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RED CELLS
From the Heart Research Institute, Iron Metabolism and
Chelation Group, Camperdown, Sydney, New South Wales,
Australia; the Department of Medicine, Royal Brisbane
Hospital, Herston, Brisbane, Queensland, Australia; and
The Lady Davis Institute for Medical Research, Montreal, Quebec,
Canada.
Friedreich ataxia (FA) is caused by
decreased frataxin expression that results in mitochondrial iron (Fe)
overload. However, the role of frataxin in mammalian Fe metabolism
remains unclear. In this investigation we examined the function of
frataxin in Fe metabolism by implementing a well-characterized model of
erythroid differentiation, namely, Friend cells induced using dimethyl
sulfoxide (DMSO). We have characterized the changes in frataxin
expression compared to molecules that play key roles in Fe metabolism
(the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization ( Friedreich ataxia (FA) is an inherited neurodegenerative
condition1,2 with an incidence of 1:30 000 in the
European population.3 The gene FRDA that is
defective in FA encodes a mitochondrial protein known as
frataxin.2,3 In 97% of individuals with FA the defect in
this gene is due to a GAA triplet repeat expansion in intron 1 (chromosome 9q13) that results in a marked decrease in gene
expression.2,3 Over the last few years evidence has accumulated to suggest that frataxin plays a role in mitochondrial iron
(Fe) metabolism.3-24 However, the function of mammalian
frataxin remains unclear.24
Experiments using the yeast, Saccharomyces cerevisiae, led
to a model that may explain the pathogenesis of FA.10,12
The yeast gene YFH1 is homologous to the human gene
FRDA,12 and encodes a mitochondrial protein
(Yfh1p) involved in Fe homeostasis and
respiration.5,9,10,12 When the YFH1 gene was
deleted, a marked accumulation of mitochondrial Fe occurred, resulting in loss of mitochondrial DNA and impaired
respiration.6,7,12,13 Thus, this led to a hypothesis that
the accumulated Fe resulted in the production of free radicals that
damage biologic targets. When YFH1 was reintroduced into the
yeast, mitochondrial Fe was exported back out into the cytosol as free
non-heme-bound Fe.10,12 Therefore, Yfh1p appeared to
regulate Fe export from mitochondria in yeast cells.10
However, Yfh1p may not be an Fe transporter, because it has no
transmembrane sequences necessary for this role.10
Both the human and mouse frataxin genes (FRDA and
Frda, respectively), are highly expressed in tissues that are rich
in mitochondria, including heart, liver, and skeletal
muscle.5,14 Consistent with the yeast knockout model,
fibroblasts from FA patients were more sensitive than normal cells to
oxidant stress,24 and contained higher levels of
mitochondrial Fe.16,21 Further, Fe accumulation and
reductions of mitochondrial DNA, complex I/II/III, and aconitase have
been noted in the heart4,11 and central nervous system of
patients with FA.22,23 Other studies in FA patients
showing Fe deposits within heart myofibrils18,19 and
defective mitochondrial respiration7,8 also suggest
mitochondrial Fe overload. Considering these observations, it is
significant that antioxidants have successfully treated the
cardiomyopathy of FA.25
If frataxin plays a role in Fe metabolism, an understanding of the
pathogenesis of FA will require the examination of mitochondrial Fe
processing pathways. The best characterized function of mammalian mitochondria in Fe metabolism comes from studies in erythroid cells,
where it is involved in the production of heme for hemoglobin synthesis.24,26 In these cells, the Fe-transport protein
transferrin (Tf) binds to the transferrin receptor (TfR) and is
endocytosed. Iron is released from Tf by a decrease in endosomal pH and
traverses the membrane via the transporter, Nramp2 (also known as the
divalent metal ion transporter 1).24 The Fe is then
targeted to the mitochondrion where it is incorporated into
protoporphyrin IX (PIX) to form heme (for reviews, see Becker and
Richardson24 and Ponka26). This molecule is
then exported out of the mitochondrion to combine with globin chains to
form hemoglobin. The rate-limiting step in the heme synthesis pathway
is the acquisition of Fe from Tf.26 When the heme
biosynthesis pathway is inhibited using isonicotinic acid hydrazide (an
inhibitor of The role of frataxin in Fe and heme metabolism remains unknown. It is
possible to suggest a hypothesis that the decreased levels of frataxin
in patients with FA may lead to some defect in either the process of Fe
incorporation into PIX, or Fe influx or export, which results in
mitochondrial Fe accumulation. Recently, it has been suggested that the
yeast frataxin homologue (Yfh1p) may polymerize to store Fe-like
ferritin.34 Another study suggested that an anionic
surface patch on frataxin could oxidize Fe++ to
Fe+++ when this molecule combines with itself or
another ligand.35 In this way, frataxin may accumulate Fe
similarly to ferritin.35 However, structural and
functional studies of mammalian frataxin have shown that this molecule
cannot bind Fe directly and does not polymerize to sequester
Fe.36 Indeed, frataxin shares little homology with other
known molecules involved in Fe or heme metabolism.36 The
clustering of conserved residues onto one face of frataxin suggests an
interaction with potential ligands and may indicate that frataxin acts
indirectly on Fe metabolism.36
In the present study we have assessed the role of frataxin in Fe and
heme metabolism by implementing a well-characterized model of erythroid
differentiation, namely, Friend cells induced with dimethyl sulfoxide
(DMSO).37-41 This model has previously been used to assess
the role of other molecules involved in Fe and heme
metabolism.37-41 We demonstrate that DMSO induction of erythroid differentiation and hemoglobinization results in a marked decrease in frataxin expression. In addition, incubation of uninduced Friend cells and other cell types with PIX resulted in down-regulation of frataxin protein levels, suggesting a possible regulatory role of
frataxin in Fe and heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest the decrease in frataxin levels during erythroid differentiation favors
mitochondrial Fe sequestration and heme synthesis. These results may
explain why there are no marked effects of decreased frataxin
expression in the erythroid cells of FA patients; that is, decreased
frataxin expression may physiologically facilitate Fe uptake and heme synthesis.
Cell treatments and reagents
Preparation of 59Fe-transferrin
Cell culture The mouse Friend erythroleukemia cell lines 707 and 745 as well as the human SK-N-MC neuroepithelioma cell line were obtained from the American Type Culture Collection (ATCC; Rockville, MD). Friend cells were induced to differentiate into hemoglobin-producing cells by incubation with 1.5% DMSO.The mouse fibroblast cell line, LMTK Assays to measure Fe uptake and Fe incorporation into heme Friend cells were incubated in the presence or absence of DMSO for 24 to 120 hours. After this, aliquots of cells (1 × 107) were placed in tubes and incubated with 59Fe-Tf (10 µM) for 1 to 120 minutes at 37°C.37 After appropriate incubation times the tubes were plunged into ice and the cells washed 3 times in a large excess of ice-cold phosphate-buffered saline.27 Incorporation of 59Fe into heme was performed by standard procedures.27 Radioactivity was measured using a -scintillation counter (LKB Wallace 1282 Compugamma, Turku, Finland).
Northern blot analysis Northern blot analysis was performed as described previously.44 The Frda probe consisted of an 870-base pair (bp) fragment from the coding region of murine Frda cloned into the pT7T3pac plasmid (from Dr R. Williamson, Murdoch Institute, Melbourne, Australia). The TfR probe of 800 bp was obtained from the coding region of the murine TfR cloned into the vector pUC18 (from Dr G. Anderson, The Queensland Institute of Medical Research, Brisbane, Queensland). The Nramp2 probe consisted of 1.7 kb of coding sequence from murine Nramp2 cloned into the pMT2 plasmid (from Dr N. C. Andrews, Howard Hughes Medical Institute, Children's Hospital, Boston, MA). The -globin probe of 612-bp was excised from
the pMG5 vector (from Dr P. Ponka). The -actin probe
consisted of a 1.4-kb fragment of -actin complementary
DNA (cDNA) cloned into pBluescript SK (ATCC catalogue
no. 37997).
Western blot analysis All procedures used for Western blotting were the same as those described previously.45 The monoclonal antibody (MoAb) against frataxin (clone 1G2) was from Chemicon International (Temecula, CA), and the MoAb against-actin (clone AC-15) was from
Sigma. The TfR MoAb (clone H68.4) was purchased from Zymed
Laboratories (San Francisco, CA).
Statistics Data were compared using the Student paired t test. Results were considered statistically significant when P < .05.
Frda (frataxin) expression is down-regulated during erythroid differentiation Friend cells exposed to DMSO undergo erythroid differentiation resulting in an increase in Fe uptake from Tf, heme synthesis, and hemoglobinization.26,37-41 Our initial studies examined the effect of DMSO on 59Fe uptake from 59Fe-Tf (10 µM) and the incorporation of 59Fe into heme by the Friend 707 cell line (Figure 1). Cells were incubated with 1.5% DMSO or control media for incubation times of 24 to 120 hours and the effect on 59Fe uptake and incorporation into heme assessed by incubating cells with 59Fe-Tf for 1 to 120 minutes (Figure 1A,B). Our studies showed that after a 24-hour incubation with DMSO there was little effect on 59Fe uptake from 59Fe-Tf and incorporation of 59Fe into heme, as shown previously (Figure 1A).40 However, after a 96-hour incubation with DMSO, there was a significant (P < .001) increase in 59Fe uptake and incorporation of 59Fe into heme when compared to control cells for time points from 30 to 120 minutes (Figure 1B). Similar results to those found in Figure 1B were also found after incubating cells with DMSO for 48, 72, and 120 hours (data not shown). These data examining Fe uptake are similar to those found by others using Friend cells.40,50
To examine the possible role of frataxin in Fe or heme metabolism,
experiments were designed to assess the effect on Frda messenger RNA (mRNA) levels by inducing erythroid differentiation in
Friend 707 cells (Figure 2). To further
investigate the function of frataxin, we also assessed changes in
expression of
A 120-hour incubation with DMSO resulted in a marked decrease in
Frda mRNA levels (Figure 2B) compared to cells incubated with medium alone. After normalization to In agreement with the decrease in Frda mRNA levels (Figure
2B), Western blot analysis also demonstrated that frataxin protein levels were decreased after induction of erythroid differentiation with
DMSO in Friend 707 cells (Figure 3). In
all experiments a band at approximately 18 kd was identified that was
in good agreement with the relative molecular mass of frataxin
reported previously.6 The decrease in frataxin protein
levels began after 24 hours of induction with DMSO, with the greatest
decrease becoming clear between 72 and 120 hours (Figure 3). Further,
the effect of erythroid differentiation on decreasing frataxin protein
levels was not specific for the Friend cell clone, with similar results
being observed in the 707 and 745 cell lines (Figure
4). All subsequent experiments
described were performed using the Friend 707 cell line.
Collectively, these data above indicate that the induction of erythroid
differentiation results in a decrease in frataxin expression at both
the mRNA and protein level. This decrease is inversely related to
hemoglobinization (as judged by cell pellet color) and the expression
of DMSO does not nonspecifically down-regulate Frda gene expression Control experiments were performed to ensure that the effect of DMSO on Frda expression was not due to nonspecific down-regulation of gene expression (Figure 5). In these studies, nonerythroid LMTK fibroblast or RAW264.7 macrophage cell lines were
incubated in the presence or absence of DMSO for 120 hours and then
Frda mRNA levels assessed (Figure 5). Incubation with DMSO
had no effect on Frda mRNA expression when compared to cells
exposed to control medium (Figure 5). These results indicate that
Frda expression was not nonspecifically down-regulated
by DMSO.
Kinetic analysis of the effects of DMSO on the
expression of Frda, Nramp2,
TfR,
In contrast to Frda mRNA expression, normalized TfR mRNA levels significantly (P < .0001) increased up to 25-fold (3 experiments) in the presence of DMSO between 24 and 48 hours compared to initial levels (time 0; Figure 6C). The levels of TfR mRNA then markedly decreased as the incubation was continued up to 120 hours (Figure 6C). In contrast, in the absence of DMSO, normalized TfR mRNA levels significantly (P < .001) decreased more than 6-fold (3 experiments) as the incubation increased from 0 to 120 hours (Figure 6C). From Figure 6D, it is clear that there are 2 Nramp2 transcripts at 2.3 and 3.1 kb in Friend cells, as observed in previous studies for murine tissues and cell lines.46,47 Although the precise identification of these 2 transcripts is uncertain, they may correspond to the Nramp2 splice variants with and without the iron-responsive element (IRE; 2.3 and 3.0 kb, GenBank accession codes AF029758 and L33415, respectively). The change in expression of the Nramp2 transcripts after incubation with DMSO was generally similar (Figure 6D). In the presence or absence of DMSO, densitometric analysis showed there was a slight increase in the normalized expression of both Nramp2 transcripts in the first 24 hours. In the presence of DMSO, this initial increase in Nramp2 mRNA expression was then followed by a significant (P < .01) decrease in 3 experiments (Figure 6D). In one typical experiment (representative of 3) in the presence of DMSO, the 2.3-kb and 3.1-kb Nramp2 transcripts decreased 3.7-fold and 2.5-fold, respectively, comparing initial levels and those found after a 120-hour incubation (Figure 6D). In the absence of DMSO, the levels of both the 3.1-kb and 2.3-kb transcripts peaked at 72 hours and then decreased to initial levels or slightly below initial levels, respectively, at 120 hours (Figure 6D). The expression of Nramp2 and TfR mRNA was important to assess because their protein products act as partners involved in Fe uptake from Tf,48,49 which is increased on erythroid differentiation.40,50 In accordance with previous studies,38 we found that the
expression of The effect of iron chelators, iron donors, and the heme precursor,
PIX, on the expression of
Frda, TfR,
Nramp2,
Friend cells were incubated for 96 hours with control medium or medium containing 1.5% DMSO and then exposed for a further 20 hours to either control medium, DFO (100 µM), PIH (50 µM), FAC (100 µg/mL), hemin (50 µM), or PIX (50 µM) in the presence or absence of 1.5% DMSO. Levels of Frda mRNA (Figure 7B) and Nramp2 mRNA (Figure 7D) were significantly (P < .003) greater under all conditions in cells not induced with DMSO in 4 experiments. Densitometric analysis (not shown) demonstrated that none of the modulatory agents above had any significant effect on normalized Frda mRNA levels when compared to the control (Figure 7B). In contrast, DFO increased TfR mRNA levels in uninduced cells, whereas it had less effect on TfR mRNA expression in cells incubated with DMSO (Figure 7C). A similar but less marked effect was also seen for PIH. The Fe donors, FAC and hemin, had a greater effect at reducing TfR mRNA in uninduced than induced Friend cells (Figure 7C). The fact that DFO, FAC, and hemin affected TfR expression provided a positive control that showed these agents acted in accordance with their reported effects to remove or donate Fe to cells.41,44,47,54-56,60 Studies on the effect of DFO and FAC on the expression of
Frda mRNA compared to TfR mRNA were also examined
in 5 nonerythroid cell lines available in our laboratory
(LMTK None of the Fe chelators, Fe donors, or PIX, had any significant effect on normalized Nramp2 mRNA levels compared to the control in 4 separate experiments (Figure 7D). Certainly, the level of Nramp2 mRNA was not regulated in concert with TfR mRNA (Figure 7C). This observation is of interest because like TfR mRNA, Nramp2 mRNA contains an IRE in its 3' untranslated region.49 These results are similar to those reported in our previous studies examining the effects of intracellular Fe levels on Nramp2 expression in nonerythroid cell types.44,47 Collectively, the results above suggest that Frda mRNA was not regulated by Fe in a similar way to TfR mRNA. To confirm this, we also incubated cells with the heme synthesis inhibitor, SA, which causes accumulation of mitochondrial Fe and, to a lesser extent, cytoplasmic Fe.26,27,30-33 In these studies, Friend cells were incubated for 96 hours with control medium or medium containing 1.5% DMSO and then exposed for a further 20 hours with either control medium or medium containing 2 mM SA. These experiments showed that SA had no significant effect on the expression of Frda mRNA or Nramp2 mRNA (data not shown). In contrast, SA down-regulated TfR mRNA levels of induced cells (data not shown), as also reported by others.41,61 This latter result provided a positive internal control for the inhibitory effect of SA on heme synthesis. Hence, inhibition of heme biosynthesis had no effect on Frda mRNA levels. PIX down-regulates frataxin protein levels in uninduced Friend cells and other cell types Western blot analysis was performed to assess whether DFO, FAC, PIH, hemin, PIX, or SA had any regulatory effects on frataxin protein levels. Friend cells were incubated for 96 hours with control medium or medium containing DMSO and then exposed for a further 20 hours with either control medium or medium containing DFO (100 µM), FAC (100 µg/ml), PIH (50 µM), hemin (50 µM), PIX (50 µM), or SA (2 mM) in the presence or absence of DMSO (Figure 8A). As shown in Figure 3, induction of erythroid differentiation with DMSO resulted in a marked decrease in frataxin protein levels (Figure 8A). Of all compounds tested, only incubation with PIX consistently decreased the level of frataxin protein in uninduced Friend cells over 8 independent experiments (Figure 8A). After normalization to-actin, PIX significantly
(P < .003) decreased the level of frataxin protein
2.2 ± 0.7-fold compared to the relevant control in 8 experiments (Figure 8A). For induced cells, PIX reduced frataxin
expression (Figure 8A) but this was not significant
(P > .05) in 8 experiments. All other agents tested in
induced cells also had no significant effect on frataxin
expression.
Similar to uninduced Friend cells, PIX also reduced frataxin expression
in mouse LMTK To assess if the above treatments had appropriately influenced cellular
Fe metabolism, their effects on TfR expression were also assessed by
Western blotting (Figure 9). As expected
from previous studies,41 DFO increased TfR expression in
both uninduced and induced Friend cells, but this was more pronounced
in uninduced cells (Figure 9). In contrast, FAC and hemin reduced TfR
levels in uninduced cells more than induced cells. In 5 experiments, densitometric analysis demonstrated that PIH, PIX, and SA had no
consistent nor significant effect on TfR protein levels in induced or
uninduced cells (Figure 9). The failure of PIH to increase TfR levels
is of interest and may relate to its different site of action to DFO.
Previous studies have also noted that in contrast to DFO, PIH did not
increase iron regulatory protein-IRE-binding activity.62
Interestingly, SA did not decrease TfR protein levels of induced cells
(Figure 9), and this was in contrast to results at the mRNA level (see
text above).
The role of frataxin in mitochondrial Fe homeostasis is critical to understand because the decrease in expression of this molecule leads to mitochondrial Fe accumulation that may contribute to FA.1,2,24 Furthermore, understanding the function of frataxin may result in new strategies for the treatment of this disorder.24,62 We examined the role of frataxin in Fe and heme metabolism by using a well-characterized model of erythroid differentiation, namely, Friend cells induced with DMSO.37-41 To our knowledge this is the first report showing down-regulation of
Frda expression on erythroid differentiation. In contrast, and as expected,26,37,38,41,56 expression of
TfR and On erythroid differentiation there is a marked up-regulation of
proteins involved in controlling Fe uptake (eg, TfR),41,61 heme synthesis,26,40,56,63 and hemoglobinization (eg,
The finding that erythroid differentiation resulted in down-regulation of frataxin expression provides insight into the function of this protein. Indeed, it is likely that the Fe loading and oxidative damage of the mitochondrion in FA patients is due to a decrease in frataxin levels.6,11 Considering this, and in conjunction with our data, 2 possible roles of frataxin could be suggested that lead to mitochondrial Fe sequestration: (1) down-regulation of frataxin expression may increase mitochondrial Fe import or decrease Fe export by regulating metal transporters, leading to Fe accumulation for heme synthesis, or (2) frataxin could act as a metabolic switch, resulting in the diversion of Fe from one metabolic pathway (eg, Fe incorporation into ferritin or the synthesis of Fe-S clusters) to another, namely, Fe incorporation into heme. For both of the above hypotheses, increased retention of mitochondrial Fe would occur that could favor heme synthesis.26 In support of the first hypothesis, it has been reported that Yfh1p plays a role in the release of nonheme mitochondrial Fe into the cytoplasm.10 Thus, the decrease in frataxin expression in erythroid cells may prevent mitochondrial Fe release and its use for heme synthesis rather than cytosolic Fe metabolism. Alternatively, or in combination with a decrease in mitochondrial Fe efflux, down-regulation of frataxin expression may increase mitochondrial Fe uptake. In this latter mechanism, frataxin could act as a negative regulator of a mitochondrial Fe import process. Considering this, a yeast gene known as CCC1 has been identified that inhibits mitochondrial Fe uptake.66 We suggest a similar molecule could be regulated by frataxin in mammalian cells. At this point it should be discussed that the regulation of cellular Fe metabolism is complex. The net flux of Fe coming into the cell via Nramp2 or into the mitochondrion as possibly regulated by frataxin, may be dependent on integrating the activity of multiple proteins. Thus, the observation that frataxin expression decreases as Fe uptake into heme increases does not prove direct causality. In accordance with a role of frataxin in the regulation of Fe and heme metabolism, our studies demonstrated that incubation of uninduced Friend cells with PIX resulted in a significant decrease in frataxin protein levels (Figure 8A). Because PIX is the immediate precursor of heme,26,56 an increase in the intracellular PIX concentration may act as a signal to coordinate heme synthesis with mitochondrial Fe uptake. For instance, an elevation of cellular PIX could signal a decrease in frataxin expression that leads to mitochondrial Fe sequestration for heme biosynthesis. It is of interest to note that although PIX reduced frataxin protein expression (Figure 8A), it had no significant effect on the mRNA levels of this molecule (Figure 7B). Considering the recent discussion of frataxin acting as a mitochondrial ferritin,34-36 it is relevant to note that incubation of Friend cells with the Fe donor, FAC, or the Fe chelator, DFO, did not significantly increase or decrease the level of frataxin, respectively (Figure 8A). This was in marked contrast to the known stimulatory and inhibitory effects of FAC and DFO, respectively, on ferritin protein levels.26,56,60 These data suggest that frataxin is not regulated by Fe in a manner similar to ferritin. It could be expected that a vital Fe storage molecule would respond to increased intracellular Fe levels. Hence, the lack of response of frataxin in our experiments is not consistent with an Fe storage role. In erythroid cells, excess mitochondrial Fe may be stored in the recently identified mitochondrial ferritin.67 Interestingly, depletion of frataxin in FA patients does not result in
marked defects in heme synthesis.20 Because a reduction in
frataxin expression occurs concurrently with an increase in Fe uptake,
heme synthesis, and TfR- and It is relevant to discuss that mitochondrial Fe must be in the Fe++ state to contribute to heme synthesis.26,56 This is exemplified by sideroblastic anemia, where ferric Fe accumulates and cannot be used for heme synthesis via ferrochelatase. In this study we used a well-characterized model of erythroid differentiation where incubation of Friend cells with DMSO results in initiation of heme biosynthesis, a significant increase in Fe uptake from Tf, and incorporation of Fe++ into PIX to form heme (Figure 1B).26,37,40,41,50,56,61,63-65 Hence, our experiments have focused on the role of frataxin during a physiologic response rather than a pathologic condition. Previous studies by Cossee and associates68 showed that homozygous deletions of frataxin cause lethality in a homozygous mouse knockout model without apparent Fe accumulation. In the case of a developing embryo, an inappropriate increase in mitochondrial Fe levels may not need to be marked to cause apoptosis and death. Moreover, in this latter study,68 there was no attempt to quantitatively examine mitochondrial Fe concentrations by isolating mitochondria, and it is difficult to assess if the methods used (Perl staining and electron microscopic investigation of tissues) were sensitive enough to determine a small but critical increase in Fe. Nonetheless, considering the work of Cossee68 and others,69 it cannot be excluded that frataxin deficiency triggers apoptosis by a mechanism independent of Fe accumulation. It is notable that no significant mitochondrial Fe deposition takes place in sideroblastic anemia at day 11.5 during the embryonic life of the erythroid 5-aminolevulinate synthase knockout mouse.70 Relevant to this, Cossee and colleagues68 reported that frataxin knockout mice die soon after implantation. Thus, it is likely that these authors may have missed the Fe accumulation that occurs later in development. In summary, we demonstrate that erythroid differentiation in Friend cells results in down-regulation of Frda mRNA levels and frataxin protein levels. Further, the down-regulation of frataxin expression during erythroid differentiation may favor the marked mitochondrial Fe uptake, heme synthesis, and hemoglobinization that occur. Indeed, the fact that incubation with PIX decreased frataxin protein levels suggests a role for this protein in the coordination of mitochondrial Fe and heme metabolism. Further studies on cell lines where overexpression or underexpression of frataxin is controlled by inducible promoters may establish the precise role of frataxin in Fe metabolism.
Professor Roger Dean, Dr Anna Baoutina, and members of our laboratory are kindly thanked for their detailed comments on the manuscript prior to submission. We gratefully acknowledge the expert assistance of Mr Ralph Watts in preparing the figures. Dr Jin Gao is also thanked for his help with Western blot analysis.
Submitted April 10, 2001; accepted January 14, 2002.
Supported by a PhD scholarship (to E.M.B.) from Cecily and Neville Cox of Brisbane, Queensland, and by grants from the National Health and Medical Research Council of Australia and an Australian Research Council Large Grant (to D.R.R.). We also kindly acknowledge the Heart Research Institute for financial support.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Des R. Richardson, Heart Research Institute, Iron Metabolism and Chelation Group, 145 Missenden Rd, Camperdown, Sydney, New South Wales, 2050 Australia; e-mail: d.richardson{at}hri.org.au.
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© 2002 by The American Society of Hematology.
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  M. L.-H. Huang, E. M. Becker, M. Whitnall, Y. S. Rahmanto, P. Ponka, and D. R. Richardson Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant PNAS, September 22, 2009; 106(38): 16381 - 16386. [Abstract] [Full Text] [PDF]
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 O. Nakajima, S. Okano, H. Harada, T. Kusaka, X. Gao, T. Hosoya, N. Suzuki, S. Takahashi, and M. Yamamoto Transgenic rescue of erythroid 5-aminolevulinate synthase-deficient mice results in the formation of ring sideroblasts and siderocytes Genes Cells, June 1, 2006; 11(6): 685 - 700. [Abstract] [Full Text] [PDF]
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 D. S. Kalinowski and D. R. Richardson The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer Pharmacol. Rev., December 1, 2005; 57(4): 547 - 583. [Abstract] [Full Text] [PDF]
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 F. Acquaviva, I. De Biase, L. Nezi, G. Ruggiero, F. Tatangelo, C. Pisano, A. Monticelli, C. Garbi, A. M. Acquaviva, and S. Cocozza Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2 J. Cell Sci., September 1, 2005; 118(17): 3917 - 3924. [Abstract] [Full Text] [PDF]
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 I. Napier, P. Ponka, and D. R. Richardson Iron trafficking in the mitochondrion: novel pathways revealed by disease Blood, March 1, 2005; 105(5): 1867 - 1874. [Abstract] [Full Text] [PDF]
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 B. Sturm, U. Bistrich, M. Schranzhofer, J. P. Sarsero, U. Rauen, B. Scheiber-Mojdehkar, H. de Groot, P. Ioannou, and F. Petrat Friedreich's Ataxia, No Changes in Mitochondrial Labile Iron in Human Lymphoblasts and Fibroblasts: A DECREASE IN ANTIOXIDATIVE CAPACITY? J. Biol. Chem., February 25, 2005; 280(8): 6701 - 6708. [Abstract] [Full Text] [PDF]
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 O. Stehling, H.-P. Elsasser, B. Bruckel, U. Muhlenhoff, and R. Lill Iron-sulfur protein maturation in human cells: evidence for a function of frataxin Hum. Mol. Genet., December 1, 2004; 13(23): 3007 - 3015. [Abstract] [Full Text] [PDF]
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T. Yoon and J. A. Cowan Frataxin-mediated Iron Delivery to Ferrochelatase in the Final Step of Heme Biosynthesis J. Biol. Chem., June 18, 2004; 279(25): 25943 - 25946. [Abstract] [Full Text] [PDF]
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E. Lesuisse, R. Santos, B. F. Matzanke, S. A. B. Knight, J.-M. Camadro, and A. Dancis Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1) Hum. Mol. Genet., April 15, 2003; 12(8): 879 - 889. [Abstract] [Full Text] [PDF]
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M. Cazzola, R. Invernizzi, G. Bergamaschi, S. Levi, B. Corsi, E. Travaglino, V. Rolandi, G. Biasiotto, J. Drysdale, and P. Arosio Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia Blood, March 1, 2003; 101(5): 1996 - 2000. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-aminolevulinic acid synthase)
) of 1.5% DMSO for 120 hours at 37°C. The isolated RNA
was electrophoresed on a 1.2% formaldehyde gel, transferred to nylon
membrane, and sequentially probed under high stringency conditions (see
"Materials and methods" for details). (A) Ethidium bromide staining
of the agarose gel; (B) Frda; (C) 
