523

Biochimica et Biophysica Acta, 5 4 3 ( 1 9 7 8 ) 5 2 3 - - 5 2 9 Q Elsevier/North-Holland

Biomedical Press

BBA 28650

IRON UPTAKE STUDIES ON ERYTHROID CELLS

A. B A R N E K O W

* a n d G. W I N K E L M A N N

Institut fi~r Biologic II, Lehrstuhl Mikrobiologie I, Universit~'t Tiibingen, D-74 Tiibingen, A u f der Morgenstelle 1 (F.R.G.) (Received March 15th, 1978)

Summary Iron uptake from SSFe-labelled transferrin, ferric citrate and the two fungal sideramines, ferricrocin and fusigen was studied using four erythroid cell cultures: Friend virus-transformed erythroleukemic cells (mouse), transformed bone marrow cells, Detroit-98 (human), reticulocytes (bovine), bone marrow cells (rabbit). The present comparative study reveals pronounced differences in iron uptake behaviour. Compared to transferrin, ferric citrate and the sideramines are preferred in transformed erythroid cells. In reticulocytes transferrin and ferric citrate showed a better uptake as compared to the two sideramines. Primary bone marrow cells showed nearly equal iron uptake rates using transferrin or ferricrocin.

Introduction

Various synthetic iron-chelating agents have been studied for iron transfer into erythropoietic cells [1]. It has been reported, that iron bound to transferrin is utilized by reticulocytes for hemoglobin synthesis more efficiently, than is iron bound to many small molecular iron-complexing agents [2]. In reticulocytes iron from certain chelates, such as ferric citrate, nitrilotriacetic and N-~-hydroxyethyliminodiacetic acid may, however, be utilized almost as well as transferrin-bound iron. Hemmaplardh and Morgan [3] studied the iron donor properties of several low molecular weight synthetic iron chelates in reticulocytes. They assumed, that iron from the synthetic chelates, is used for hemoglobin biosynthesis after iron exchange to intracellular transferrin. The existence of intracellular transferrin in reticulocytes has been shown using 12SI-labelled transferrin [4,5]. Although an iron transfer to intracellular transferrin may occur in reticulocytes, other iron utilization mechanisms seem to be * Present address: Institut fiir Bioehemie I, Universit~t Heidelberg, D-69 Heidelberg, l m N e u e n h e i m e r Feld 328, G.F.R.

524 involved in Friend erythroleukemic cells. Kluge et al. [6] have shown, that a subclone (F4) of Friend leukemia cells, which could be cultivated serum free for several months was inducible for hemoglobin biosynthesis after addition of iron sulfate, erythropoietin and dimethylsulfoxide. Thus transferrin-iron seems obviously n o t to be an absolute requirement for heme synthesis in these cells. In a preceding paper we have shown that Friend erythroleukemia cells may utilize microbial iron chelates for iron incorporation into hemoglobin [7]. Iron from the two fungal sideramines, ferricrocin and fusigen, was incorporated into hemoglobin to about the same extent as iron from ferric citrate. Iron from transferrin, however, led to insufficient specific activity of labelled hemoglobin, indicating a comparably slow utilization of exogenous transferrin. Therefore it was of interest to compare the uptake behaviour of Friend erythroleukemia cells [8] using different iron chelates, such as transferrin, ferric citrate, fusigen and ferricrocin. In addition iron uptake was studied on some other erythroid cell cultures. Materials and Methods Chemicals. 5SFeC13 in 0.1 M HC1 (carrier free) was purchased from Amersham Buchler, Great Britain. Transferrin (human), substantially iron free, was obtained from Sigma Chemical Co. St. Louis, Mo., U.S.A. Ferricrocin and fusigen were gifts from Professor H. Diekmann, Hannover. Preparation of SSFelabelled chelates was essentially as described previously [9]. Cell cultures. Friend virus-infected murine erythroleukemic cells (B8/3A) were kindly supplied by Professor Dr. W. Ostertag, Max-Planck-Institut filr experimentelle Medizin, Abt. Molekulare Biologie, GSttingen. Passaging of Friend cells was done all 4--5 days by adding 20 ml of fresh medium to 0.3 ml cell suspension. The culture medium was the same as for the human sternal marrow cells. Cell density was about 106 cells/ml medium. Human sternal marrow cells (Detroit-98) were obtained from Flow Laboratories, Scotland. Like the Friend cells t h e y were cultivated in minimum essential medium with Earle's salts containing 15% fetal bovine serum, 1% L-glutamine (200 mM), 1% non-essential amino acids and 100 units penicillin/ml. Trypsinisation of confluent monolayers of Detroit-98 cells was performed with 10% trypsin (1 : 250) in Ca 2÷- and Mg~÷-free balanced salt solution. Both cell cultures, the transformed Detroit-98 and the Friend erythroleukemia were incubated at 37 ° C. Reticulocytes-rich blood was obtained from the upper layer of freshly sedimented bovine red cells and washed three times with cold 0.9% NaC1 solution by sedimentation {2000 rev./min), before use for uptake measurements. Primary, non-transformed bone marrow cells were obtained from the femora of 6-month-old rabbits. The two femora were rapidly opened at both ends and flushed with serum-free medium and used after repeated sedimentation in fresh serum-free medium (3000 rev./min). All cells were counted in a counting chamber and the vitality was estimated by addition o f trypane blue. The incubation assays contained serum-free minimal essential medium and

525 the SSFe-labelled iron chelates as ferric citrate, transferrin, ferricrocin and fusigen. Incubation assays. (1) For the uptake of chelated iron into Friend erythroleukemia cells, 4~lay-old cultures were used, which had been induced to hemoglobin synthesis b y addition of 1.5% dimethylsulfoxide [7]. After repeated sedimentation (3000 rev./min) in serum-free medium cells were transfered to an 100 ml incubation vessel containing 0.2 nmol SSFe-labelled iron chelates in 25 ml serum-free minimal essential medium. The incubation was performed at 37°C in a gentle shaking water bath. At various time intervals 0.5-ml samples (10 ~ cells) were carefully layered over 2 ml ice-cold osmotic stabilizing solution containing 10% Ficoll in phosphate-buffered saline. After centrifugation (10 min at 10 000 X g, Sorvall RC2-B), the upper layer was removed by aspiration and the Ficoll layer was rinsed with 1 ml distilled water to remove residual radioactivity. After removing the Ficoll solution, the pellet was dissolved in 1 ml 1% Triton X-100 solution. For the measurement of radioactivity 0.5 ml of lysed cells was taken in 10 ml Unisolve-1 and counted in a liquid scintillation counter (Mark I, Nuclear Chicago). (2) Rabbit bone marrow cells were incubated like all the other cells at 37°C in a gentle shaking water bath. The incubation mixture consisted of 5.5 ml standard medium used b y Schulman [10], containing 0.5% bovine serum albumin and 0.136 nmol SSFe-labelled iron chelate. Cell density was 2 . l 0 T ceUs/ml. At various time intervals samples of 0.5 ml (107 cells) were removed and treated essentially as described for the Friend erythroleukemia cells. (3) Reticulocytes-rich blood was obtained from the upper layer of freshly sedimented bovine red blood cells (3000 rev./min for 20 min). The cells were washed three times b y sedimentation in isotonic 0.9% NaC1 solution and incubated in 1 ml isotonic NaC1 solution containing 0.034 nmol SSFe-labelled iron chelate. To avoid color quenching b y heme, the disappearance of radioactivity in the supernatant fluid was measured. At appropriate time intervals the samples were sedimented and 0.1 ml of the supernatant was counted for radioactivity in 10 ml Unisolve-1. (4) The monolayer cultures Detroit-98 were incubated in small scintillation vials, previously treated with concentrated H2SO4. After epithelial monolayers were obtained, the vials contained approx. 3" 105 cells. The monolayers of several vials were incubated with a solution containing 1 nmol SSFe-labelled iron chelate in serum-free medium (minimal essential medium). At times indicated, the incubation medium was carefully removed by aspiration and the epithelial cell layer was rinsed three times with serum-free medium. After adding 10 ml Unisolve-1 (Koch Light Lab., England) the samples were allowed to stand over night and counted subsequently for radioactivity. Results

All graphs are plotted on data from at least four determinations. As shown in Fig. 1, Friend erythroleukemia cells take up iron from labelled ferric citrate, ferricrocin and fusigen more rapidly than from transferrin. The extent and the differences in iron chelate uptake were found to be similar in dimethylsulfoxide-treated as well as in non-induced cell cultures. Thus chelate-iron uptake

526

~m

c~

A~A

mA

~

A

~.~.--..

b. - - - - ' - - A

Time

I

~

(h3

Fig. 1. U p t a k e o f i r o n i n t o F r i e n d v i r u s - t r a n s f o r m e d e r y t h r o l e u k e m i a cells ( B S / 3 A ) a f t e r i n d u c t i o n o f h e m o g l o b i n s y n t h e s i s . T h e cells w e r e i n c u b a t e d in s e r u m - f r e e m e d i u m containing 5 S F e . l a b e l l e d t r a n s f e r r i n ( • ) , ferric c i t r a t e (m), f e r r i c r o c i n (~) and fusigen (a) and s u b s e q u e n t l y i s o l a t e d b y s e d i m e n t a t i o n t h r o u g h 2 m 1 1 0 % F i c o l l s o l u t i o n . Results are m e a n s o f five different e x p e r i m e n t s (standard d e v i a t i o n ~ 2 % ) .

does not depend on intracellular activity of hemoglobin biosynthesis. We found only slight differences in the transport properties of ferric citrate and the two fungal sideramines. As shown in a preceding paper ferric citrate, ferricrocin and fusigen, also gave rise to very high specific activity of hemoglobin, which exceeds that o f transferrin [9]. Our results on iron chelate transport may support the view that in Friend erythroleukemia cells, transferrin is not preferably used as an iron chelate for hemoglobin synthesis. The transformed bone marrow cells of the t y p e Detroit-98 revealed similar transport behaviour (Fig. 2) with chelated iron as Friend erythroleukemia cells. The sideramines ferricrocin and fusigen were, however, somewhat less effective than ferric citrate. The superiority of small molecular weight iron chelates over transferrin was evident also in these transformed cells. Contrary to the Friend cells, the transformed bone marrow cells exhibit significant higher transferrin iron uptake during a 5 h incubation period. For comparative purposes we also studied the chelate iron uptake behaviour of non-transformed bone marrow cells. Bone marrow cells of the femora of adult rabbits were used for the transport assay immediately after preparation. As shown in Fig. 3, iron uptake from transferrin and ferricrocin is very similar. Transferrin iron uptake, however, proceeds more rapidly than ferricrocin iron uptake. In general chelate iron uptake seems to be very fast during the first minutes, and is only slowly increased during further incubation. In contrast with the transformed cells, the absolute amount of chelate iron taken up by the primary cell culture is very low.

527 100 •

D

-~ o

o u. =

50,

Time

(h)

Fig. 2. U p t a k e o f i r o n i n t o cells of a p e r m a n e n t c u l t u r e of h u m a n s t e r n a l b o n e m a r r o w cells ( D e t z o i t - 9 8 ) . Cells w e r e g r o w n in scintillation vials u n t i l c o n f l u e n c y of cells was a c h i e v e d , w a s h e d w i t h i s o t o n i c saline a n d i n c u b a t e d in s e r u m - f r e e m e d i u m c o n t a i n i n g 5 5 F e - l a b e l l e d t r a n s f e r r i n (A), ferric c i t r a t e (m), ferric r o c i n (~) a n d fusigen (o). R e s u l t s are m e a n s of five d i f f e r e n t e x p e r i m e n t s ( s t a n d a r d d e v i a t i o n < 2 % ) .

~-, 0,4~

o

r

% 0.3

0.2-

Time (h) Fig. 3. U p t a k e of i r o n i n t o b o n e m a r r o w cells of r a b b i t f z o m 5 5 F e . l a b e l l e d t r a n s f e r r i n (A) a n d f e r r i c r o c i n (~). R e s u l t s are m e a n s o f f o u r d i f f e r e n t e x p e r i m e n t s ( s t a n d a r d d e v i a t i o n < 2 % ) .

u_

~

5

5

Time (h) Fig. 4. U p t a k e o f i~on i n t o r e t i c u l o c y t e s - r i c h c a l f b l o o d . T h e ce~s were i n c u b a t e d w i t h 5 5 F e - l a b e n e d transferrL~ (A), f e r r i c c i t r a t e (11), f e r r i c r o c i n (~) and fusigen (D). R e s u l t s are means o f five d i f f e r e n t e x p e r i m e n t s ( s t a n d a r d d e v i a t i o n < 2%).

528 In reticulocytes-rich blood iron uptake was studied following the disappearance of 55Fe-labelled transferrin, ferric citrate, ferricrocin and fusigen. As shown in Fig. 4, a characteristic feature of chelate iron uptake in reticulocytes is the preference for transferrin iron. The iron uptake from the two sideramines, ferricrocin and fusigen is comparatively slow. Ferric citrate was found to be as effective as an iron d o n o r as transferrin. After incubation of 3 h, iron uptake from ferric citrate reached the values obtained with t~ansferrin. The observed iron uptake from ferric citrate proceeded more linearly than that from transferrin, which seemed to be saturated after 1 h of incubation. Discussion As previously reported b y Martinez-Medellin and Schulman [ 11] the data of the present investigation indicate, that under in vitro conditions, iron uptake from transferrin occurs in reticulocytes and bone marrow cells. Although the transformed erythroid cells also utilize iron from transferrin, iron from the microbial iron chelates and from ferric citrate was taken up in significantly higher amounts. It may be argued that the superiority of the transformed cells in iron uptake from low molecular weight iron complexes might depend on existing intracellular transferrin, as was proposed for reticulocytes [3]. It must be pointed out, however, that the stability constants of sideramines (K = 103°) [12], are in the same order of magnitude as those of transferrin iron [13]. Bates et al. [14,15] have reported times required for equimolar concentrations of three typical chelates to half-saturate the sites of human apotransferrrin. They are for nitrilotriacetate: 3 s, citrate: 8 h, ethylendiaminetetraacetate: a b o u t 4 days. In addition the stability constant of iron for transferrin is a b o u t six times greater than that measured for ferric EDTA. Thus, when identical stability constants are present, an iron exchange mechanism would occur much more slowly. In biological systems, however, the exchange rates may be faster, as iron is continuously removed from equilibrium reaction. Nevertheless the observed high iron uptake rates from sideramines like ferricrocin and fusigen cannot be interpreted on the basis of iron transfer to cellular transferrin. Transferrin-bound iron as compared to other iron chelates is not rapidly taken up by Friend erythroleukemia cells. Also in the other transformed cell type, Detroit-98, transferrin iron uptake is relatively slow. The only cells, which significantly prefer transferrin are reticulocytes. The transformed erythroid cells do not appear to require extracellular bound iron for suboptimal growth or for stimulation of hemoglobin synthesis by dimethylsulfoxide. As reported b y Kluge et al. [6], serum-free cultivated cells of Friend erythroleukemia (subclone F4), utilize iron added as iron ascorbate for hemoglobin biosynthesis when induced with dimethylsulfoxide. Thus the hemoglobin biosynthesis is not dependent on intracellular transferrin in erythroleukemia cells. As we were able to show, these cells use external low molecular weight iron chelates containing trivalent iron for hemoglobin biosynthesis [7]. Spiro et al. [16,17] have reported that ferric citrate is not a low molecular weight iron chelate at physiological pH 7.2, but behave like a polymer of an average molecular weight of 2 • l 0 s. However, in the presence of excess citrate

529

(20 times) low molecular weight species, tentatively identified as ferric dicitrate, arise. Thus ferric citrate, like the trihydroxamate iron complexes is considered as a low molecular weight chelate during our uptake measurements, whereas transferrin is a high molecular weight iron chelate containing 2 Fe/mol with a molecular weight of about 90 000 [13]. In fact, the transport studies confirm similar transport behaviour of both sideramine iron and citrate iron uptake, especially in the transformed cell cultures. On the other hand, it may be assumed, that transformed and non-transformed erythroid cells differ greatly in their membrane permeability properties for chelates, such as ferricrocin and fusigen. Thus it is likely that the iron-utilizing sites are more accessible to low molecular weight iron chelates, when the cells are transformed. The transferrin iron uptake in erythroid cells of primary cultures requires special surface receptor sites [18,19], which develop concomitantly during erythroid differentiation. The cell membrane of the transformed erythroid cells may possibly be altered, permitting preferential iron transfer from low molecular weight iron complexes. Further studies will be necessary to decide whether altered iron transport is a general characteristic of transformed erythroid cells. Acknowledgements This investigation was supported by the Deutsche Forschungsgemeinschaft. The authors are grateful to Dr. A.K. Walli for reading the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Morgan, E.H. (1971) Biochim. Biophys. A c t a 244, 103--116 Princiotto, J.V., Rubin, M., Shashaty, G.C. and Zapolski, E.J. (1964) J. Clin. Invest. 43, 825--833 Hemmaplardh, D. and Morgan, E.H. (1974) Biochim. Biophys. A c t a 373, 84--99 Morgan, E.H. and Appleton, T.C. (1969) Nature 223, 1371--1372 Sly, D.A., Grohlich, D. and B e z k o r o v a i n y , A. (1975) Biochim. Biophys. Acta 385, 36--40 Klu~e, N., Gaedicke, G., Steinhelder, G., Dube~ S. and Ostertag, W. (1974) Exp. Cell Res. 88, 257-263 Barnekow, A. and W i n k e l m a n n , G. (1976) Exp. Hematol. 4, 70--74 Friend, C. and Scher, W. (1975) Ann. N.Y. Acad. Sci. 243, 155--163 Barnekow, A., Winkelmann, G. and Z~Lhner, H. (1974) Arch. Microbiol. 100, 329--340 Schulman, H.M. (1967) Biochim. Biophys. Acta 148, 251--255 Martinez-Medellin, J. and Sehulman, H.M. (1972) Biochim. Biophys. A c t a 264, 272--284 Anderegg, G., L'Eplatenier, F. and S e h w a r z e n b a c h , G. (1963) Helv. Chim. Acta 46, 1400--1409 Aisen, P. (1973) in Inorganic Biochemistry, Vol. 1, pp. 280--303, Elsevier A m s t e r d a m Bates, G.W., Billups, C. and Saltman, P. (1967) J. Biol. Chem. 242, 2810--2815 Bates, G.W., Billups~ C. and Saltman, P. (1967) J. Biol. Chem. 242, 2816--2821 Spiro, T.G., Pape, L. and Saltman, P. (1967a) J. Am. Chem. Soc. 89, 5555--5559 Spiro, T.G., Bates, G. and Saltman, P. (1967b) J. Am. Chem. Soc. 89, 5559--5562 Jandl, J.H., Inman, I.K., Simmons, R.L. and Allen, D.W. (1959) J. Clin. Invest. 38, 161--185 Fielding~ J. and Speyer, B.E. (1974) Biochim. Biophys. A c t a 363, 387--396