Biochimica et Biophysica Acta, 1093 (1991) 20-28 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100175B
20
BBAMCR 12935
The uptake of inorganic iron complexes by human melanoma cells Des R i c h a r d s o n a n d Erica B a k e r Department of Physiology, University of Western Australia, Nedlands. Western Australia (Australia)
(Received 6 August 1990) (Revised manuscript received 19 December 1990)
Key words: Melanotransferrin; Transferrin; Iron; (Melanoma cell)
The human melanoma cell line, SK-MEL-28, expresses high levels of melanotransferrin. The uptake of inorganic iron (Fe) complexes compared to transferrin.bound Fe by these cells has been investigated to determine whether melanotransferrin has a role in ice uptake. The mechanisms of ICe uptake have been characterised using SgFe complexes of citrate, nitrilotriacetate, desferrioxamine, and SgFe added to Eagle's minimum essential medium (MEM) and compared with human transferrin Off) labelled with S9Fe and iodine-125. Iron uptake from the ICe complexes of citrate, nitrilotriacetate and MEM were similar, and far greater than that from Tf at the same ICe concentration (2.5/~M). Ammonium chloride and a monodonal antibody to the transferrin receptor (42/6), had no effect on the uptake of ICe from inorganic ICe complexes, suggesting that receptor-mediated endocytosis of "If was not involved. The monoclonal antibody, 96.5, specific for melanotransferrin did not alter total Fe uptake but slightly increased the proportion of ICe internalised, possibly due to the modulation of the antigen by the antibody. However, from the time required for modulation to occur (~ 2 h), the small increase in internalisation observed and the fact that no increase in total cell ICe occurred, it is suggested that melanotransferrin has little role in ICe uptake.
Introduction The mechanism of cellular iron (Fe) uptake from membrane impermeable and permeable Fe chelates is being studied in several laboratories [1-3] in an effort to understand the process of Fe transport across the membrane after Fe release from transferrin (Tf). An active transport process probably mediated by a membrane-bound Fe-binding protein(s) has been proposed from several recent studies in reticulocytes [1-3]. With regard to the membrane transport of Fe, we are investigating the role of a membrane-bound Fe-binding molecule known as p97, or melanotransferrin (MTf), which is found predominantly in human melanoma cells [4]. Melanotransferrin has several characteristics in common with serum Tf. These are: (i) it is a sialoglycoprotein (13~ carbohydrate); (ii) it has 37-39% se-
Abbreviations: FCS, fetal calf serum; MEM, Eagle's minimum essential medium; BSS, balanced salt solution; NTA, nitrilotriacetate; BSA, bovine serum albumin; gPR, gram of protein; Tf, transferrin; MTf, melanotransferrin. Correspondence: E. Baker, Department of Physiology, University of Western Australia, Nedlands, Western Australia, 6009, Australia.
quence homology with human serum Tf, human lactoferrin and chicken Tf; (iii) it has a high interdomain sequence homology (46%), which is greater than that found for other Tfs (33-41%); (iv) the MTf gene is on chromosome 3, as are those for Tf and the Tf receptor (TfR); and (v) the purified protein can bind Fe from Fe(III) citrate complexes [5-9]. Recent studies by the authors [10,11] have identified a saturable, temperaturedependent, Fe-binding component on the membrane of melanoma cells, with properties consistent with those attributed to MTf. Seligman et ai. [12] have suggested that MTf may be important in Fe uptake from non-Tf-bound Fe in the plasma. In addition, the observation that MTf bound Fe from Fe complexes of citrate [6], encouraged us to investigate the possible role of this membrane-bound protein in Fe uptake from small molecular weight (Mr) Fe complexes. The mechanisms of Fe and Tf uptake have been determined by standard procedures similar to those described previously [13,14], using pronase to separate internalised (pronase-resistant) and membrane-bound (pronase-sensitive) Fe and Tf. The results indicate that melanoma cells can accumulate Fe from inorganic Fe chelates much more efficiently than from diferric Tf. The mechanism of Fe uptake probably does not involve
21 the receptor-mediated endocytosis of Tf as both NH4C1, and the monoclonal antibody (MoAb), 42/6, which inhibits binding of Tf to the TfR [15], had no effect. A MoAb specific for MTf (96.5), did not affect total Fe uptake and only caused a slight increase in the proportion of Fe which was internalised, which may be due to the modulation of MTf by the MoAb. However, from the time required for modulation to occur ( - 2 h), the small extent of internalisation observed and the fact that there was no increase in total Fe uptake, it is suggested that MTf has little role in the uptake of Fe from inorganic Fe complexes. Materials and Methods
Reagents Iron-59 (as ferric chloride in 0.1 M HCl) and iodine125 (as sodium iodide) were purchased from Amersham International, U.K. Pronase was purchased from Boehringer-Mannheim, Mt. Waverley, Australia. Eagle's modified minimum essential medium (MEM) as Autopow and fetal calf serum (FCS) were supplied by Flow Laboratories, Annandale, Australia. Penicillin (Crystapen-BenzylpeniciUin sodium B.P.) was obtained from Glaxo, Boronia, Australia. Bovine serum albumin (BSA; 9870 pure, fatty acid free), human apo-Tf, L-glutamine and Hepes was obtained from Sigma, St. Louis, MO, U.S.A. Non-essential amino acids (100 × concentrate) and trypsin-versene solution (1 x ) were obtained from Commonwealth Serum Laboratories, Melbourne, Australia. Balanced salt solution (BSS) was prepared by the method of Hanks and Wallace [16]. All other chemicals were of analytical reagent quality.
Protein purification and labelling Human apoTf was prepared and labelled with 59Fe and iodine-125 by the methods of Hemmaplardh and Morgan [17] and McFarlane [18], as described previously [10]. Monoclonal antibodies specific for MTf (96.5 and 133.1) and the TfR (42/6) were generous gifts from Dr. J.P. Brown (Oncogen, Seattle, U.S.A.) and Dr. I. Trowbridge (Salk Institute, San Diego, U.S.A.), respectively.
Cell culture The human melanoma cell line, SK-MEL-28 (American Type Culture Collection, Rockville, MD), was used as these cells have the highest concentration of MTf of all cell types studied (300 000-380 000 sites/cell; Refs. 5, 19). Cells were grown as described previously and subcultured using 1 mM EDTA in calcium/ magnesium free PBS [10,20,21]. Procedures used to check cell viability and dedifferentiation of the cell line were the same as described previously [10].
Experimental procedures Preparation of inorganic iron complexes. Initial experiments examined the uptake of Fe from small molecular weight Fe chelates. The chelating agents, desferrioxamine (DFO), nitrilotriacetic acid (NTA) and citrate were mixed at a 0.9, 10 and 100-fold molar excess to iron-59, respectively, to prevent hydrolytic polymerisation of Fe [22]. The Fe chelate was then added to MEM which had been supplemented with 17o non-essential amino acids, BSA (5 mg/ml) and Hepes (20 mM; pH 7.4). It should be noted that under the experimental conditions used, a complex equilibrium would probably exist between the Fe in the added chelators and those already present in MEM. Indeed, it was deemed worthwhile to examine whether 59Fe directly added to MEM could donate Fe(III) ion to cells. The MEM contains many good Fe chelating agents at high concentration, including amino acids, sugars, vitamins, bicarbonate and phosphate (Flow Laboratories Catalogue). Considering only the contribution from amino acids and glucose, there was greater than a 6000-fold molar excess of chelating agent to Fe at an Fe concentration of 2.5 #M. Hence, the polymerisation of Fe would be unlikely. Indeed, MSssbauer spectroscopy studies (St. Pierre, Richardson, Baker and Webb, unpublished data) demonstrated that polynuclear Fe complex formation was very low using this protocol and that Fe was not taken up by cells as polynuclear Fe complexes. It is prudent to note that such : mixture of chelating agents may be more physiologically relevant, as Fe complexes of maino acids, sugars, vitamins and to a lesser degree, proteins, have been proposed as candidate ligands involved in the intracellular labile Fe pool and non-Tf-bound Fe in serum [23,24]. Similar reasoning has been applied in studying zinc(II) uptake by human fibroblasts [25]. In some experiments the uptake of ferric and ferrous Fe (2.5 #M) from sucrose (0.27 M; pH 6.5) during a 30 rain incubation was investigated. Solutions were prepared as described by Morgan [3]. These solutions were prepared immediately before use. All subsequent procedures were the same as those described below for experiments done in MEM.
Washing procedure to deplete cells of bovine transfertin. An important prerequisite in these studies on the uptake of small Mr Fe complexes was to ensure that the incubation medium was free of Tf. Melanoma cells were grown in the presence of 3070 FCS which contained bovine Tf, which can bind to the human TfR [26]. Several workers have depleted cells of Tf by three separate 15 rain incubations at 37°C [2,3]. Previous work in melanoma cells using human Tf [11] has shown that most intracellular Tf is released within 30 min of incubation. To ensure depletion of bovine Tf, three separate 30-45 min incubation periods at 37°C in MEM were used.
22 The observation that NH4CI (15 mM) and the monoclonal antibody, 42/6, which sterically inhibits the binding of Tf to the human TfR [15], had no effect on Fe uptake from inorganic Fe complexes indicated that this protocol effectively removed bovine Tf from the cell. Uptake of iron and transferrin. To measure the uptake by melanoma cells of Tf, Tf-bound Fe and Fe-bound to small Mr chelating agents, the medium was replaced after 24-48 h of culture with prewarmed/pregassed (95~ air; 5~ CO2) MEM containing BSA (5 mg/ml), 20 mM Hepes (pH 7.4) and the radiolabel. All experiments were performed on totally confluent plates. At the end of the incubation with the radiolabel, the plates were placed on ice, the medium decanted and the cell monolayer washed four times with ice-cold BSS. Increasing the number of times the plates were washed prior to the addition of pronase did not substantially reduce membrane-bound Fe or internalised Fe. As described previously [10], the amount of radioactivity internalised by the cells was measured by incubation with pronase (1 mg/ml) for 30 rain at 4°C, to separate membrane-bound Fe and Tf [27]. Longer incubations in pronase, up to 150 rain, did not increase the amount of Fe or Tf released. Subsequent procedures were the same as those described previously [10]. The distribution of intracellular Fe into ferritin, stromal-mitochondrial membrane and ferritin-free cytosol was determined as described previously [11]. Effect of monoclonal antibodies on iron uptake. Cell plates which had been washed to remove bovine Tf (see above) were preincubated with a saturating concentration of the MoAb (5/tg/ml; Ref. 29) for up to 24 h. After the required preincubation period the medium was replaced with medium containing MEM-SgFe complexes or SgFe-t2Sl-Tf and the same concentration of MoAb. All subsequent procedures were the same as those described above. Effect of ammonium chloride on iron uptake. Washed cells were preincubated with MEM containing NH4CI (15 mM) for 15 rain, then MEM-iron-59 or SgFe-t2SI-Tf was then added to a final Fe concentration of 2.5/~M and the incubation continued for up to 2 h. All subsequent procedures were the same as those described above. U p t a k e o f Fe is expressed a s / L m o l o f Fe p e r g r a m o f
protein (gPR). The data are expressed as mean 4-S.E. (number of experiments) with 2-12 replicates in each experiment. Experimental data were compared using the Student's t-test. Results were considered statistically significant when P < 0.05. Results
General characteristics of iron uptake The internalised uptake of Fe from complexes of citrate, nitrilotriacetate and MEM was linear with time
1.5" 0
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Fig. 1. The uptake of iron from inorganic iron complexes and transferrin as a function of time. Cells were incubated for up to 2 h in the presence of the small molecular weight iron complexes and Fe-trans-
ferdn at an iron concentrationof 2.5/~M. The cellswerethen washed and treated with pronase. (a) Internalised iron uptake from the iron complexes of citrate, MEM, NTA and from diferric Tf. (b) The proportion of iron internalised for iron complexesof citrate, MEM, NTA and from diferric Tf. (c) Membraneiron uptake from the iron complexes of citrate, MEM, NTA and from diferric Tf. Results are expressed as mean±S.E. (two experiments; four determinations).
up to 2 h (Fig. la). The Fe complexes of citrate and MEM appear slightly more effective as Fe donors than the Fe complex of NTA. Quantitatively, Fe uptake from small M r complexed Fe was far greater than that from Tf (Fig. la). As the Fe complexes of MEM were considered more physiologically relevant than citrate and NTA (see above), and since the Fe donating properties were similar, MEM-complexed Fe was used to further examine the mechanism of Fe uptake. The rate of internalised Fe uptake from MEM-Fe complexes was 440000_ 40000 [6] atoms Fe/cell per min, which was approx. 5.5-times greater than Fe uptake from diferric Tf (80 000 _ 7000 [8] atoms Fe/cell
23 per min; see Ref. 10) at the same Fe concentration of 2.5 /~M (Fig. la). The rates of uptake of Fe from complexes of NTA and citrate were similar to those described for MEM chelated Fe. After 3 h, approx. 31% of the original amount of Fe added as the Fe complex of MEM had been taken up, whereas only 5% of the Fe added as diferric Tf was taken up. In addition, the internalisation of Fe from small Mr complexes occurred more rapidly than that from Tf (Fig. lb). The internalisation of small Mr chelated Fe into the cell was markedly temperature-dependent as demonstrated for the internalisation of Tf-bound Fe [10]. After 2 h incubation at 4°C, internalised Fe was approx. 10% of that seen at 37°C. The Fe which appeared internalised at 4°C may be largely due to non-specific adsorption of Fe from the pronase fraction to the cell pellet. Iron uptake in the membrane compartment showed
biphasic kinetics for all Fe complexes (Fig. lc) and the amount of Fe found in the membrane compartment was far greater than that found when the Fe donor was Tf. The subcellular distribution of Fe from diferric Tf and from MEM-complexed Fe into ferritin, stromalmitochondrial membrane and the ferritin-free cytosol was also examined. Iron uptake into the ferritin fraction was linear with time between 30 min and 3.5 h for both sources of Fe (Fig. 2a). Quantitatively far more Fe was incorporated into the subcellular compartments when MEM-complexed Fe was used as the Fe donor compared to diferric Tf. However, distribution of Fe between these compartments from both Fe donors was very similar (Fig. 2b). The proportion of Fe found in the stromal-mitochondrial membrane decreased markedly with time, with a corresponding increase in the amount found in ferritin (Fig. 2b). After 3 h incubation, 38% of Fe was in
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Fig. 2. The uptake and subcellular distribution of iron. Cells were incubated for up to 3.5 h in the presence of MEM-iron complexes and diferric Tf at an Fe concentration of 2.5 ~M. The cells were then washed and treated with pronase. (a) The quantitative Fe uptake from diferric Tf (ferritin-free cytosol, o; ferritin, ®; stromal-mitochondrial membrane, a) and MEM-iron complexes (ferritin-free cytosol, O; ferritin, I ; stromai-mitochondrial membrane, A) into subcellular compartments. (b) The distribution of iron in the subcellular compartments of ferritin (ll), ferritin-free cytosol (O) and stromal-mitochondrial membrane (4). Results are means of duplicates.
24 0,7:
TABLE I
I~1 S u c r o s e
The proportion of iron in~'ernalisedinto the cell and the ferritin, ferritinfree cytosol and stromai-mitochondrial (strom.-mit.) membrane compartments from the iron(Ill) complexes of desferrioxamine, citrate, nitriIotriacetic acid and minimum essential medium compared to that from diferric transferrin at the same iron concentration (2.5 ttM) after a 24 h incubation
I--I M e r n
re 0.6 13. U~
I~. 0.5 Fs is ii
0
~ 0.4
Results are expressed as mean+S.E. (2 experiments; 16 determinations)
i~1 Ii ~ I I1
x~ 0.3
Iron complex
Tf-Fe(lll) DFO.Fe(lll) Cit-Fe(lil) NTA-Fe(lll) MEM-Fe(ilI)
lntemalised iron
Subcellular distribution of internalised iron
~" 0.2-
(~ total)
Ferritin
e-
964.1 85 4.1 96 :t: 1 92 4.1 87 4. 2
48+1 47 + 1 47 4- 2 45 + 1 44 4-1
Ferritinfree cytosol
Strom.-mit. membrane
294.1 25 ::1:2 29 + 1 31 4.1 30+ 1
23+1 28 + 1 23 + 1 22 + 1 25 + 1
ferritin, 48~ was in the ferritin-free cytosol and 14~ in the stromal-mitochondrial membrane fraction. Almost all Fe taken up was internalised for all Fe donors between 2 h and 24 h (85-96~; of total; Table I). The subcellular distribution of internalised Fe was very similar for all Fe donors (Table I). By 24 h the proportion of Fe in ferritin and the stromal-mitochondrial membrane had increased to about 46~ and 24~, respectively, while the proportion of Fe in the ferritin-free cytosol had markedly decreased to about 29~; of the total. Examining the amounts of Fe internalised and its subcellular distribution, the Fe complexes of MEM, citrate and NTA donated greater than 3-times more Fe than that obtained from diferric Tf alone (Table II), citrate being slightly more effective than the Fe complexes of NTA and MEM. In contrast, ferrioxamine
TABLE !!
Iron uptake into the total cell, ferritin, ferritin-free cytosol and stromal. mitochondrial membrane from the iron(Ill) complexes of desferrioxamine, citrate, nitrilotriacetic acid and minimum essential medium corn. pared to the iron uptake from diferric transferrin at the same iron concentration (2.3 pM) after a 24 h incubation Results are expressed as ~ control (mean+S.E.; 2 experiments; 16 dcter~ir, a:ions). Iron complex
Tf-Fe~m) DFO-Fe(ilI) cit-Fe(m) NTA-FemO MEM-Fe(III)
Transferrin iron uptake Internalised Ferritin iron-59
Ferritin-free Strom.-Mit. cytosol membrane
100± 1 88+- 1 349+- 10 325=!= 6 321+ 5
100+- 4 81+ 7 351 +- 17 352+-15 328+-13
100+- 2 85+ 3 333 +- 15 302+-11 294+- 5
100+- 3 111+- 5 354+- 18 329+-17 364+-23
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F'e.3 F'e.3 F'e.2 NTA NTA Cit Cit .F-e.3 .Fe .3 .F.e"3. F.e.3 Iron complex Fig. 3. The internalised iron uptake after 30 rain incubation in M E M (pH 7.4) or 0.27 M sucrose (pH 6.5) with diferric Tf, Fe(III) ion, Fe(ll) ion (sucrose only) and the Fe(lll) complexes of citrate and nitrilotriacetate at an iron concentration of 2.5 pM. Results are mean 4-S.E. (eight experiments) for Tf in M E M and mean :t: S.E. (two experiments; 4-24 determinations) for the remaining Fe chelates.
was slightly less effective than diferric Tf as an Fe donor (Table II). Iron uptake in sucrose
Comparing Fe uptake in MEM (pH 7.4) and sucrose (pH 6.5) after 30 rain of incubation, the amounts of Fe internalised from Tf and chelated Fe(lll) ion were similar irrespective of the medium (Fig. 3). However, internalised Fe uptake from the Fe complexes of citrate and NTA in sucrose was approx, half that found in MEM. Of all Fe donors tested, the Fe(ll) ion complex of sucrose was the most effective at increasing internalised Fe uptake.
The effect of ammonium chloride on iron uptake Ammonium chloride was used to examine the mechanism of Fe uptake from inorganic Fe complexes and diferric Tf. Ammonium chloride had no effect on the uptake of Fe from inorganic Fe complexes. In contrast, ammonium chloride reduced Fe uptake from Tf (0.1 mg/ml) to 28+1% [2] of the control after a 2 h incubation. The effect on Fe uptake of monoclonal antibodies specific for the transferrin receptor and melanotransferrin Effect on the uptake of inorganic iron complexes. Initial experiments examined the effect on the amount of Fe internalised from MEM-Fe complexes by preincubating cells with MoAbs for 1, 3, 5 and 24 h, after which the medium was replaced with medium containing the MoAb and MEM-59Fe chelates for a 2 h incuba-
25 150. o 14C ~ Con I~ 965 I~ 1331
13C
12( 11( 100 [ ~ 90 24 h
3h 5h Preincubation time
lh
1001" b
80
$ "" 60 4O _¢ 20
/
/ /
& Control A 965
[3 1331 [] 4 2 / 6
0
60 Time (rain)
90
lzO
Fig. 4. The internalised iron uptake from MEM-iron-59 complexes in the presence of monoclonal antibodies (5 #g/ml) specific for melanotransferrin (96.5, 133.1) and the transferrin receptor (42/6). (a) The internalised iron uptake from cells that were preincubated with antibodies 96.5 and 133.1 for 1-24 h. The medium was then replaced with medium containing MEM-iron-59 complexes and the same concentration of antibody for 1 h. Results are mean+ S.E. (one experiment; five determinations). (b) The internalised iron uptake from cells that were preincubated for 2 h in the presence of antibodies 96.5, 133.1 and 42/6. The medium was then replaced with medium containing MEM-iron-59 complexes and the same concentration of antibody and incubated for up to 2 h. Results are mean (two experiments: four determinations).
tion. After a 1 h preincubation with the MoAbs, Fe uptake in the presence of the M o A b s was not significantly different from that of the control (Fig. 4a). However, after preincubation for 3.5 and 24 h internalisation in the presence of M o A b 96.5 was significantly increased to 130% ( P < 0.025), 133% ( P < 0.01) and 145% of the control, respectively (Fig. 4a), although there was no significant change in total cell Fe. In contrast, M o A b 133.1 had no significant effect on Fe uptake when compared to the control. In other experiments t h e proportion of Fe internalised from M E M chelated 5 Fe was compared after the cells had been preincubated with M o A b s 96.5, 133.1 and 4 2 / 6 for 2 h. Only M o A b 96.5 increased the internalisation of Fe to greater than that seen for the control (Fig. 4b). There was no significant change in total cellular Fe in the presence of any of the MoAbs. Effect on uptake of transferrin bound iron. After preincubation with MoAbs 96.5 and 133.1 for 3 h, the cells were exposed to medium containing diferric. Tf (0.1 m g / m l ) and the same concentration of MoAb. Both M o A b s significantly ( P < 0.005) increased the a m o u n t of n o n - T f - b o u n d Fe present on the membrane (Table III), resulting in a significant increase in the molar ratio of Fe to T f in the m e m b r a n e compartment in the presence of M o A b s 133.1 ( P < 0 . 0 2 5 ) and 96.5 ( P < 0.005) when compared to the control. Monoclonal antibody 96.5 significantly decreased both m e m b r a n e Fe ( P < 0.05) and Tf ( P < 0.005) uptake, whereas M o A b 133.1 had no effect (Table Ill). Neither M o A b had any significant effect on internalised Fe or T f uptake, or the molar ratio of Fe to Tf in the internalised and total cellular compartments (Table III). The M o A b 96.5 significantly ( P < 0.025) decreased total Tf uptake to 85% of the control, whereas total F e uptake decreased slightly to 95% of the control.
TABLE I!1 The effect of preincubation with monoclonal antibodies specific for melanotransferrin on iron and transferrin uptake Cell plates were preincubated for 3 h with a saturating concentration of antibody (5/~g/ml; Hellstrom et al., 1983). The medium was then replaced with medium containing radiolabelled Tf (0.1 mg/ml) and the same concentration of antibody and incubated for an additional 2 h. Results are expressed as a percentage of the control (mean +-S.E.; five determinations). Non-Tf bound membrane iron was calculated by subtracting iron due to membrane bound transferrin from measured total membrane iron assuming that each transferrin molecule on the membrane had two iron atoms bound. Treatment
Control •96.5 133.1
Membrane-bound % Control Fe Tf 100+-4 90+2" 110+6
100+6 76+4"** 102+7
Total % Control
Molar Non-Tf Bound Fe Fe: Tf Ratio
Tot. Fe Tot. Tf
100+-4 129+-7"** 135+-7 ***
100+-2 100+4 95+-2 85+-3"* 103+-1 103+-5
2.72+0.05 3.23+-0.11"** 2.95+_0.07 **
Tot. Fe: Tf
22+- ! 24+1 22+-1
i nternalised % Control
Int. Fe : Tf
Int. Fe
int. Tf
100+-2 96+-2 102+-2
100+2 56+1 102+-2 53+-2 105+-2 55 4-1
Asterisks indicate significant differences from the corresponding control * P < 0.05; * * P < 0.025; * * *P < 0.005.
26
Discussion Uptake and incorporation of iron from small molecular weight iron chelates General characteristics of iron uptake. Melanoma cells can take up Fe from chelates of citrate, NTA and MEM and incorporate it intracelluiady more efficiently than Fe obtained from Tf at the same Fe concentration. In contrast, uptake of Fe from ferrioxamine was less efficient than Tf, as seen in other cell types [30-32]. Once Fe was internalised from small M r complexes, it followed a similar metabolic route to Fe which had been donated from Tf. The weak base, NH4CI, which increases intravesicular pH [33-35], had no effect on the uptake of Fe from small Mr Fe complexes, in contrast to its inhibition of Fe uptake from diferric Tf. This suggests that the uptake of Fe from small Mr complexes was not mediated by the receptor-mediated endocytosis of Tf. This is also supported by experiments using the MoAb 42/6, which blocks Tf binding to the TfR [15], but had no effect on Fe uptake from the Fe chelates. Comparisons with other cell types. Reticulocytes and spleen erythroid calls can take up Fe for heme synthesis from small Mr chelators, such as those of the pyridoxal isonicotinoyl hydrazone class, although less efficiently than Tf-bound Fe [2,32,36]. In addition, the efficient uptake of small Mr Fe chelates by hepatocytes [37-42] and leukemic cell lines [43-45] has been well documented. However, in some cell types Fe chelates could only partially replace the growth promoting potential of Tf [46,47]. As Fe uptake from small Mr Fe chelates appeared to vary with cell type [48,49], possibly some cells cannot obtain enough Fe from these chelates to sustain optimal growth. In addition, there does not appear to be any correlation between the ability to incorporate Fe from Fe chelates and MTf expression
[4]. Uptake of Fe(II) ion. Experiments investigating the uptake of Fe in sucrose have demonstrated that in melanoma cells the uptake of the Fe(II) ion is preferred to the uptake of F¢(III) ion. A similar higher uptake of F¢(II) compared to F¢(III) occurs in reticulocytes [1,3] and the liver [42,50]. There is evidence for the presence of an F¢(II) membrane carder [1,3,42,50-52], but again there is no correlation with MTf expression [4]. Mechanism of iron uptake from small molecular weight iron chelates Uptake of Fe could occur by diffusion of a neutral Fe complex through the membrane or via an active transport mechanism [3]. Some cells are capable of removing Fe from the Fe complexes of the pyridoxal isonicotinoyl hydrazone class of chelators [2,49,53] which have a very high affinity for Fe(III) [54,55]. This
suggests that an active mechanism is required to remove Fe from the chelate so that it can be used by the cell. In melanoma cells, the presence of MTf which can bind Fe from citrate-Fe chelates [6], suggested that this protein also may have a role in Fe uptake from small Mr chelates. However, if this is the case, it appears that l~'~'f does not donate its bound Fe via a mechanism analogous to that found for serum Tf, viz., invoh,ing internalisation via endocytosis and a decrease in intravesicular pH, as NHnC1 has no effect on the uptake of Fe. In addition, the observation that many normal and neoplastic cells that apparently do not have MTf [4] preferentially take up Fe from small M r Fe chelates rather than Tf-bound Fe, argues against a specific role for MTf in Fe uptake from inorganic Fe complexes (see below). Iron uptake from small Mr chelates may be of functional significance for melanoma cells. The amount of non-Tf-bound Fe in the plasma of humans under normal conditions is very low (0.6-1/~M; Ref. 41). However, considering the efficacy of the non-Tf-bound Fe uptake process compared to the Tf-mediated Fe uptake process at an Fe concentration of 2.5 /~M, it may be suggested that non-Tf-bound Fe could be an important source of Fe for these cells. In addition, as melanoma cells invade tissues they secrete a variety of enzymes [56-53] which attack normal tissues resulting in cell injury and necrosis. Upon damage to normal cells, the cell's cytoplasmic pool of Fe complexes [23,24] would be released. From the data presented the melanoma cell would benefit, efficiently taking up the released Fe chelates.
Effect of monoclonal antibodies on iron uptake from inorganic iron complexes and diferric transfirrin Uptake of iron from inorganic iron complexes. The MoAbs specific for MTf were used to examine whether they could cause modulation of the antigen on the cell membrane [59-61] and thus perturb MTf-dependent Fe uptake. The MoAb 96.5 which is specific for MTf [4], had no significant effect on total cell uptake, but significantly increased the proportion of Fe internalised. Neither of the MoAbs 42/6 (specific for the human TfR) or 133.1 (specific for another MTf epitope) had any effect. These observations suggested that MTf was modulated by MoAb 96.5, increasing the internalisation of Fe by endocytosis of the membrane containing both the antigen-antibody complex and 59Fe. Modulation and internalisation of-MTf by MoAb 96.5 has also been suggested in previous studies [19,62,63]. A minimum preincubation exposure period of 2 h was required before an increase in the internalisation of Fe was apparent, in agreement with previous observations [63]. Increasing the preincubation period to 24 h resulted in only a small further increase in the inter-
27
nalisation of Fe, suggesting that even after a long exposure period to MoAb 96.5 the cell was still expressing MTf on its membrane, consistent with previous investigations [29,63]. Considering the length of the preincubation period required before modulation of MTf occurred (i.e., 2 h), the extent of internahsation observed (30-45% greater than the control) and the fact that there was no significant change in total cell Fe, it is suggested that MTf does not have a significant role in Fe uptake from small M r Fe chelates. These observations are supported by other data [11] which suggested that membrane non-Tfbound Fe was not utilised by the cell. Iron uptake from transferrin. As MoAb 96.5 appeared to modulate MTf, it was possible to use it as a tool to determine if it had any link to Fe transport from Tf. Previous investigations [10,11] demonstrated that an Fe-binding component was present in the cell membrane which became labelled with 59Fe after incubation with 59Fe-~25I-Tf. If this component was MTf, it should be modulated by MoAb 96.5. Both MoAbs 96.5 and 133.1 significantly increased the molar ratio of Fe to Tf in the membrane, there being a significant increase in the amount of non "bound Fe present compared to the control. This ooservation could be explained by an increase in the expression of MTf on the membrane following initial internalisation of the MoAb-antigen complex, as has been demonstrated in previous studies [63]. The MoAb 96.5 resulted in a significant decrease in the total amount of Tf in the membrane compartment and hence total cell Tf uptake was reduced. Since human serum Tf has high homology with MTf [8], these results could be explained by a cross-reaction of MoAb 96.5, binding to a similar epitope on the Tf molecule and preventing the uptake of Tf by the TfR. However, neither MoAb 96.5 or 133.1 affected internalised Fe or Tf uptake. Considering that 96.5 can modulate MTf, these data suggested that MTf does not have a significant role in the process of Fe uptake from Tf, but has access to Fe released from Tf. Acknowledgements D.R.R. thanks the National Health and Medical Research Council (NH& MRC) of Australia for a Biomedical Postgraduate Scholarship. E.B. thanks the N H &MRC for a research project grant (No. 880524). Dr. J.P. Brown and Dr. I. Trowbridge are thanked for their kind gifts of MoAb. References 1 Egyed, A. (1988) Br. J. Haematol. 68, 483-486. 2 Fuchs, O., Borova, J., Hradilek, A. and Neuwirt, J. (1988) Biochim. Biophys. Acta 969, 158-165.
3 Morgan, E.H. (1988) Biochim. Biophys. Acta 943, 428-439. 4 Woodbury, R.G., Brown, J.P., Yeh, M.-Y., Hellstrom, I. and Hellstrom, K.E. (1980) Proc. Natl. Acad. Sci. USA 77(4), 21832187. 5 Brown, J.P., Nishiyama, K., Hellstrom, I. and Hellstrom, K.E. (1981) J. Immunol. 127, 539-546. 6 Brown, J.P., Hewick, R.H., Hellstrom, I., Hellstrom, K.E., Doolittle, R.F. and Dreyer, W.J. (1982) Nature 296, 171-173. 7 Plowman, G.D., Brown, J.P., Enns, C.A., Schroder, J., Nikinmaa, B., Sussman, H.H., Hellstrom, K.E. and Hellstrom, I. (1983) Nature 303, 70-72. 8 Brown, J.P., Rose, T.M. and Plowman, G.D. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., eds.), pp. 39-46, Elsevier Science Publishers, Amsterdam. 9 Rose, T.M., Plowman, G.D., Teplow, D.B., Dreyer, W.J., Hellstrom, K.E. and Brown, J.P. (1986) Proc. Natl. Acad. Sci. USA 83, 1261-1265. 10 Richardson, D.R. and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12. 11 Richardson, D.R. and Baker, E. (1990) Biochim. Biophys. Acta, 1091, 294-304. 12 Seligman, P.A., Butler, C.D., Massey, E.J., Kaur, J.A., Brown, ,LP., Plowman, G . D . . Miller, Y. and Jones, C (1986) Am. J. Hum. Genet. 38, 540-548. 13 Karin, M. and Mintz, B. (1981) J. Biol. Chem. 256, 3245-3252. 14 lacopetta, B.J. and Morgan, E.H. (1983) J. Biol. Chem. 258 (15), 9108-9115. 15 Trowbridge, I.S. and Lopez, F. (1982) Proc. Natl,. Acad. Sci USA 79, 1175-1179. 16 Hanks, J.H. and Wallace, K.E. (1949) Proc. Soc. Exp. Biol. Med. 71,196-200. 17 Hemmaplardh, D. and Morgan, E.H. (1976) Int. J. Appl. Radiat. Isot. 27, 89-92. 18 McFarlane, A.S. (1958) Nature 182, 53-60. 19 Caseilas, P., Brown, J.P., Gros, O., Gros, P., Hellstrom. I., Jansen, F.K., Poncelet, P., Roncucci, R., Vidal, H. and Hellstrom, K.E. (1982) Int. J. Cancer 30, 437-443. 20 Lindmo, T., Davies, C., Fodstad, O. and Morgan, A.C. (1984) Int. J. Cancer 34, 507-512. 21 Fodstad, O., Aamdal, S., McMenamin, M., Nesland, J.M. and Pihi, A. (1988) Int. J. Cancer 41,442-449. 22 Spiro, (3. and Saltman, P. (1969) Struct. Bond. 6, 116-156. 23 Jacobs, A. (1977) in Iron Metabolism, Ciba Foundation Symposium 51, New Series, pp. 91-106, Elsevier, Exerpta Medica, Amsterdam. 24 May, P.M. and Williams, D.R. (1980) in Iron in Biochemistry and Medicine (Jacobs, A. and Worwood, M., eds.), Vol. II, Chapt. 1, Acad. Press, London., 25 Ackland, H.L., Danks, D.H. and McArdle, H.J. (1988) J. Cell. Physiol. 135, 521-526. 26 Sussman, H.H., Stein, B.S. and Tsavaler, L. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., eds.), pp. 143-153, Elsevier Science Publishers, Amsterdam. 27 Trinder, D., Morgan, E. and Baker, E. (1986) Hepatology 6 (5), 852-858. 28 White, G.P., Bailey-Wood, R. and Jacobs, A. (1975) Clin. Sci. Mol. Med. 50, 145-152. 29 Hellstrom,, I., Brown, J.P. and Hellstrom, K.E, (1983) Int. J. Cancer 31, 553-555. 30 Morgan, E.H. and Baker, E. (1988) Ann. N.Y. Acad. Sci. 526, 65-82. 31 Gerlanc, M.D., Zapolski, E.J., Rubin, M. and Princiotto. J.V. (1970) Blood 35 (4), 493-495. 32 Morgan, E.H. (1971) Biochim. Biophys. Acta 244, 103-116.
28 33 Ohkuma, S. and Peele, B. (1978) Prec. Natl. Acad. Sci. USA 75, 3327. 34 Armstrong, N.J. and Morgan, E.H. (1983) Biochim. Biophys. Acta 762, 175-186. 35 Glass, J. and Nunez, M.T. (1986) J. Biol. Chem. 261 (18), 82988302. 36 Princiotto, J.V., Rubin, M., Shashaty, (3.C. and Zapolski, E.J. (1964) J. Clin. Invest. 43 (5), 825-833. 37 White, G.P. and Jacobs, A. (1978) Biochim. Biophys. Acta 543, 217-225. 38 Page, M.A., Baker, E. and Morgan, E.H. (1984) Am. J. Physiol. 246, (326-(333. 39 (3rootveld, M., Bell, J.D., Halliwell, B., Aruoma, O.I., Bomford, A. and Sadler, P.J. (1989) J. Biol. Chem. 264 (8), 4417-4422. 40 Wheby, M.S. and Jones, L.C. (1963) J. Clin. Invest. 42, 1007-1016. 41 Bfissot, P . , Wright, T.L., Ma, W.-L. and Weisiger, A. (1985) J. Clin, Invest. 76, 1463-1470. 42 Wright, T.L., Brissot, P., Ma, W.-L. and W~isiger, R.A. (1986) J. Biol. Chore, 261 (23), 10909-10914. 43 Fibach, E., Konijn, A.M., Baumingor, R.E, Ofer, S. and Rachmilewitz, E,A, (1987) J. Cell. Physiol, 130, 460-465. 44 Titeux, M,, Testa, U., Louache, F., Thomopoulos, P., Rochant, H. and Breton-(3orius, J. (1984) J. Cell. Physiol. 121, 251-256. 45 Basset, P., Zwiller, I., Revel, M.O. and Vincendon, (3. (1985) Carcinogenesis 6 (3), 355-359. 46 Percz.infante, V. and Mather, J.P. (1982) Exp. Cell Res. 142, 325-332. 47 Mendelsohn, J, Trowbridge, i. and Castagnola, J. (1983) Blood 62. 821-826. 48 Taetle, R., Hone.ysctt, J.M. and Trowbridge, I.S. (1983) Int. J. Cancer 32, 343-349.
49 Ekblom, P., Landschulz, W. and Andersson, L.C. (1986) Scand. J. Haematol. 36. 258-262. 50 Wright, T.L., Fits, J.G. and Weisiger, R.A. (1988) J. Biol. Chem. 263 (4), 1842-1847. 51 Thorstensen, K. (1988) J. Biol. Chem. 263 (32), 16837-16841. 52 Thorstensen, K. and Romslo, I. (1988) J. Biol. Chem. 263 (18), 8844-8850. 5"~ Ponka, P., Schulman, H.M. and Wilzynska, A. (1982) Biochim. Biophys. Acta. 718, 151-156. 54 Richardson, D.R., Hefter, (3.T., May, P.M., Baker, E. and Webb, J. (1990) Biol. Metals 2, 161-167. 55 Vitolo, H.L., Clare, B., Hefter, (3.T., May, P.M. and Webb, J. (1991) Inorgan. Chim. Acta, in press. 56 Liot*.a, L.A., Tryggvason, S., (3arbisa, I., Hart, C.M., Foltz, C.M. and Shafie, S. (1980) Nature 284, 67-68. 57 Strauli, P. (1980) in Proteinases and Tumor Invasion, (Strauli, P., Barrett, A.J. and Baici, A., eds.), p. 15, Raven Press, New York. 58 Sloane, B.F., Dunn, J.R. and Horn, K.V. (1981) Science 212, 1151-1152. 59 Old, L.J., Stockert, E., Boyse, E.A. and Kim, J.H. (1968) J. Exp. Med. 127, 523-539. 60 Shawler, D.L., Miceli, M.C., Wormsley, S.B., Royston, l. and Diilman, R.O. (1984) Cancer Res. 44, 5921-5927. 61 Ritz, J., Pesando, J.M., Notis-McConarty, J. and Schlossman, S.F. (1980) J. Immunol. 125, 1506-1514. 62 Rowland, (3.F., Axton, C.A., Baldwin, R.W., Brown, J.P., Corvalen, J.R.F., Embleton, M.J., Gore, V.A., Hellstrom, I., Hellstrom, K.E., Jacobs, E., Marsden, C.H., Pimm, M.V., Simmonds, R.(3. and Smith, W. (1985) Cancer Immunol. Immunother. 19, 1-17. 63 Wang, B.S., Lumanglas, A.L., Silva, S., Ruszala-Malion, V. and Duff, F.E. (1987) Ceil. Immunol. 106, 12-21.
20
BBAMCR 12935
The uptake of inorganic iron complexes by human melanoma cells Des R i c h a r d s o n a n d Erica B a k e r Department of Physiology, University of Western Australia, Nedlands. Western Australia (Australia)
(Received 6 August 1990) (Revised manuscript received 19 December 1990)
Key words: Melanotransferrin; Transferrin; Iron; (Melanoma cell)
The human melanoma cell line, SK-MEL-28, expresses high levels of melanotransferrin. The uptake of inorganic iron (Fe) complexes compared to transferrin.bound Fe by these cells has been investigated to determine whether melanotransferrin has a role in ice uptake. The mechanisms of ICe uptake have been characterised using SgFe complexes of citrate, nitrilotriacetate, desferrioxamine, and SgFe added to Eagle's minimum essential medium (MEM) and compared with human transferrin Off) labelled with S9Fe and iodine-125. Iron uptake from the ICe complexes of citrate, nitrilotriacetate and MEM were similar, and far greater than that from Tf at the same ICe concentration (2.5/~M). Ammonium chloride and a monodonal antibody to the transferrin receptor (42/6), had no effect on the uptake of ICe from inorganic ICe complexes, suggesting that receptor-mediated endocytosis of "If was not involved. The monoclonal antibody, 96.5, specific for melanotransferrin did not alter total Fe uptake but slightly increased the proportion of ICe internalised, possibly due to the modulation of the antigen by the antibody. However, from the time required for modulation to occur (~ 2 h), the small increase in internalisation observed and the fact that no increase in total cell ICe occurred, it is suggested that melanotransferrin has little role in ICe uptake.
Introduction The mechanism of cellular iron (Fe) uptake from membrane impermeable and permeable Fe chelates is being studied in several laboratories [1-3] in an effort to understand the process of Fe transport across the membrane after Fe release from transferrin (Tf). An active transport process probably mediated by a membrane-bound Fe-binding protein(s) has been proposed from several recent studies in reticulocytes [1-3]. With regard to the membrane transport of Fe, we are investigating the role of a membrane-bound Fe-binding molecule known as p97, or melanotransferrin (MTf), which is found predominantly in human melanoma cells [4]. Melanotransferrin has several characteristics in common with serum Tf. These are: (i) it is a sialoglycoprotein (13~ carbohydrate); (ii) it has 37-39% se-
Abbreviations: FCS, fetal calf serum; MEM, Eagle's minimum essential medium; BSS, balanced salt solution; NTA, nitrilotriacetate; BSA, bovine serum albumin; gPR, gram of protein; Tf, transferrin; MTf, melanotransferrin. Correspondence: E. Baker, Department of Physiology, University of Western Australia, Nedlands, Western Australia, 6009, Australia.
quence homology with human serum Tf, human lactoferrin and chicken Tf; (iii) it has a high interdomain sequence homology (46%), which is greater than that found for other Tfs (33-41%); (iv) the MTf gene is on chromosome 3, as are those for Tf and the Tf receptor (TfR); and (v) the purified protein can bind Fe from Fe(III) citrate complexes [5-9]. Recent studies by the authors [10,11] have identified a saturable, temperaturedependent, Fe-binding component on the membrane of melanoma cells, with properties consistent with those attributed to MTf. Seligman et ai. [12] have suggested that MTf may be important in Fe uptake from non-Tf-bound Fe in the plasma. In addition, the observation that MTf bound Fe from Fe complexes of citrate [6], encouraged us to investigate the possible role of this membrane-bound protein in Fe uptake from small molecular weight (Mr) Fe complexes. The mechanisms of Fe and Tf uptake have been determined by standard procedures similar to those described previously [13,14], using pronase to separate internalised (pronase-resistant) and membrane-bound (pronase-sensitive) Fe and Tf. The results indicate that melanoma cells can accumulate Fe from inorganic Fe chelates much more efficiently than from diferric Tf. The mechanism of Fe uptake probably does not involve
21 the receptor-mediated endocytosis of Tf as both NH4C1, and the monoclonal antibody (MoAb), 42/6, which inhibits binding of Tf to the TfR [15], had no effect. A MoAb specific for MTf (96.5), did not affect total Fe uptake and only caused a slight increase in the proportion of Fe which was internalised, which may be due to the modulation of MTf by the MoAb. However, from the time required for modulation to occur ( - 2 h), the small extent of internalisation observed and the fact that there was no increase in total Fe uptake, it is suggested that MTf has little role in the uptake of Fe from inorganic Fe complexes. Materials and Methods
Reagents Iron-59 (as ferric chloride in 0.1 M HCl) and iodine125 (as sodium iodide) were purchased from Amersham International, U.K. Pronase was purchased from Boehringer-Mannheim, Mt. Waverley, Australia. Eagle's modified minimum essential medium (MEM) as Autopow and fetal calf serum (FCS) were supplied by Flow Laboratories, Annandale, Australia. Penicillin (Crystapen-BenzylpeniciUin sodium B.P.) was obtained from Glaxo, Boronia, Australia. Bovine serum albumin (BSA; 9870 pure, fatty acid free), human apo-Tf, L-glutamine and Hepes was obtained from Sigma, St. Louis, MO, U.S.A. Non-essential amino acids (100 × concentrate) and trypsin-versene solution (1 x ) were obtained from Commonwealth Serum Laboratories, Melbourne, Australia. Balanced salt solution (BSS) was prepared by the method of Hanks and Wallace [16]. All other chemicals were of analytical reagent quality.
Protein purification and labelling Human apoTf was prepared and labelled with 59Fe and iodine-125 by the methods of Hemmaplardh and Morgan [17] and McFarlane [18], as described previously [10]. Monoclonal antibodies specific for MTf (96.5 and 133.1) and the TfR (42/6) were generous gifts from Dr. J.P. Brown (Oncogen, Seattle, U.S.A.) and Dr. I. Trowbridge (Salk Institute, San Diego, U.S.A.), respectively.
Cell culture The human melanoma cell line, SK-MEL-28 (American Type Culture Collection, Rockville, MD), was used as these cells have the highest concentration of MTf of all cell types studied (300 000-380 000 sites/cell; Refs. 5, 19). Cells were grown as described previously and subcultured using 1 mM EDTA in calcium/ magnesium free PBS [10,20,21]. Procedures used to check cell viability and dedifferentiation of the cell line were the same as described previously [10].
Experimental procedures Preparation of inorganic iron complexes. Initial experiments examined the uptake of Fe from small molecular weight Fe chelates. The chelating agents, desferrioxamine (DFO), nitrilotriacetic acid (NTA) and citrate were mixed at a 0.9, 10 and 100-fold molar excess to iron-59, respectively, to prevent hydrolytic polymerisation of Fe [22]. The Fe chelate was then added to MEM which had been supplemented with 17o non-essential amino acids, BSA (5 mg/ml) and Hepes (20 mM; pH 7.4). It should be noted that under the experimental conditions used, a complex equilibrium would probably exist between the Fe in the added chelators and those already present in MEM. Indeed, it was deemed worthwhile to examine whether 59Fe directly added to MEM could donate Fe(III) ion to cells. The MEM contains many good Fe chelating agents at high concentration, including amino acids, sugars, vitamins, bicarbonate and phosphate (Flow Laboratories Catalogue). Considering only the contribution from amino acids and glucose, there was greater than a 6000-fold molar excess of chelating agent to Fe at an Fe concentration of 2.5 #M. Hence, the polymerisation of Fe would be unlikely. Indeed, MSssbauer spectroscopy studies (St. Pierre, Richardson, Baker and Webb, unpublished data) demonstrated that polynuclear Fe complex formation was very low using this protocol and that Fe was not taken up by cells as polynuclear Fe complexes. It is prudent to note that such : mixture of chelating agents may be more physiologically relevant, as Fe complexes of maino acids, sugars, vitamins and to a lesser degree, proteins, have been proposed as candidate ligands involved in the intracellular labile Fe pool and non-Tf-bound Fe in serum [23,24]. Similar reasoning has been applied in studying zinc(II) uptake by human fibroblasts [25]. In some experiments the uptake of ferric and ferrous Fe (2.5 #M) from sucrose (0.27 M; pH 6.5) during a 30 rain incubation was investigated. Solutions were prepared as described by Morgan [3]. These solutions were prepared immediately before use. All subsequent procedures were the same as those described below for experiments done in MEM.
Washing procedure to deplete cells of bovine transfertin. An important prerequisite in these studies on the uptake of small Mr Fe complexes was to ensure that the incubation medium was free of Tf. Melanoma cells were grown in the presence of 3070 FCS which contained bovine Tf, which can bind to the human TfR [26]. Several workers have depleted cells of Tf by three separate 15 rain incubations at 37°C [2,3]. Previous work in melanoma cells using human Tf [11] has shown that most intracellular Tf is released within 30 min of incubation. To ensure depletion of bovine Tf, three separate 30-45 min incubation periods at 37°C in MEM were used.
22 The observation that NH4CI (15 mM) and the monoclonal antibody, 42/6, which sterically inhibits the binding of Tf to the human TfR [15], had no effect on Fe uptake from inorganic Fe complexes indicated that this protocol effectively removed bovine Tf from the cell. Uptake of iron and transferrin. To measure the uptake by melanoma cells of Tf, Tf-bound Fe and Fe-bound to small Mr chelating agents, the medium was replaced after 24-48 h of culture with prewarmed/pregassed (95~ air; 5~ CO2) MEM containing BSA (5 mg/ml), 20 mM Hepes (pH 7.4) and the radiolabel. All experiments were performed on totally confluent plates. At the end of the incubation with the radiolabel, the plates were placed on ice, the medium decanted and the cell monolayer washed four times with ice-cold BSS. Increasing the number of times the plates were washed prior to the addition of pronase did not substantially reduce membrane-bound Fe or internalised Fe. As described previously [10], the amount of radioactivity internalised by the cells was measured by incubation with pronase (1 mg/ml) for 30 rain at 4°C, to separate membrane-bound Fe and Tf [27]. Longer incubations in pronase, up to 150 rain, did not increase the amount of Fe or Tf released. Subsequent procedures were the same as those described previously [10]. The distribution of intracellular Fe into ferritin, stromal-mitochondrial membrane and ferritin-free cytosol was determined as described previously [11]. Effect of monoclonal antibodies on iron uptake. Cell plates which had been washed to remove bovine Tf (see above) were preincubated with a saturating concentration of the MoAb (5/tg/ml; Ref. 29) for up to 24 h. After the required preincubation period the medium was replaced with medium containing MEM-SgFe complexes or SgFe-t2Sl-Tf and the same concentration of MoAb. All subsequent procedures were the same as those described above. Effect of ammonium chloride on iron uptake. Washed cells were preincubated with MEM containing NH4CI (15 mM) for 15 rain, then MEM-iron-59 or SgFe-t2SI-Tf was then added to a final Fe concentration of 2.5/~M and the incubation continued for up to 2 h. All subsequent procedures were the same as those described above. U p t a k e o f Fe is expressed a s / L m o l o f Fe p e r g r a m o f
protein (gPR). The data are expressed as mean 4-S.E. (number of experiments) with 2-12 replicates in each experiment. Experimental data were compared using the Student's t-test. Results were considered statistically significant when P < 0.05. Results
General characteristics of iron uptake The internalised uptake of Fe from complexes of citrate, nitrilotriacetate and MEM was linear with time
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ferdn at an iron concentrationof 2.5/~M. The cellswerethen washed and treated with pronase. (a) Internalised iron uptake from the iron complexes of citrate, MEM, NTA and from diferric Tf. (b) The proportion of iron internalised for iron complexesof citrate, MEM, NTA and from diferric Tf. (c) Membraneiron uptake from the iron complexes of citrate, MEM, NTA and from diferric Tf. Results are expressed as mean±S.E. (two experiments; four determinations).
up to 2 h (Fig. la). The Fe complexes of citrate and MEM appear slightly more effective as Fe donors than the Fe complex of NTA. Quantitatively, Fe uptake from small M r complexed Fe was far greater than that from Tf (Fig. la). As the Fe complexes of MEM were considered more physiologically relevant than citrate and NTA (see above), and since the Fe donating properties were similar, MEM-complexed Fe was used to further examine the mechanism of Fe uptake. The rate of internalised Fe uptake from MEM-Fe complexes was 440000_ 40000 [6] atoms Fe/cell per min, which was approx. 5.5-times greater than Fe uptake from diferric Tf (80 000 _ 7000 [8] atoms Fe/cell
23 per min; see Ref. 10) at the same Fe concentration of 2.5 /~M (Fig. la). The rates of uptake of Fe from complexes of NTA and citrate were similar to those described for MEM chelated Fe. After 3 h, approx. 31% of the original amount of Fe added as the Fe complex of MEM had been taken up, whereas only 5% of the Fe added as diferric Tf was taken up. In addition, the internalisation of Fe from small Mr complexes occurred more rapidly than that from Tf (Fig. lb). The internalisation of small Mr chelated Fe into the cell was markedly temperature-dependent as demonstrated for the internalisation of Tf-bound Fe [10]. After 2 h incubation at 4°C, internalised Fe was approx. 10% of that seen at 37°C. The Fe which appeared internalised at 4°C may be largely due to non-specific adsorption of Fe from the pronase fraction to the cell pellet. Iron uptake in the membrane compartment showed
biphasic kinetics for all Fe complexes (Fig. lc) and the amount of Fe found in the membrane compartment was far greater than that found when the Fe donor was Tf. The subcellular distribution of Fe from diferric Tf and from MEM-complexed Fe into ferritin, stromalmitochondrial membrane and the ferritin-free cytosol was also examined. Iron uptake into the ferritin fraction was linear with time between 30 min and 3.5 h for both sources of Fe (Fig. 2a). Quantitatively far more Fe was incorporated into the subcellular compartments when MEM-complexed Fe was used as the Fe donor compared to diferric Tf. However, distribution of Fe between these compartments from both Fe donors was very similar (Fig. 2b). The proportion of Fe found in the stromal-mitochondrial membrane decreased markedly with time, with a corresponding increase in the amount found in ferritin (Fig. 2b). After 3 h incubation, 38% of Fe was in
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Translerrin
iron
80
80
60
60
20
0
4O
--.-I
~- - - ~ . a ~ a _ . . _ .
2O
~
I
I
I
1
2
3
0 Time
o ....
o__--o
. . . . .
°----°
&
. . . .
0 3
A
-
(h)
100
40
.
Iron chelates
0--.0~ D~ 0
~]
A
I
I
I
1
2
3
(h)
Fig. 2. The uptake and subcellular distribution of iron. Cells were incubated for up to 3.5 h in the presence of MEM-iron complexes and diferric Tf at an Fe concentration of 2.5 ~M. The cells were then washed and treated with pronase. (a) The quantitative Fe uptake from diferric Tf (ferritin-free cytosol, o; ferritin, ®; stromal-mitochondrial membrane, a) and MEM-iron complexes (ferritin-free cytosol, O; ferritin, I ; stromai-mitochondrial membrane, A) into subcellular compartments. (b) The distribution of iron in the subcellular compartments of ferritin (ll), ferritin-free cytosol (O) and stromal-mitochondrial membrane (4). Results are means of duplicates.
24 0,7:
TABLE I
I~1 S u c r o s e
The proportion of iron in~'ernalisedinto the cell and the ferritin, ferritinfree cytosol and stromai-mitochondrial (strom.-mit.) membrane compartments from the iron(Ill) complexes of desferrioxamine, citrate, nitriIotriacetic acid and minimum essential medium compared to that from diferric transferrin at the same iron concentration (2.5 ttM) after a 24 h incubation
I--I M e r n
re 0.6 13. U~
I~. 0.5 Fs is ii
0
~ 0.4
Results are expressed as mean+S.E. (2 experiments; 16 determinations)
i~1 Ii ~ I I1
x~ 0.3
Iron complex
Tf-Fe(lll) DFO.Fe(lll) Cit-Fe(lil) NTA-Fe(lll) MEM-Fe(ilI)
lntemalised iron
Subcellular distribution of internalised iron
~" 0.2-
(~ total)
Ferritin
e-
964.1 85 4.1 96 :t: 1 92 4.1 87 4. 2
48+1 47 + 1 47 4- 2 45 + 1 44 4-1
Ferritinfree cytosol
Strom.-mit. membrane
294.1 25 ::1:2 29 + 1 31 4.1 30+ 1
23+1 28 + 1 23 + 1 22 + 1 25 + 1
ferritin, 48~ was in the ferritin-free cytosol and 14~ in the stromal-mitochondrial membrane fraction. Almost all Fe taken up was internalised for all Fe donors between 2 h and 24 h (85-96~; of total; Table I). The subcellular distribution of internalised Fe was very similar for all Fe donors (Table I). By 24 h the proportion of Fe in ferritin and the stromal-mitochondrial membrane had increased to about 46~ and 24~, respectively, while the proportion of Fe in the ferritin-free cytosol had markedly decreased to about 29~; of the total. Examining the amounts of Fe internalised and its subcellular distribution, the Fe complexes of MEM, citrate and NTA donated greater than 3-times more Fe than that obtained from diferric Tf alone (Table II), citrate being slightly more effective than the Fe complexes of NTA and MEM. In contrast, ferrioxamine
TABLE !!
Iron uptake into the total cell, ferritin, ferritin-free cytosol and stromal. mitochondrial membrane from the iron(Ill) complexes of desferrioxamine, citrate, nitrilotriacetic acid and minimum essential medium corn. pared to the iron uptake from diferric transferrin at the same iron concentration (2.3 pM) after a 24 h incubation Results are expressed as ~ control (mean+S.E.; 2 experiments; 16 dcter~ir, a:ions). Iron complex
Tf-Fe~m) DFO-Fe(ilI) cit-Fe(m) NTA-FemO MEM-Fe(III)
Transferrin iron uptake Internalised Ferritin iron-59
Ferritin-free Strom.-Mit. cytosol membrane
100± 1 88+- 1 349+- 10 325=!= 6 321+ 5
100+- 4 81+ 7 351 +- 17 352+-15 328+-13
100+- 2 85+ 3 333 +- 15 302+-11 294+- 5
100+- 3 111+- 5 354+- 18 329+-17 364+-23
f~ f/ f=,
m
OA-
Tf
Z
1"
ri rr sl
ff/ iii IJl /// i!1 f // !/.,,
I1 f .o ~..e ,.-s ,rl f .~ , " ~'
/IJ /11 /// i/i iii
¢'1 J'~ ,%1 ~'1 Is
///
r i
7//
" ~/ J,I
T
TY
F'e.3 F'e.3 F'e.2 NTA NTA Cit Cit .F-e.3 .Fe .3 .F.e"3. F.e.3 Iron complex Fig. 3. The internalised iron uptake after 30 rain incubation in M E M (pH 7.4) or 0.27 M sucrose (pH 6.5) with diferric Tf, Fe(III) ion, Fe(ll) ion (sucrose only) and the Fe(lll) complexes of citrate and nitrilotriacetate at an iron concentration of 2.5 pM. Results are mean 4-S.E. (eight experiments) for Tf in M E M and mean :t: S.E. (two experiments; 4-24 determinations) for the remaining Fe chelates.
was slightly less effective than diferric Tf as an Fe donor (Table II). Iron uptake in sucrose
Comparing Fe uptake in MEM (pH 7.4) and sucrose (pH 6.5) after 30 rain of incubation, the amounts of Fe internalised from Tf and chelated Fe(lll) ion were similar irrespective of the medium (Fig. 3). However, internalised Fe uptake from the Fe complexes of citrate and NTA in sucrose was approx, half that found in MEM. Of all Fe donors tested, the Fe(ll) ion complex of sucrose was the most effective at increasing internalised Fe uptake.
The effect of ammonium chloride on iron uptake Ammonium chloride was used to examine the mechanism of Fe uptake from inorganic Fe complexes and diferric Tf. Ammonium chloride had no effect on the uptake of Fe from inorganic Fe complexes. In contrast, ammonium chloride reduced Fe uptake from Tf (0.1 mg/ml) to 28+1% [2] of the control after a 2 h incubation. The effect on Fe uptake of monoclonal antibodies specific for the transferrin receptor and melanotransferrin Effect on the uptake of inorganic iron complexes. Initial experiments examined the effect on the amount of Fe internalised from MEM-Fe complexes by preincubating cells with MoAbs for 1, 3, 5 and 24 h, after which the medium was replaced with medium containing the MoAb and MEM-59Fe chelates for a 2 h incuba-
25 150. o 14C ~ Con I~ 965 I~ 1331
13C
12( 11( 100 [ ~ 90 24 h
3h 5h Preincubation time
lh
1001" b
80
$ "" 60 4O _¢ 20
/
/ /
& Control A 965
[3 1331 [] 4 2 / 6
0
60 Time (rain)
90
lzO
Fig. 4. The internalised iron uptake from MEM-iron-59 complexes in the presence of monoclonal antibodies (5 #g/ml) specific for melanotransferrin (96.5, 133.1) and the transferrin receptor (42/6). (a) The internalised iron uptake from cells that were preincubated with antibodies 96.5 and 133.1 for 1-24 h. The medium was then replaced with medium containing MEM-iron-59 complexes and the same concentration of antibody for 1 h. Results are mean+ S.E. (one experiment; five determinations). (b) The internalised iron uptake from cells that were preincubated for 2 h in the presence of antibodies 96.5, 133.1 and 42/6. The medium was then replaced with medium containing MEM-iron-59 complexes and the same concentration of antibody and incubated for up to 2 h. Results are mean (two experiments: four determinations).
tion. After a 1 h preincubation with the MoAbs, Fe uptake in the presence of the M o A b s was not significantly different from that of the control (Fig. 4a). However, after preincubation for 3.5 and 24 h internalisation in the presence of M o A b 96.5 was significantly increased to 130% ( P < 0.025), 133% ( P < 0.01) and 145% of the control, respectively (Fig. 4a), although there was no significant change in total cell Fe. In contrast, M o A b 133.1 had no significant effect on Fe uptake when compared to the control. In other experiments t h e proportion of Fe internalised from M E M chelated 5 Fe was compared after the cells had been preincubated with M o A b s 96.5, 133.1 and 4 2 / 6 for 2 h. Only M o A b 96.5 increased the internalisation of Fe to greater than that seen for the control (Fig. 4b). There was no significant change in total cellular Fe in the presence of any of the MoAbs. Effect on uptake of transferrin bound iron. After preincubation with MoAbs 96.5 and 133.1 for 3 h, the cells were exposed to medium containing diferric. Tf (0.1 m g / m l ) and the same concentration of MoAb. Both M o A b s significantly ( P < 0.005) increased the a m o u n t of n o n - T f - b o u n d Fe present on the membrane (Table III), resulting in a significant increase in the molar ratio of Fe to T f in the m e m b r a n e compartment in the presence of M o A b s 133.1 ( P < 0 . 0 2 5 ) and 96.5 ( P < 0.005) when compared to the control. Monoclonal antibody 96.5 significantly decreased both m e m b r a n e Fe ( P < 0.05) and Tf ( P < 0.005) uptake, whereas M o A b 133.1 had no effect (Table Ill). Neither M o A b had any significant effect on internalised Fe or T f uptake, or the molar ratio of Fe to Tf in the internalised and total cellular compartments (Table III). The M o A b 96.5 significantly ( P < 0.025) decreased total Tf uptake to 85% of the control, whereas total F e uptake decreased slightly to 95% of the control.
TABLE I!1 The effect of preincubation with monoclonal antibodies specific for melanotransferrin on iron and transferrin uptake Cell plates were preincubated for 3 h with a saturating concentration of antibody (5/~g/ml; Hellstrom et al., 1983). The medium was then replaced with medium containing radiolabelled Tf (0.1 mg/ml) and the same concentration of antibody and incubated for an additional 2 h. Results are expressed as a percentage of the control (mean +-S.E.; five determinations). Non-Tf bound membrane iron was calculated by subtracting iron due to membrane bound transferrin from measured total membrane iron assuming that each transferrin molecule on the membrane had two iron atoms bound. Treatment
Control •96.5 133.1
Membrane-bound % Control Fe Tf 100+-4 90+2" 110+6
100+6 76+4"** 102+7
Total % Control
Molar Non-Tf Bound Fe Fe: Tf Ratio
Tot. Fe Tot. Tf
100+-4 129+-7"** 135+-7 ***
100+-2 100+4 95+-2 85+-3"* 103+-1 103+-5
2.72+0.05 3.23+-0.11"** 2.95+_0.07 **
Tot. Fe: Tf
22+- ! 24+1 22+-1
i nternalised % Control
Int. Fe : Tf
Int. Fe
int. Tf
100+-2 96+-2 102+-2
100+2 56+1 102+-2 53+-2 105+-2 55 4-1
Asterisks indicate significant differences from the corresponding control * P < 0.05; * * P < 0.025; * * *P < 0.005.
26
Discussion Uptake and incorporation of iron from small molecular weight iron chelates General characteristics of iron uptake. Melanoma cells can take up Fe from chelates of citrate, NTA and MEM and incorporate it intracelluiady more efficiently than Fe obtained from Tf at the same Fe concentration. In contrast, uptake of Fe from ferrioxamine was less efficient than Tf, as seen in other cell types [30-32]. Once Fe was internalised from small M r complexes, it followed a similar metabolic route to Fe which had been donated from Tf. The weak base, NH4CI, which increases intravesicular pH [33-35], had no effect on the uptake of Fe from small Mr Fe complexes, in contrast to its inhibition of Fe uptake from diferric Tf. This suggests that the uptake of Fe from small Mr complexes was not mediated by the receptor-mediated endocytosis of Tf. This is also supported by experiments using the MoAb 42/6, which blocks Tf binding to the TfR [15], but had no effect on Fe uptake from the Fe chelates. Comparisons with other cell types. Reticulocytes and spleen erythroid calls can take up Fe for heme synthesis from small Mr chelators, such as those of the pyridoxal isonicotinoyl hydrazone class, although less efficiently than Tf-bound Fe [2,32,36]. In addition, the efficient uptake of small Mr Fe chelates by hepatocytes [37-42] and leukemic cell lines [43-45] has been well documented. However, in some cell types Fe chelates could only partially replace the growth promoting potential of Tf [46,47]. As Fe uptake from small Mr Fe chelates appeared to vary with cell type [48,49], possibly some cells cannot obtain enough Fe from these chelates to sustain optimal growth. In addition, there does not appear to be any correlation between the ability to incorporate Fe from Fe chelates and MTf expression
[4]. Uptake of Fe(II) ion. Experiments investigating the uptake of Fe in sucrose have demonstrated that in melanoma cells the uptake of the Fe(II) ion is preferred to the uptake of F¢(III) ion. A similar higher uptake of F¢(II) compared to F¢(III) occurs in reticulocytes [1,3] and the liver [42,50]. There is evidence for the presence of an F¢(II) membrane carder [1,3,42,50-52], but again there is no correlation with MTf expression [4]. Mechanism of iron uptake from small molecular weight iron chelates Uptake of Fe could occur by diffusion of a neutral Fe complex through the membrane or via an active transport mechanism [3]. Some cells are capable of removing Fe from the Fe complexes of the pyridoxal isonicotinoyl hydrazone class of chelators [2,49,53] which have a very high affinity for Fe(III) [54,55]. This
suggests that an active mechanism is required to remove Fe from the chelate so that it can be used by the cell. In melanoma cells, the presence of MTf which can bind Fe from citrate-Fe chelates [6], suggested that this protein also may have a role in Fe uptake from small Mr chelates. However, if this is the case, it appears that l~'~'f does not donate its bound Fe via a mechanism analogous to that found for serum Tf, viz., invoh,ing internalisation via endocytosis and a decrease in intravesicular pH, as NHnC1 has no effect on the uptake of Fe. In addition, the observation that many normal and neoplastic cells that apparently do not have MTf [4] preferentially take up Fe from small M r Fe chelates rather than Tf-bound Fe, argues against a specific role for MTf in Fe uptake from inorganic Fe complexes (see below). Iron uptake from small Mr chelates may be of functional significance for melanoma cells. The amount of non-Tf-bound Fe in the plasma of humans under normal conditions is very low (0.6-1/~M; Ref. 41). However, considering the efficacy of the non-Tf-bound Fe uptake process compared to the Tf-mediated Fe uptake process at an Fe concentration of 2.5 /~M, it may be suggested that non-Tf-bound Fe could be an important source of Fe for these cells. In addition, as melanoma cells invade tissues they secrete a variety of enzymes [56-53] which attack normal tissues resulting in cell injury and necrosis. Upon damage to normal cells, the cell's cytoplasmic pool of Fe complexes [23,24] would be released. From the data presented the melanoma cell would benefit, efficiently taking up the released Fe chelates.
Effect of monoclonal antibodies on iron uptake from inorganic iron complexes and diferric transfirrin Uptake of iron from inorganic iron complexes. The MoAbs specific for MTf were used to examine whether they could cause modulation of the antigen on the cell membrane [59-61] and thus perturb MTf-dependent Fe uptake. The MoAb 96.5 which is specific for MTf [4], had no significant effect on total cell uptake, but significantly increased the proportion of Fe internalised. Neither of the MoAbs 42/6 (specific for the human TfR) or 133.1 (specific for another MTf epitope) had any effect. These observations suggested that MTf was modulated by MoAb 96.5, increasing the internalisation of Fe by endocytosis of the membrane containing both the antigen-antibody complex and 59Fe. Modulation and internalisation of-MTf by MoAb 96.5 has also been suggested in previous studies [19,62,63]. A minimum preincubation exposure period of 2 h was required before an increase in the internalisation of Fe was apparent, in agreement with previous observations [63]. Increasing the preincubation period to 24 h resulted in only a small further increase in the inter-
27
nalisation of Fe, suggesting that even after a long exposure period to MoAb 96.5 the cell was still expressing MTf on its membrane, consistent with previous investigations [29,63]. Considering the length of the preincubation period required before modulation of MTf occurred (i.e., 2 h), the extent of internahsation observed (30-45% greater than the control) and the fact that there was no significant change in total cell Fe, it is suggested that MTf does not have a significant role in Fe uptake from small M r Fe chelates. These observations are supported by other data [11] which suggested that membrane non-Tfbound Fe was not utilised by the cell. Iron uptake from transferrin. As MoAb 96.5 appeared to modulate MTf, it was possible to use it as a tool to determine if it had any link to Fe transport from Tf. Previous investigations [10,11] demonstrated that an Fe-binding component was present in the cell membrane which became labelled with 59Fe after incubation with 59Fe-~25I-Tf. If this component was MTf, it should be modulated by MoAb 96.5. Both MoAbs 96.5 and 133.1 significantly increased the molar ratio of Fe to Tf in the membrane, there being a significant increase in the amount of non "bound Fe present compared to the control. This ooservation could be explained by an increase in the expression of MTf on the membrane following initial internalisation of the MoAb-antigen complex, as has been demonstrated in previous studies [63]. The MoAb 96.5 resulted in a significant decrease in the total amount of Tf in the membrane compartment and hence total cell Tf uptake was reduced. Since human serum Tf has high homology with MTf [8], these results could be explained by a cross-reaction of MoAb 96.5, binding to a similar epitope on the Tf molecule and preventing the uptake of Tf by the TfR. However, neither MoAb 96.5 or 133.1 affected internalised Fe or Tf uptake. Considering that 96.5 can modulate MTf, these data suggested that MTf does not have a significant role in the process of Fe uptake from Tf, but has access to Fe released from Tf. Acknowledgements D.R.R. thanks the National Health and Medical Research Council (NH& MRC) of Australia for a Biomedical Postgraduate Scholarship. E.B. thanks the N H &MRC for a research project grant (No. 880524). Dr. J.P. Brown and Dr. I. Trowbridge are thanked for their kind gifts of MoAb. References 1 Egyed, A. (1988) Br. J. Haematol. 68, 483-486. 2 Fuchs, O., Borova, J., Hradilek, A. and Neuwirt, J. (1988) Biochim. Biophys. Acta 969, 158-165.
3 Morgan, E.H. (1988) Biochim. Biophys. Acta 943, 428-439. 4 Woodbury, R.G., Brown, J.P., Yeh, M.-Y., Hellstrom, I. and Hellstrom, K.E. (1980) Proc. Natl. Acad. Sci. USA 77(4), 21832187. 5 Brown, J.P., Nishiyama, K., Hellstrom, I. and Hellstrom, K.E. (1981) J. Immunol. 127, 539-546. 6 Brown, J.P., Hewick, R.H., Hellstrom, I., Hellstrom, K.E., Doolittle, R.F. and Dreyer, W.J. (1982) Nature 296, 171-173. 7 Plowman, G.D., Brown, J.P., Enns, C.A., Schroder, J., Nikinmaa, B., Sussman, H.H., Hellstrom, K.E. and Hellstrom, I. (1983) Nature 303, 70-72. 8 Brown, J.P., Rose, T.M. and Plowman, G.D. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., eds.), pp. 39-46, Elsevier Science Publishers, Amsterdam. 9 Rose, T.M., Plowman, G.D., Teplow, D.B., Dreyer, W.J., Hellstrom, K.E. and Brown, J.P. (1986) Proc. Natl. Acad. Sci. USA 83, 1261-1265. 10 Richardson, D.R. and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12. 11 Richardson, D.R. and Baker, E. (1990) Biochim. Biophys. Acta, 1091, 294-304. 12 Seligman, P.A., Butler, C.D., Massey, E.J., Kaur, J.A., Brown, ,LP., Plowman, G . D . . Miller, Y. and Jones, C (1986) Am. J. Hum. Genet. 38, 540-548. 13 Karin, M. and Mintz, B. (1981) J. Biol. Chem. 256, 3245-3252. 14 lacopetta, B.J. and Morgan, E.H. (1983) J. Biol. Chem. 258 (15), 9108-9115. 15 Trowbridge, I.S. and Lopez, F. (1982) Proc. Natl,. Acad. Sci USA 79, 1175-1179. 16 Hanks, J.H. and Wallace, K.E. (1949) Proc. Soc. Exp. Biol. Med. 71,196-200. 17 Hemmaplardh, D. and Morgan, E.H. (1976) Int. J. Appl. Radiat. Isot. 27, 89-92. 18 McFarlane, A.S. (1958) Nature 182, 53-60. 19 Caseilas, P., Brown, J.P., Gros, O., Gros, P., Hellstrom. I., Jansen, F.K., Poncelet, P., Roncucci, R., Vidal, H. and Hellstrom, K.E. (1982) Int. J. Cancer 30, 437-443. 20 Lindmo, T., Davies, C., Fodstad, O. and Morgan, A.C. (1984) Int. J. Cancer 34, 507-512. 21 Fodstad, O., Aamdal, S., McMenamin, M., Nesland, J.M. and Pihi, A. (1988) Int. J. Cancer 41,442-449. 22 Spiro, (3. and Saltman, P. (1969) Struct. Bond. 6, 116-156. 23 Jacobs, A. (1977) in Iron Metabolism, Ciba Foundation Symposium 51, New Series, pp. 91-106, Elsevier, Exerpta Medica, Amsterdam. 24 May, P.M. and Williams, D.R. (1980) in Iron in Biochemistry and Medicine (Jacobs, A. and Worwood, M., eds.), Vol. II, Chapt. 1, Acad. Press, London., 25 Ackland, H.L., Danks, D.H. and McArdle, H.J. (1988) J. Cell. Physiol. 135, 521-526. 26 Sussman, H.H., Stein, B.S. and Tsavaler, L. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R. and Mazurier, J., eds.), pp. 143-153, Elsevier Science Publishers, Amsterdam. 27 Trinder, D., Morgan, E. and Baker, E. (1986) Hepatology 6 (5), 852-858. 28 White, G.P., Bailey-Wood, R. and Jacobs, A. (1975) Clin. Sci. Mol. Med. 50, 145-152. 29 Hellstrom,, I., Brown, J.P. and Hellstrom, K.E, (1983) Int. J. Cancer 31, 553-555. 30 Morgan, E.H. and Baker, E. (1988) Ann. N.Y. Acad. Sci. 526, 65-82. 31 Gerlanc, M.D., Zapolski, E.J., Rubin, M. and Princiotto. J.V. (1970) Blood 35 (4), 493-495. 32 Morgan, E.H. (1971) Biochim. Biophys. Acta 244, 103-116.
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49 Ekblom, P., Landschulz, W. and Andersson, L.C. (1986) Scand. J. Haematol. 36. 258-262. 50 Wright, T.L., Fits, J.G. and Weisiger, R.A. (1988) J. Biol. Chem. 263 (4), 1842-1847. 51 Thorstensen, K. (1988) J. Biol. Chem. 263 (32), 16837-16841. 52 Thorstensen, K. and Romslo, I. (1988) J. Biol. Chem. 263 (18), 8844-8850. 5"~ Ponka, P., Schulman, H.M. and Wilzynska, A. (1982) Biochim. Biophys. Acta. 718, 151-156. 54 Richardson, D.R., Hefter, (3.T., May, P.M., Baker, E. and Webb, J. (1990) Biol. Metals 2, 161-167. 55 Vitolo, H.L., Clare, B., Hefter, (3.T., May, P.M. and Webb, J. (1991) Inorgan. Chim. Acta, in press. 56 Liot*.a, L.A., Tryggvason, S., (3arbisa, I., Hart, C.M., Foltz, C.M. and Shafie, S. (1980) Nature 284, 67-68. 57 Strauli, P. (1980) in Proteinases and Tumor Invasion, (Strauli, P., Barrett, A.J. and Baici, A., eds.), p. 15, Raven Press, New York. 58 Sloane, B.F., Dunn, J.R. and Horn, K.V. (1981) Science 212, 1151-1152. 59 Old, L.J., Stockert, E., Boyse, E.A. and Kim, J.H. (1968) J. Exp. Med. 127, 523-539. 60 Shawler, D.L., Miceli, M.C., Wormsley, S.B., Royston, l. and Diilman, R.O. (1984) Cancer Res. 44, 5921-5927. 61 Ritz, J., Pesando, J.M., Notis-McConarty, J. and Schlossman, S.F. (1980) J. Immunol. 125, 1506-1514. 62 Rowland, (3.F., Axton, C.A., Baldwin, R.W., Brown, J.P., Corvalen, J.R.F., Embleton, M.J., Gore, V.A., Hellstrom, I., Hellstrom, K.E., Jacobs, E., Marsden, C.H., Pimm, M.V., Simmonds, R.(3. and Smith, W. (1985) Cancer Immunol. Immunother. 19, 1-17. 63 Wang, B.S., Lumanglas, A.L., Silva, S., Ruszala-Malion, V. and Duff, F.E. (1987) Ceil. Immunol. 106, 12-21.
