先天性鉄芽球性貧血の原因遺伝子の同定及びin vitroでの鉄芽球の解析

先天性鉄芽球性貧血の原因遺伝子の同定及びin

vitroでの鉄芽球の解析

著者

張替 秀郎

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(東北大学医学部附属病院検査部軍師

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研究組織

研究代表者:張替秀郎(東北大学医学部附属病院講師)

研究分担者:古山軒道(東北大学大学院医学系研究科助手)

研究軽費

研究発表

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合計

平成11年度 ﾃ# x冷 0 ﾃ# x冷 平成12年度 ﾃ x冷 0 ﾃ x冷 平成13年度 ﾃ3 x冷 0 ﾃ3 x冷 総1計 ｢ﾃc x冷 o一 ﾃc x冷

(1)学会誌等

1) _馳_rigae H, Nakajima 0, Suwabe N, Yokoyama H, Furuyama K, Sasaki T,

Kaku M, Yamamoto M, Sas.sa S･ High content of iron and oxidative status of erythroid-specirlC 81aminolevulinate synthase (ALAS2)-deflC ient definitive

erythroblasts.発表予定

2) Akagi R, Nishitani C, Hari竺ae H, Horie Y, Garbaczewski L, Hassoun A, Mercelis 良, Verstraeten L, Sassa S. Molecular analysis

of8-aminolevulinate dehydratase deficiency ln a Patient with an unusual late-onset porphyria. Blood. 96;3618-23, 2000.

3)山本雅之,中島修,古山和道,張替秀郎,林典夫.遺伝性鉄芽球

性貧血の分子診断とモデルマウス臨床血液41:540-543, 2001

(2)口頭発表

1)弔替秀郎,中島修,古山和道,佐々木毅,佐々茂,山本雅之 へム

欠損成体型赤芽球の形質解析 第63回日本血液学会総会

平成13年3月

2) IIari竺ae H, Nakajima 0, Suwabe N, Furuyama K, Sasaki T, Kaku M, Yamanoto M, Sassa S. Differential role of the erythroid-specific 8-aminolevulinate synthase in primitive and definitive erythropoiesis.

American society of hematology 42th amual meetlng. December 2000.

3) Harigae 汁, Furuyama K, Kondo K, Kimura A, Sasaki T, Kaku M, Hayashi

N, Yamamoto M, Sassa S.Two novel mutations of 8-aminolevulinate

synthase-2 gene in inherited and non-inherited sideroblastic anemia. American society

Aberrant iron accumulation and oxidized status of erythroid-specific

8-aminolevulinate synthase (ALAS2)-deficient definitive erythroblasts

Hideo HarigaeI", osamu NakajimaZ, Naruyoshi SuwabeZ, HisayLiki Yokoyama3･,

Kazumichi Furuyama4, Takeshi Sasaki3, Mitsuo KakuII MasaytJki Yamamoto2, and

Shigeru SassaS

lDepartment of Molecular Diagnostics, 3Rheumatology and Hematology, 4 Biochemistry, Tohoku Universlty School of Medicine, Sendai, Japan, 2center for Tsukuba Advanced Research Alliance･ Tsukuba Universlty, Tsukuba, Japan, 5The Rockefeller Universlty, New York, NY, USA

Running title: Iron accumulation in alas21null erythroblasts

Supported in part by Grant-in-Aid fTrom the Ministry of Education, Science and Culture of Japan(to HH),and USPHA grant DK 32890 (to SS)

a total text word count: 2597 an abstract text word count: 242

Category: Red Cells

Correspondence to:

Hideo Harigae, M.D.

Department of Molecular Diagnostics Tohoku University School of Medicine Aoba-ku, Sendai 980-8574, Japan

Tel:01 1-81-22-717-7373; Fax:01 1-81-22-717-7390

e-mail:[email protected]

-1-Abstract

alas2 encodes the erythroid specific 8-aminolevulinate synthase (ALAS2), the first

enzyme in heme biosynthesis in erythroid cells. In vivo in mice, alas2-null prlmitive

erythroblasts showed a maturation a汀eSt aS Well as massive cytoplasmic iron accumulation.

However, the effect of ALAS2 deficiency on definitive erythropoleSis still remains unclear,

because alas2-null mice died z'n utero by embryonic day 1 1.5 befわre definitive erythropoleSis

develotp'ed. To clarifythe effect ofheme deficiency on differentiation of definitive erythroid

cells, ES cells lacking the alas2 gene were induced to differentiate specifically Into erythroid

lineage t'n vitro, and phenotypes of definitive erythroblasts were examined･ In comparison to

red cell pellets of wild-りpe erythroblasts, alas2-null definitive erythroblasts were totally

white due to a marked deficiency of heme, although the morphology was similar to wild-type

erythroblasts･ Consistent with their similar morphology, levels of erythroid-Specific gene

expression such as GATA-1, NF-E2 and TERl 19 in alas2-null definitive erythroblasts were

also similar to those of wild-りpe Cells, indicatlng that both alas2-null and wild-りpe

erythroblasts developed to the stage of mature definitive erythroblasts･ In contrast to their

similar morphology, however, iron content in alas21null definitive erythroblasts was twice

more than that of wt-ld-りpe Cells. Consistent with the aberrant increase in iron content,

alas2-null definitive erythroblasts were more oxidized compared with wild-type erythroblasts

as judged by increased formation of peroxidized metabolites. These rlndings suggest that

alas2 deficiency does not influence erythroid differentiation per se, but induces aberrant iron

accumulation and an oxidized condition in derlnitive erythroblasts.

e-mai I : [email protected] p

ー2-Introduction

Heme, the prosthetic group of hemeproteins, is essential for the function of all

aerobic cells･ Approximately 85% of heme is synthesized in erythroid tissues and utilized

for hemoglobinformation (1). Besides the main function as an oxygen carrier in

hemoglobin, heme also plays several important roles in erythroid cells, and its deficiency has

been shown to result in dysregulation of gene expression of pTOteins (2), and apoptosis of

erythroid cells (3,4).

Erythroid specific 8-aminolevulinate synthase (ALAS2) gene is the first enzyme in

the heme biosynthetic pathway, which is exclusively expressed in erythroid cells (5,6).

During erythroid di飴rentiation of Friend virus-transformed murine erythroleukemia cells,

ALAS2 mRNA increased dramatically and resulted in marked increase in heme synthesis, as

well as in increased expression of genes for erythroid-specific proteins and f♭r enzymes in the

heme biosynthetic pathway (7,8). In humans, mutations of the alas2 gene, which is located

at Xpl 1 ･2 1, have been shown to be responsible f♭r X-linked sideroblastic anemia (XLSA), an

X chromosome-linked hypochromic and microcytlC anemia characterized by rlnged

sideroblasts (9-12). It is thought that ALAS2 deficiency in XLSA results in a decreased

supply ofprotoporphyrin IX which in tum elicits an iron overload in erythroblasts. Such an

iron overload may likely induce iron一mediated oxi血tive changes in the cell, which may

shorten the cellular lifTe span. All these findings polnt tO the critical role ofALAS2 0n

erythroid differentiation as weu as on the survival oferythroid cells.

In vertebrates, hematopoleSis orlgmateS im the yolk sac, which then mlgrateS tO liver,

finally to the bone marrow (13,14). Erythroid cells in the yolk sac and erythroid cellsin

-3-liverand the bone marrow, are termed primitive and definitive erythroid cells, respectively,

and they display different characteristics and their growth and survivals are controlled by

different growth factors (15-19). In 'our previous study, we demonstrated that alas2-null

prlmitive erythroid cells resulted in a maturation arrest as well as in massive cytoplasmic iron

accumulation in vt'vo in mice, indicatlng thefundamental role of alas2 in cell diffTerentiation

and iron homeostasis in primitive er叩hropoiesis (20)･ However㌔ the role of ALAS2 0n

definitive erythropoleSis remained unclear, because alas2-null animals died in utero by 1 I.5

embryonic days because of severeanemia (20). In order to studythe effect of the lack of

ALAS2 and resultant heme deficiency on definitive erythropoleSis in this study, We used a

culture system in which both prlmitive and definitive erythroid cells can be separately

obtained at different times in culture, using specific cytokines and OP9 stromalcells (21)･

Our results demonstrated that mutation of ALAS2 leads to abe汀ant iron accumulation and

hyperoxidized status in definitive erythroblasts.

ー4-Materials and Methods

Cell Culture

alis2-null ES cells (20) and wild 6pe ES cells were cultivated with OP9, which is an M-CSF

deficient mouse stromal cell line, and were induced to differentiate to erythroid cells as

described previously withminor modinlCations (21)･ Namely, ES cells were plated on to

con fluent OP9 cells at a concentration of 5xl03 per well in a 6-well plate uslng α-MEM

supplemented with 20pM of P-mercaptoethanol, 2U/miof human erythropoietin, 50ng/ml of

murine SCF (Gibco Lifetec Oriental, Rockeville, MD), and long/ml of VEGF (Peprotec,

Rocky Hills, NJ)･ Humanerythropoietin was kindly provided by Chugai Pharmaceutical Co.,

Tokyo, Japan･ On day 3 ofculture, halfofthe culture media was replenished with the fresh

medium and total cells were haⅣested on day 5 by trypslnlZation･ All cells collected were

transferred to a P100 culture dish covered with connuently grownOP9 cells, With the same

culture media but without VEGF･ To collect primitive erythroblasts, all noating cells were

haⅣested on day 8･ To collect de血itive erythroblasts, total cells were collected on血y 10

by pIPetting but without trypslnlZation and transfhed to a new OP9 cell-coated culture dish,

thenincubation was continued to day 14 when all noating cells were collected.

Reverse-transcriptase polymerase chain reaction (RT-PCR)

Total RNA was extracted by the guanidine-phenol method (22)･ CDNA was synthesized

from 2pg of totalRNA in 20pl of the reaction mixture containing O･5pg oligo(dt)12118 200

units or Moloney mu血e leukemia vims reverse transcriptase (SuperscriptTMII: Gibco BRL,

Gaithersburg, MD), 20mM Tris-HCl (pH 9･4), 50mM KCl, 2･5mM MgC12 , 0.5mM oreach

dNTP and 10mM DTT･ Then target genes were amplirled by PCR withSpeciflC PrumerS

-5-uslng ll⊥l ofcDNA solution as a template･ Sequences of each primers are asfollows.

GATA- I sense: 5 '-ACTCGTCATACCACTAAGGT,

anti-sense : 5 '-AGTGTCTGTAGGCCTCAGCT

ev globin sense: 5'-AACCCTCATCAATGGCCTGTGG,

anti-sense: 5 '-TCAGTGGTACTTGTGGGACAGC,

P-major globin sense: 5'- ATGGTGCACCTGACTGATGCTG,

anti-sense : 5 '-GGTTTAGTGGTACTTGTGAGcc,

Nramp2 sense: 5 '-GGTTCTGACATGCAGGAAGT,

anti-sense: 5 '-CAAAGACATTGATGATGAAG,

ALAS2 sense: 5 '-GATCCAAGGCATTCGCAACA,

anti-sense; 5 I-GATGGCCTGCACATAGATGC,

ALAS 1 sense: 5 LCATCTTCACCACCTCCTTGCCACCA,

anti-sense : 5 '-CTATGTGGGTATGGTAATGGCCTGGG,

H0- I sense.･5 I-ACGCATATACCCGCTACCTG,

anti-sense : 5 '-AAGCTGAGAGTGAGGACCCA,

β-actin sense: 5 '-GTGACGAGGCCCAGAGCAAG,

anti-sense : 5 '-AGGGGCCGGACTCATCGTAC,

plow Cytometric Analysis (FACS)

Floating cells collected on day 8 and day 14 were incubated with TERl 19 (Phamingen, Sam

Diego, CA), which is an antibody against erythroid-specific antigen (23), and anti-CD71,

which is an antibody for transferrin receptor (Caltag, Burlingame, CA). For apoptosis

analysis, day 14 erythroblasts were incubated with Anexin V and propidium iodide (PI),

accordirlg tO the manufacture's protocol (MBL Co., Ltd., Nagoya, Japan). Then flow

cytometric analysis was perfb-ed using FACS Calibur (Becton Dickinson, Lincoln Park,

NJ).

Heme assay

Floatlng Cells were haⅣested on day 14 and heme content was dete-ined仙orometrically

using 105 cells per assay as described previously (24). All determinationswere made in

triplicates.

Iron content

For iron quantification, cells were collected by centrifugation and cell pellet was dissolved in

nitric acid solution,and used for atomic absorption SPeCtrOmetric analysts uSlng Z-5000 type

polarized Zeeman atomic absorption spectrometer (20).

Measurement of intracellular reactive oxygen intermediates

Day 14 erythroblasts were washed in PBS, then incubated with

lOpM 2･,7-dichlorodihydronuorescein (DCFHDA: Sigma, St Louis, MO) for 30 min in physiological

saline contalnlng 2% FBS･ Cellular nuorescence, developed by products formed from

DCFHDA by oxidation, was determined by now cytometry (25).

-7-Results

Morphology of alas21null erythroblasts

First, erythroid cells haⅣested on day 8 or day 14 Were cytospun and stained with

May-Gruenwald Giemsa solution.Asshown in Fig. I , a/as2-null erythroblasts exhibited the

morphology of various stages of erythroblasts which was very similar to that of wild-Ewe

e.rythroblasts･ Namely, most of alas2-null erythroblasts were found differentiated to

polychromatophilic or orthochromatic stages, which are of a smaller size with more

condensed chromatin than proerythroblasts. Although the morphology of alas2-null

erythroblasts per se was not different from that of wt'ld-りpe erythroblasts, the cell pellet of

alas2-null erythroblasts were completely colorless which reflects their heme derlCiency (Fig.

le, the right tube). Heme content, as determined by nuorometry, was 1620i45pmol/106cells,

and 1 20±1 5pmol/ 1 06, fTor wild-りpe, and alas21null definitive erythroblasts, respectively･

Expression of genes which are erythroid-specific or irLVOIved in iron metabolism

Next, levels of erythroid-specific gene expression were examined by RTIPCR and

FACS･AsShown in Fig. 2a, P-major globin mRNA, an adult-type globin, was dominant in

deflnitive erythroid cells harvested on day 14, whereas ev globin mRNA, an embryonic-type

globin, was dominant on day 8. β-major globin mRNA in wt'ld-りpe erythroblasts was only

weakly detectable on day 8, and the level in alas2-null erythroblasts was even lower. The

expression level of GATA- I and NF-E2 mRNA was similar fわr both alas2-null definitive and

wild-りpe erythroblasts (Fig. 2a). By flow cytometric analysis, TERl19, a marker of well

differentiated erythroblasts beyond CFU-E, was also fわund to be expressed at a similar level

in alas2-null definitive erythroblasts, asinwi/d-type definitive erythroblasts (Fig. 3). In

-8-contrast, the expression level of this protein in prlmitive erythroblasts was markedly

suppressed in alas2-null cells as compared with wild-type cells (Fig. 3), consistent with

findings in in vivo in a/as21targeted mice (20)･ These results suggest that, in contrast to

alas2-null prlmitive erythroblasts which show maturation arrest, alas2-null definitive

erythroblasts do not show a maturation arrest, despite their heme deficiency･

Heme and iron metabolism in alas21muII derlnitive erythroblasts

ln addition to erythroid-specific genes, expression of genes involved in heme and iron

metabolism was also examined･ Since exon 8 through 10 of the alas2 gene was replaced by

a neomycln resistant cassette in alas2-null erythroblasts, the corresponding portion could not

be amplified by RT-PCR using primer pairs which anneal with exon 7 and exon 10 (Fig. 2b)

(26)･ Interestingly, ALAS I mRNA was highly expressed in alas21null erythroblasts, while it

was not detectable in wild-ore erythroblasts (Fig. 2b). Conversely, H0-1 mRNA, which

encodes the rate-limltlng enZyme in the heme catabolic pathway and is inducible by heme,

was decreased in a/as2-null erythroblasts, Consistent with their heme deficiency (Fig. 2b).

Levels of NRAMP2 mRNA and transferrin receptor protein, which are involved in iron

metabolism, were similarfor bothalas2-null and wt'ld-ore definitive erythroblasts (Fig. 2b

andFig.3).

Next, iron content of definitive erythroblasts was examined･ Siderotic granules,

characteristic of erythroblasts of patients withXLSA, were not observed in alas2-null

definitive erythroblasts by standard iron staining (data not shown), however, iron content of

alas2-null definitive erythroblasts determined by atomic absorption SPeCtrOmetric analysts,

was slgnificantly increased compared to wild-ore definitive erythroblastsI Thus this finding lS

ー9-similar tothat observed in in vivo alas2-null primitive erythroblasts (Fig. 4). This finding

clearly indicatesthat mutation in ALAS2 leads to aberrant iron accumulation in definitive

erythroblasts.

0Ⅹidized status of alas2-null erythroblasts

Since accumulation of iron causes peroxidation of membrane lipids of erythrocytes,

alas2-null erythroid cells may contain a higher level of oxidized substances aS compared with

wt'ld-ore erythroid cells･ This question was examined by now cytometric analysis, using

definitive erythroblasts harvested on day14･Asshown in Fig･ 5, nuorescence intensity

renecting the oxidized products of DCFHDAfollowlng Incubation withcells was higher in

alas2-null erythroblasts than wt'ld-ore erythroblasts･ Next, to study the effect of an oxidized

status of alas2-null erythroblasts on cell aglng, the number of apoptotic cells was examined

by flow cytometricanalysISuslng Anexin V as an indicator for apoptosis･ Significant

fraction of alas2-null erythroblasts on day 14 were apoptotic, however,the percentage of

apoptotic alas2-null erythroblasts was not different from that of wild-りpe erythroblasts,

because the slgnificant number of wild-Ore erythroblasts were apoptotic which renect

completed maturation of cells (data not shown).

-10-Discussion

XLSA has been shown to be due to a mutation of the alas2 gene, and various

mutations have been reported i品the catalytic domain of the ALAS2 protein (10-12).

Abnormal iron accumulation in prImitive erythroblasts in alas2-null mice proved

experimentally that ALAS2 deficiency in fact results in aberrant iron metabolism and is

responsible for sideroblastic anemia in vivo･ However, alas2-null mice died L･n utero by

embryonic day ll･5 before definitive erythropoleSis developed, therefore, the effect of

ALAS2 deficiency on definitive erythropoleSis still remained unclear･ In this study, we

prepared alas2-null definitive erythroblasts uslnganin vitro differentiation system with OP9

stromal cells･ In alas2-null definitive erythroblasts, both morphology and expression of

erythroid-Specific genes were similar to those found in wild-ore definitive erythroblasts,

though the cell pellet of alas2-null definitive erythroblasts was completely colorless,

suggestlng a lack of heme･ In fact, chemical quantification of cellular heme content by

fluorometry demonstrated that alas21null definitive erythroblasts contained less than10%

●

heme found in wild-りpe Cells･ Furthermore, iron content of alas2-null definitive

erythroblasts was approximately 3-fold higher thanthat of wild-りpe cells, a finding similar to

those in alas2-null primitive erythroid cells in vivo in mice･ These results clearly establish

that ALAS2 deficiency results in aberrant iron metabolism bothin prlmitiveand definitive

erythropoiesis, and confirm that ALAS2 deficiency is responsible for the development of

sideroblasts and severe anemia in XLSA.

An important aspect in t'n vitro culture should also be noted that siderotic granules,

the biochemical hallmark of XLSA in in vivo in patients, were not detected in alas2lnull

Ill-definitive erythroblasts･While certain differentiated erythroblasts, e･g･, Polychromatophilic

or orthochromatic erythroblasts, developed in culture, enucleated erythrocytes were not fbund･

Erythrocytes take up and achieve a 7000 fわld greater iron concentration in cells than plasma

which occurs at a very late stage of differentiation (I). Perhaps in viEro in culture,

environment to support full erythroid differentiation may be inadequate･ Altematively,

uplike alas2-null erythroblasts, residual ALAS activity in patients wi,th XLSA may contribute

to drive iron into mitochondria･ It should also be noted that ALASl mRNA is upregulated in

alas2-null erythroblasts, renectlng de-repression of alasI gene expression by heme deficiency,

while this level must be yet insufrlCient fわr the support fわr iron transport into mitochondria in

erythroblasts.

To date, there has not been any report which describes the induction of sideroblasts in f〃

vL'tro culture uslng hematopoletic cells of patients with XLSA･ This finding does not

indicate, however, that no sideroblasts can be developed uslng ln Vitro culhre, since

sideroblasts were detected in BFU-E from bone marrow cells of patients with primary

●

acquired sideroblastic anemia (PASA) (27, 28). Thus, E'n vt'lro culture system is capable for

supportlng development of sideroblasts, at least of the PASA origin･ However, PASA is an

acquired clonal disorder and is not due to a slngle gene mutation as is the case with XLSA,

thus the mechanism for the fわrmation of sideroblasts must be different between these

disorders･While alas21null erythroblasts accumulate an excessive amount of iron,

erythroblasts or PASA may be perhaps more aggressive in iron accumulation into their

mitochondria than erythroblasts of XLSA.

Iron isknown to produce active hydroxyl radicalsthat are highly toxic to cells･ It

ー12-has been shown thatferritin iron can also partlCIPate in the generation of reactive oxygen

species, and cause oxidative tissue damages (29, 30). In this study, we found that alas2-null

erythroblasts produced more oxidized conversion from DCFHDA than did wE･ld-ore cells.

Consistent with our findings, erythrocytes in sickle cellanemia and thalassemia are known to

contain excess amounts or iron as well as peroxidized lipids in the membrane. Thus, similar

to sickle cell anemia and thalassemia erythrocytes, erythrocyte! in XLSA may also be more

susceptible to oxidative damages than intact erythrocytes, and such a mechanism may also

contribute to the aggravation of anemia in XLSA･ If so, treatment or XLSA with

iron-chelating agents and/or antioxidants may be useful and such therapy should be evaluated for

its efficacy.

In conclusion, Our study is the first which systematically examined and described the

characteristics of null definitive erythroblasts. Aberrant iron accumulation in

alas2-null definitive erythroblasts unequlVOCally substantiates the notion that ALAS2 deficiency lS

responsible fわr the development ofsideroblasts and severe anemia in patients with XLSA.

-13-Acknowledgment

Authors thank Ms･ Y Nishiyama, K･ Sato, K Kozawa, A Aizawa, Dr･ T Miura f♭r their

teclmical assistance. Authors are alsoJgrateful to Dr･ T Nakano for providing OP9 cells･

-14-Referen ces

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in erythroid cells. Blood. 1997;g9:1-25.

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[Review】. Trends Biochem S°i. 1995;20: 105-108.

3･ Pasanen AVO, Salmi M, Vuopio P, Tenl1unen R･ Heme biosypthesis in sideroblastic anemia･

Int J Biochem. 1980;12: 969-974.

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6･ Kappas A･, Sassa S, Galbraith RA, Nordmam Y･ The Porphyria in The Metabolic and

Molecular Basis or I血erited Disease (eds Scriver, CR, Beaudet, AL, Sly, WS 良 Valle, D)

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ー18-Legends to Figures

Figure l･  Morphology of Cultured erythroid cells

Wild-りpe and alas21null ES cells were cultured with OP9 cells and floating cells

were collected on day 8 and day 14l May-Gruenwald-Giemsa stainof (a) wild-Ewe and

alas2-null (b) erythroblasts on day 8, (C) wild-ore and (d) alas2Tnull erythroblasts on day 14.

Original magnification is x400･ (e) cell pellets of wild-ore (le氏) and alas2-null (right)

erythroid cells.

Figure2･  Expression of erythroid-specirIC genes and genes involved in

biosynthesis and catabolism

Total RNA was extracted fTrom harvested cells, and 2pg of total RNA was

reverse-transcribed･ Then target genes were amplified by PCR with specific prlmerS aS described in

Materials and Methods･ (a) erythroid-Specific genes (b) genes involved in heme biosynthesis

and catabolism, and in iron transport･

Figure 3･  Expression ofTERl19 and transferrin receptor examined by FACS

(a) wt'ldやpe erythroblasts on day 8, (b) alas2-null erythroblasts on day 8, (C) wt'ld-type

erythroblasts on day 14, (d) alas2-null erythroblasts on day 14.

Figure 4･ Iron content of dertnitive erythroblasts

lron content of cells haⅣested on day 14 was determined by atomic absorptlOn

-19-spectrometry as described previously (20). Iron content was- 1 15±10･6mg and 389･ 1±1 1･4ng

/lx 106 cells,for wild一昨e and alas2-null cells, respectively･

Figure 5.  0Ⅹidized state of alas2-rLull dertnitive erythroblasts comparedwith

wild-type erythroblasts

Cells harvested on day 14 Were incubated and cellular nuorescence intenslty Was

examined by FACS. alas2-null erythroblasts contain more oxidized cells than wild-type

erythroblasts, as indicated by a shift of peak toward right for alas2-null definitive

erythroblasts (b) compared to wt'ld-type cells (a). The number of positive cells are 60･55%

vs 38.79%,for alas2-null (d) and wild-type cells (C), respectively.

-20-鶴

C, +

WT ALAS2(-)

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(a) WT.002 '_i, 白謦粫剃梯 ･::､遇､'f;,'':::I. ･姓:- I.r.i:1.< ..滋.i:..LIE -I;;機と義盛FF:- ■■■ ■ 44-JW 呈 爾r .JVよJ ･ヽ■ こ.ち ､i:LI. 冢ﾈ6Rﾓ｢ 占0'"''j'占1川‥j■占2日''';占吉川'l1"64 TFR FITC (C) Ei ⊂〉 ｣.■■■■ 【むコ ⊂〉

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Lr:41tE.:･.-!i:;心P芦 -261 E:･亡FH-D A 石工P･:-.い心P蒜 DCFトトD A

(l爪l OE=L O爪,TfjP O的 0

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