell Hb levels even in the absence of might be essential for stabilizing or reversing the toxic effects ofDFP (although increased, these levels were signiﬁcantly lower in labile iron, in some pathologies it might be equally important tothe absence of DFP than in its presence). This result provides render the mobilized iron available for metabolic reuse. This is
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1697especially important in those conditions in which regional iron hemoglobin synthesis when presented together with DFP or afteraccumulation is accompanied by regional or systemic deprivation. DFP’s removal (Figure 7).All the chelators in clinical use have been designed for massive The direct bioavailability of DFP-bound iron was investigatedremoval and excretion of iron from organs of iron-overloaded using hemoglobin synthesis by cells in culture as a model. DFP-Fepatients.2,10,33 Here we assessed the potential application of an iron supported hemoglobin synthesis even in the absence of transferrinchelator in clinical use (DFP) as a shuttling agent capable of (Figure 7), consistent with a similar activity of 1:1 complexes ofredistributing iron within and among cells, while satisfying certain iron with salicylaldehyde isonicotinoyl hydrazone.43 More impor-basic requirements. tant, DFP enhanced the transfer of iron from iron-loaded macro- Permeability across cell membranes in the free and iron- phages to preerythroid cells for hemoglobin synthesis, without thebound form. The high membrane permeability of the iron-free intermediary presence of transferrin (Figure 7). Such direct dona-form of DFP is well documented, as shown by its capacity to tion to intracellular iron-requiring or iron-metabolizing enzymesrapidly access and deplete intracellular labile iron pools.36,37 On the may not be a necessary feature if iron is efﬁciently transferred fromother hand, the ability of DFP-Fe complexes to traverse intracellu- the shuttle to circulating transferrin. However, it may be desirablelar and plasma membranes of cells has not been studied in or even essential in the brain, where the iron-binding capacity ofdetail.20,21,36,37 One indication is the capacity of orally administered transferrin in the CSF is negligible and has been estimated in theDFP to increase serum transferrin saturation12 and labile iron11 submicromolar range.29,30within an hour after intake in thalassemia patients, which is Low toxic potential of iron-shuttling agent complexes.attributable to the rapid exit of DFP-Fe complexes from cells into A major concern in the use of chelators is the formation of ironthe circulation. At the intracellular level, it is shown in this work complexes that undergo redox cycling and thereby catalyze ROSthat DFP can facilitate the removal of labile iron from nuclei generation. This tendency is present in chelators with relatively low(Figure 3), from endosomes (Figure 4), and from mitochondria redox potentials, such as those based on amine- and carboxyl-(Figure 6), and can transfer iron to acceptors in the mitochondria liganding groups but not with the bidentate 3:1 complexing(Figure 3), nuclei (Figure 4), and extracellular medium (Figure 6). hydroxypyridone DFP.20,21,44,45 However, speciation studies10 andThe transfer might involve dissociation of the DFP-Fe complexes recent examination of ROS production42 indicated that complete abolition of labile iron by DFP is attained only at DFP:Fe(III) ratiosand/or formation of ternary complexes with other ligands. close to 5:1. Whether DFP levels reached therapeutically are Ability to compete effectively for intracellular labile iron with consistently high enough to avoid formation of substoichiometricother intermediate afﬁnity species. Labile iron that is likely to be redox-active complexes is difﬁcult to predict. This is particularlyaccessible to DFP is assumed to be bound to several possible important in susceptible, DNA-containing organelles such asligands (eg, citrate, ATP) and to be in dynamic equilibrium between nuclei and mitochondria. The same applies to intracellular signal-Fe(II) and Fe(III) as dictated by the redox status of the intracellular ing pathways activated by oxidative stress that could be generatedenvironment.38,39 DFP competes effectively for labile cell iron both by DFP-mediated redistribution of cell iron.in vitro and in vivo,32 and in individuals with normal iron balance it Permeability across the blood-brain barrier (BBB) for thera-raises serum iron levels, indicating mobilization of tissue iron.12 peutic applications to neurodegenerative diseases. The capacityThis was also observed with isolated cells (Figures 3,4), where of an iron-shuttling agent to redistribute iron in the central nervousDFP readily chelated iron bound to the EDTA analog CALG in system (CNS) may be an essential requirement for iron redistribu-endosomes and nuclei and was indicated in other systems.40 An tion in Parkinson disease, Friedreich ataxia, or NBIA. A high rate ofundesirable property of a chelating molecule would be interference BBB penetration is expected for free DFP, based on its lowwith normal cellular iron metabolism by overchelation. In this molecular weight (below 300) and lipophilicity. This was con-respect, DFP ought to be used at restricted doses, as at high levels it ﬁrmed by direct measurement of DFP accumulation in the perfusedappears to inhibit iron-requiring tyrosine and tryptophan rat brain.15,16 In rats given DFP doses several times higher thanhydroxylases.10,14 analogous doses given to thalassemia patients, brain tyrosine and Ability to donate iron to physiologic acceptors. The rationale tryptophan hydroxylase activities were signiﬁcantly inhibited, asbehind DFP-mediated redistribution of iron leans on DFP’s ability measured by the accumulation of 3,4-dihydroxyphenylalanineto transfer the metal to extracellular transferrin, thereby minimizing (DOPA) and 5-hydroxytryptophan, respectively, presumably viaundesired iron loss by excretion via the urinary or biliary pathways. coordination to iron bound by these enzymes.14 Yet, despiteWe surmised that under physiologic conditions iron transfer to aTf DFP’s brain accessibility, major effects on CNS function have notis likely to occur (1) spontaneously, as the pFe value of Fe(III) for been reported.32,44transferrin is 3 orders of magnitude higher than for DFP41 (Table 1), Whether DFP can alter the iron balance in the brain by shuttlingand (2) directly as Fe(III), because the thermodynamically favored iron across the BBB remains an open question. The absence of ironDFP-Fe complexes are (DFP)2-Fe(III) and (DFP)3-Fe(III).10,25,42 In loading of the CNS in iron-overload conditions indicates thatvitro studies based on an indirect analytical method11 corroborated neither transferrin-bound nor non–transferrin-bound iron is able toearlier observations that a single oral dose of 3 g DFP raised within freely cross the BBB.35 Because DFP treatment has not been6 hours the transferrin saturation (determined by urea-gel electro- reported to be associated with increased iron loading of the CNS inphoresis) of a healthy individual from a normal level of 20% to thalassemia patients, it is conceivable that DFP-Fe complexes do80%.12 In this work we provided direct demonstration of iron not readily permeate the BBB. The permeabilities of free andtransfer from DFP-Fe to aTf at physiologic concentrations (Figure iron-bound DFP may differ due to differences in their molecular5). Using spectrophotometric and ﬂuorimetric methods, we showed weights (473 for 3DFP:1Fe compared with 139 for DFP). In athat efﬁcient transfer occurs at various DFP:Fe ratios and within recent study, 6 months of treatment with DFP was shown to result1 to 2 hours in culture conditions. The resulting holotransferrin in a decrease in the iron-associated magnetic resonance imagingformed from DFP-Fe was biologically active, as it was recognized (MRI) transverse relaxation rate (R2*) signals in dentate nuclei ofby the transferrin receptor (Figure 6) and provided iron to cells for Friedreich ataxia patients.13 Whether this decrease was caused by
From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.1698 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3egress of DFP-Fe complexes from the brain or by redistribution of iron loss.48,49 Optimization of such strategies with novel chelatorsiron from an area of accumulation to areas of lower concentration for treating the various conditions of regional iron accumulationwithin the brain is unclear. demand further laboratory and clinical work. We show that redistribution of iron in multiple directions and tomultiple recipients is experimentally feasible, particularly fromareas of accumulation to areas of iron need, via donation to Acknowledgmentstransferrin or by direct metal donation. A modality of iron chelationbased on iron redistribution has several therapeutic implications. In This work was supported by the Association Française contre lesthe CNS, it could potentially be used to relocate local iron deposits Myopathies, the Israel Science Foundation (ISF), the EEC Frame- work 6 (LSHM-CT-2006-037296 Euroiron1) and French-Israelithat may be an important factor in the etiology of several Organization for Research in Neuroscience (AFIRN). Z.I.C. is theneurodegenerative diseases. In ACD, it could be used to release Sergio and Adelina Della Pergolla Professor of Life Sciences.iron trapped within macrophages. Current experimental approachesto alleviation of ACD tend to be directed at depressing the levels ofhepcidin, the iron-regulator protein responsible for macrophage Authorshipiron retention. However, recent evidence for hepcidin-independentdown-regulation of the iron exporter ferroportin by the inﬂamma- Contribution: Y.-S.S. and W.B. performed experimentation andtory mediators46 could obviate such an approach. As shown in this analysis; A.M. and Z.I.C. designed and supervised the research. Allwork, an alternate or complementary approach could be based on authors contributed to the writing of the paper.iron-shuttling agents that relocate misdistributed iron and safely Conﬂict-of-interest disclosure: The authors declare no compet-bypass impaired internal routes of iron trafﬁcking. Early pilot ing ﬁnancial interests.studies with iron chelators in rheumatoid arthritis patients with Correspondence: Z. I. Cabantchik, Alexander Silberman Insti-ACD47 provided clinical evidence that agents like DFP might be tute of Life Sciences, Hebrew University of Jerusalem, Safraadopted as part of a short-term strategy of regional metal detoxiﬁ- Campus at Givat Ram, Jerusalem, Israel 91904; e-mail:cation and systemic relocation that generates no signiﬁcant urinary email@example.com.References 1. Hershko C, Link G, Konijn AM, Cabantchik ZI. of catechol-O-methyltransferase (COMT) as well 24. 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From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 169936. Zanninelli G, Glickstein H, Breuer W, et al. Chela- ing labile iron in cells and biological ﬂuids. Anal susceptibility to chelation. Blood. 2003;102: tion and mobilization of cellular iron by different Biochem. 2002;304:1-18. 2670-2677. classes of chelators. Mol Pharmacol. 1997;51: 41. Aisen P, Listowsky I. Iron transport and storage 46. Ludwiczek S, Aigner E, Theurl I, Weiss G. Cyto- 842-852. proteins. Annu Rev Biochem. 1980;49:357-393. kine-mediated regulation of iron transport in hu-37. Link G, Konijn AM, Breuer W, Cabantchik ZI, Her- 42. Devanur LD, Neubert H, Hider RC. The Fenton man monocytic cells. Blood. 2003;101:4148- shko C. Exploring the “iron shuttle” hypothesis in activity of iron(III) in the presence of deferiprone. 4154. chelation therapy: effects of combined deferox- J Pharm Sci. Prepublished August 27, 2007, as amine and deferiprone treatment in hypertrans- 47. Vreugdenhil G, Swaak AJ, de Jeu-Jaspers C, DOI 10.1002/jps.21039. fused rats with labeled iron stores and in iron- van Eijk HG. Correlation of iron exchange be- 43. Garrick LM, Gniecko K, Hoke JE, al-Nakeeb A, tween the oral iron chelator 1,2-dimethyl-3-hy- loaded rat heart cells in culture. J Lab Clin Med. Ponka P, Garrick MD. Ferric-salicylaldehyde 2001;138:130-138. droxypyrid-4-one(L1) and transferrin and possible isonicotinoyl hydrazone, a synthetic iron chelate, antianaemic effects of L1 in rheumatoid arthritis.38. Breuer W, Shvartsman M, Cabantchik ZI. Intra- alleviates defective iron utilization by reticulo- Ann Rheum Dis. 1990;49:956-957. cellular labile iron. Int J Biochem Cell Biol. Pre- cytes of the Belgrade rat. J Cell Physiol. 1991; published on March 19, 2007, as DOI 10.1016/ 146:460-465. 48. Spivak JL. Iron and the anemia of chronic dis- j.biocel.2007.03.010. 44. Singh S, Khodr H, Taylor MI, Hider RC. Thera- ease. Oncology (Williston Park). 2002;16:Suppl39. Kruszewski M. Labile iron pool: The main deter- peutic iron chelators and their potential side- 10:25-33. minant of cellular response to oxidative stress. effects. Biochem Soc Symp. 1995;61:127-137. 49. Vreugdenhil G, Swaak AJ, Kontoghiorghes GJ, Mutat Res. 2003;29:81-92. 45. Esposito BP, Breuer W, Sirankapracha P, van Eijk HG. Efﬁcacy and safety of oral iron che-40. Esposito BP, Epsztejn S, Breuer W, Cabantchik ´ Pootrakul P, Hershko C, Cabantchik ZI. Labile lator L1 in anaemic rheumatoid arthritis patients ZI. A review of ﬂuorescence methods for assess- plasma iron in iron overload: redox activity and [letter]. Lancet. 1989;2:1398-1399.