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CELLULAR AND MOLECULAR

Resistance to the Antineoplastic Agent Gallium Nitrate Results in Marked Alterations in Intracellular Iron and Gallium Trafficking: Identification of Novel Intermediates

Children's Cancer Institute Australia for Medical Research, the Iron Metabolism and Chelation Program, Randwick, Sydney, New South Wales, Australia (N.P.D., Y.S.R., D.R.R.); and Division of Neoplastic Diseases, Medical College of Wisconsin, Milwaukee, Wisconsin (C.R.C.)

Received November 24, 2005; accepted December 21, 2005.

	Abstract

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Gallium (Ga) shows significant antitumor activity by markedly interfering with iron (Fe) metabolism, and ⁶⁷Ga is used as a radio-imaging agent for cancer detection. Therefore, the mechanisms involved in ⁶⁷Ga uptake, metabolism, and resistance are critical to understand. The development of tumor lines that are gallium-resistant suggests ⁶⁷Ga uptake may be different in these cells, providing an opportunity for understanding intracellular ⁶⁷Ga and ⁵⁹Fe transport and gallium resistance. In this study, gallium-resistant cells were used to assess ⁶⁷Ga and ⁵⁹Fe uptake using native polyacrylamide gel electrophoresis autoradiography. In contrast to the common view that ⁶⁷Ga and ⁵⁹Fe use the same uptake pathways, we show that the trafficking of these two metal ions is different in cells either resistant (R) or sensitive (S) to gallium. Indeed, in contrast to ⁵⁹Fe, little ⁶⁷Ga is incorporated into ferritin, with most present as a labile ⁶⁷Ga pool. We also report unique changes in ⁶⁷Ga and ⁵⁹Fe trafficking between R and S cells. In particular, in R cells, there was a distinct transferrin-transferrin receptor 1-hemochromatosis protein (HFE) complex (band B) not observed in S cells. Furthermore, because HFE regulates iron and gallium uptake, the two Tf-TfR1-HFE complexes in R cells may be involved in reduced ⁶⁷Ga and ⁵⁹Fe uptake compared with S cells. In S cells, a novel iron-binding intermediate (band D) was identified that was not present in R cells and may be a "sensitivity factor" to gallium. In contrast to the general view that ⁶⁷Ga and ⁵⁹Fe use the same or similar uptake pathways, we show that their distribution and trafficking is markedly different in R and S cells.

Interestingly, Ga(III) binds to the iron-transport protein transferrin (Tf) and is delivered to cells via receptor-mediated endocytosis (Larson et al., 1980). The Tf receptor (TfR1) on the cell membrane binds Tf and is subsequently internalized into an endosome and the iron transported into the cell (Chitambar and Zivkovic, 1987; Richardson and Ponka, 1997). Hence, iron and gallium do share some common aspects in terms of their uptake into cells. The product of the hemochromatosis gene HFE competes with Tf for binding to the TfR1 and regulates both iron and gallium uptake (Parkkila et al., 1997; Roy et al., 2000; Chitambar and Wereley, 2003). The fact that tumor cells express much higher TfR1 levels than their normal counterparts results in greater gallium acquisition by cancer cells (Chitambar and Zivkovic, 1987). This accounts, at least in part, for the selective antitumor activity of gallium and the ability of ⁶⁷Ga to act as a radio-imaging agent (Chitambar and Zivkovic, 1987).

Gallium nitrate is approved for the treatment of hypercalcemia of malignancy, and more recently, there has been renewed interest in the use of gallium for the treatment of cancer (for review, see Chitambar, 2004). Indeed, several clinical trials have demonstrated that gallium nitrate has clinical efficacy in bladder cancer and non-Hodgkin's lymphoma (Chitambar, 2004). In addition to this, ⁶⁷Ga-citrate is used as a well known radio-imaging agent for tumor detection (Chitambar, 2004). Considering these important therapeutic uses, it is therefore critical to understand gallium uptake and its metabolism by tumor cells.

The resistance of cancer cells to antineoplastic agents is a major problem, and resistance to gallium therapy has been described previously (Warrell et al., 1983; Chitambar et al., 1997). Human leukemic cell lines have been developed that are resistant to the growth inhibitory effects of gallium nitrate (Chitambar and Seligman, 1986; Chitambar et al., 1990; Chitambar and Wereley, 1997, 1998). However, the precise mechanisms of gallium resistance are not known. Because gallium markedly interferes with iron metabolism, it is thought that gallium resistance may involve alterations in cellular gallium and iron trafficking (Chitambar et al., 1990; Chitambar and Wereley, 1997). Until now, there have been no studies directly comparing the trafficking of ⁵⁹Fe or ⁶⁷Ga between gallium-resistant and -sensitive cells. Examining these alterations may be useful in elucidating the mechanisms of iron and gallium trafficking, as well as the process of gallium resistance. In addition, such knowledge could also lead to more effective radio imaging of tumors.

We demonstrate for the first time using sensitive native PAGE ⁵⁹Fe or ⁶⁷Ga autoradiography (Richardson et al., 1996) that resistance to gallium nitrate results in changes in cellular ⁵⁹Fe and ⁶⁷Ga trafficking. Moreover, in contrast to the general view that ⁶⁷Ga and ⁵⁹Fe use the same or similar pathways (Chitambar and Zivkovic, 1987), we show that their intracellular distribution and trafficking is markedly different in HL60 cells resistant (R) and sensitive (S) to gallium.

	Materials and Methods

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Cell Treatments. Gallium nitrate was obtained from Aldrich Chemicals (Milwaukee, WI), and horse spleen Ftn was from Sigma Chemical Co. (St. Louis, MO). The antibodies used were obtained from the following sources: anti-human Ftn antibody (DakoCytomation; Carpinteria, CA); anti-divalent metal transporter 1 (DMT1) antibody (Alpha Diagnostic International, San Antonio, TX); anti-TfR1 antibody (Zymed Laboratories, San Francisco, CA); anti-cyclin D1 antibody (Santa Cruz, San Diego, CA), and HFE antiserum (William S. Sly, St. Louis University, St. Louis, MO).

Cell Culture. The HL60 cell lines S and R to gallium (Chitambar et al., 1990) were grown using established techniques (Richardson and Baker, 1990). Briefly, cells were grown in RPMI (Invitrogen, Mt. Waverley, VIC, Australia) containing 10% fetal bovine serum (FBS), 1% nonessential amino acids (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 100 U/ml penicillin (Invitrogen), and 0.28 µg/ml Fungizone (Squibb Pharmaceuticals, Montreal, QC, Canada). To maintain resistance in the R clone, these cells were routinely grown in the presence of gallium nitrate (137 µM) (Chitambar et al., 1990). Cells were grown in an incubator (Forma Scientific, Marietta, OH) at 37°C in a humidified atmosphere of 5% CO₂/95% air. Cellular growth and viability were assessed by phase contrast microscopy and trypan blue staining.

Cellular Proliferation. The growth of cells in the presence and absence of gallium nitrate was measured using the MTT assay, as described previously (Richardson et al., 1995b). MTT color formation was directly proportional to the number of viable cells measured by trypan blue staining (Richardson et al., 1995b).

Western Blot Analysis. Western blot analysis was performed as described previously (Le and Richardson, 2004). The mouse monoclonal anti-human -actin antibody was from Sigma (clone AC-15) and used at a 1/15,000 dilution. The mouse anti-human TfR1 antibody was from Zymed Laboratories (San Francisco, CA). The secondary antibody used was an anti-mouse antibody (1:10,000 dilution; Sigma) conjugated with horseradish peroxidase.

Preparation of ⁵⁹Fe-¹²⁵I-Transferrin or ⁶⁷Ga-Transferrin. Human apotransferrin (Sigma) was labeled with ⁵⁹Fe and ¹²⁵I (Dupont NEN, Boston, MA) or ⁶⁷Ga-citrate (ANSTO/Ari, Lucas Heights, NSW, Australia) to produce ⁵⁹Fe-¹²⁵I-Tf or ⁶⁷Ga-Tf, using standard techniques (Chitambar and Zivkovic, 1987; Chitambar et al., 1990; Richardson and Baker, 1990).

Determination of Intracellular Iron Distribution Using Native ⁵⁹Fe/⁶⁷Ga Autoradiography. Native PAGE autoradiography was performed using established techniques (Richardson et al., 1996). Briefly, to deplete R cells of gallium nitrate, they were washed in RPMI containing 10% FBS and then incubated overnight in this media containing no gallium nitrate and washed twice. To ensure the same conditions, S cells were treated likewise. For uptake experiments, cells were incubated for 1 h in RPMI without FBS and washed to deplete bovine Tf. The cells were then labeled at 7 x 10⁵/ml with ⁶⁷Ga-Tf or ⁵⁹Fe-Tf ([protein] = 1 µM; [metal] = 2 µM) or ⁵⁹Fe-citrate (molar ratio of iron to citrate is 1:100; [Fe] = 2 µM) in RPMI without FBS. Cells were harvested, washed three times, and lysed (Richardson et al., 1996). Samples were centrifuged at 14,000 rpm for 45 min at 4°C to separate the stromal-mitochondrial membrane (SMM) fraction from the cytosol. The SMM contains membranes and organelles, including mitochondria and lysosomes (Patel and Rickwood, 1995). The SMM was further disrupted to examine ⁵⁹Fe or ⁶⁷Ga distribution as described previously (Richardson et al., 1996).

Cytosolic fractions and/or SMM fractions were loaded onto a 5% native PAGE gel to give equal amounts of protein (100 µg) across all samples. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Because a native gel is used to preserve protein integrity and metal binding, molecular weight markers are not implemented because they do not run in proportion to molecular weight. Electrophoresis was performed at 15 mA per gel for 2 to 3 h at 4°C (Richardson et al., 1996). Gels were subsequently dried at 80°C, and autoradiography was performed. Bands on X-ray film were quantified by scanning densitometry using a laser densitometer and analyzed by Kodak Biomax I Software (Kodak Ltd., Rochester, NY).

⁵⁹Fe-¹²⁵I-Tf Uptake. The ⁵⁹Fe and ¹²⁵I-Tf uptake were determined via established methods using Pronase (Sigma) to separate membrane and internalized ⁵⁹Fe and ¹²⁵I-Tf (Richardson and Baker, 1990; Richardson et al., 1995a). Briefly, cells were incubated with ⁵⁹Fe-¹²⁵I-Tf (1 µM) for up to 120 min at 37°C. The culture plates were then placed on a tray of ice, and the cells were washed three times with ice-cold phosphate-buffered saline. The cells were subsequently incubated with the general protease Pronase (1 mg/ml) for 30 min at 4°C. The cells were removed from the plates using a plastic spatula and centrifuged at 14,000 rpm for 1 min. This procedure resulted in the separation of Pronase-insensitive (internalized) ⁵⁹Fe and ¹²⁵I in the cell pellet, whereas the Pronase-sensitive (membrane-bound) ⁵⁹Fe and ¹²⁵I remained in the supernatant. Previous studies have demonstrated that this latter technique is valid for measuring internalized and membrane-bound ⁵⁹Fe-¹²⁵I-Tf uptake by cells (Richardson and Baker, 1990). The supernatant and pellet were separated and placed in separate counting tubes. Radioactivity was measured on a -scintillation counter (Wallace, Compugamma, Finland).

Antibody "Supershifts". Supershifts were performed in 10 µl of 1.5% Triton X-100 containing 1.5 µg of antibody and 50 µg of cell lysate. The mixture was incubated at 25°C for 1 h and then loaded onto the gel.

Statistical Analysis. Experimental data were compared using Student's t test. Results were expressed as mean or mean ± S.D. (number of experiments) and considered statistically significant when p < 0.05.

	Results

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Resistance to Gallium Results in Marked Alterations in Iron Uptake. Intracellular trafficking of iron and gallium is thought to be similar (Chitambar and Zivkovic, 1987; Chitambar et al., 1988), but at the molecular level, it is poorly understood. To better characterize these pathways and understand the resistance of leukemia cells to gallium, we investigated the intracellular iron distribution in R and S cells (Chitambar et al., 1990) using native PAGE ⁵⁹Fe autoradiography (Richardson et al., 1996; Richardson and Ponka, 1997; Vyoral and Petrak, 1998).

The gallium-resistant cells were generated by exposure to incremental doses of gallium nitrate over 9 to 12 months as described previously (Chitambar et al., 1990). To test the resistance of the clone used, cellular proliferation was assessed (Fig. 1A). The gallium nitrate concentration that inhibited growth by 50% was 430 µM for S cells and 2820 µM for R cells. Therefore, R cells were approximately 6.6-times more resistant to gallium nitrate-induced growth inhibition than S cells (Fig. 1A).

View larger version (28K): [in this window] [in a new window]   Fig. 1. R HL60 cells were 6.6-fold more resistant than S cells to the growth inhibitory effects of gallium and take up less 59Fe from Tf relative to S cells. A, cells were plated at 15,000 cells per well (100 µl) in the presence of gallium nitrate (0–10,000 µM). An MTT assay was then performed using S cells () and R cells () after 72 h of incubation (see Materials and Methods for details). The results shown are the mean ± S.D. of three experiments. B, the expression of the TfR1 is greater in S cells than R cells as performed by Western blot analysis. The results shown are a typical experiment of three performed. C to F, R and S cells were incubated with 59Fe-125I-Tf ([Tf] = 1 µM; [Fe] = 2 µM) for 10 min to 2 h at 37°C, and the membrane-bound and internalized fractions were separated and analyzed on a -scintillation counter for 59Fe and 125I-Tf levels (see Materials and Methods for details). The total () 125I-Tf or 59Fe per cell is shown as well as the internalized () and membrane-bound () 125I-Tf or 59Fe. The results shown are means of duplicate determinations in a typical experiment from three separate experiments performed.

Considering that gallium binds to Tf and is delivered to cells via the TfR1 (Richardson and Ponka, 1997), we examined whether resistance to gallium may perturb TfR1 expression (Fig. 1B). Expression of TfR1 protein was markedly and significantly (p < 0.0001) higher at the protein level in S cells compared with R cells (Fig. 1B). This led us to investigate the uptake of ⁵⁹Fe-¹²⁵I-Tf by cells. To investigate this, R and S cells were incubated with ⁵⁹Fe-¹²⁵I-Tf (1 µM) for 10 min to 2 h at 37°C. As shown in Fig. 1, C and D, total, internalized, and membrane uptake of ¹²⁵I-Tf was biphasic with time in R and S cells. In contrast, the uptake of total and internalized ⁵⁹Fe was linear with time (Fig. 1, E and F). The kinetics of ⁵⁹Fe and ¹²⁵I-Tf uptake were consistent with receptor-mediated endocytosis of Tf (Richardson and Baker, 1990). There were clear differences in the total and internalized uptake kinetics of ⁵⁹Fe-¹²⁵I-Tf in R and S cells (Fig. 1, C–F). In fact, the total uptake of ⁵⁹Fe per cell in R cells after a 2-h incubation was significantly (p < 0.05) less than that in S cells, namely 75 ± 2% of the ⁵⁹Fe taken up by S cells (compare Fig. 1, E and F). Importantly, this decrease in ⁵⁹Fe uptake may be at least in part due to the lower expression of TfR1 in R cells compared with S cells. This is not a clone-specific effect, since similar changes have been identified in other cell lines (e.g., CCRF-CEM leukemia cells) that have been made resistant to gallium (Chitambar and Wereley, 1997).

The intracellular distribution of ⁵⁹Fe (Fig. 2A) was then examined by incubating cells with ⁵⁹Fe-Tf (1 µM) for 2 to 24 h at 37°C and implementing native PAGE ⁵⁹Fe autoradiography (Fig. 2, A, C, and D). In the cytosol, an upper band in the gel designated as "A" was observed in R and S cells (Fig. 2, A and C), but after six experiments, this band displayed significantly (p < 0.05) different gel migration characteristics between the two cell types. Densitometric analysis revealed that in the cytosol, between 2 to 6 h band A was 1.5- to 2-fold more intense (p < 0.01) in S cells than R cells (Fig. 2B). However, after 24 h, band A was not clearly discernible above the increasing lane background and may have disappeared (Fig. 2A). Band A was consistent with a band previously identified as the Tf-TfR1 complex in K562 cells (Vyoral and Petrak, 1998). The identity of this band was further explored below and confirmed to contain the TfR1 (see Fig. 4). After 24 h, a faint band denoted by the arrow was observed in R and S cells above the band A (Fig. 2A).

View larger version (50K): [in this window] [in a new window]   Fig. 2. R and S cells display differences in 59Fe trafficking following incubation with 59Fe-Tf. R and S HL60 cells were incubated with 59Fe-Tf ([Tf] = 1 µM; [Fe] = 2 µM) for 2 to 24 h at 37°C and then washed. The cytosolic (A) and SMM fractions (D) were then isolated and native PAGE 59Fe autoradiography was performed (see Materials and Methods for details). Densitometric analyses of bands A and C are also shown for S cells ( and ) and R cells ( and ) for the cytosolic fraction (B) and the SMM fraction (E). C, the alterations in 59Fe distribution after the addition of 1 mM DFO to a lysate derived from cells incubated with 59Fe-Tf for 6 h, indicating that DFO removes 59Fe from the LIP but no other compartment. The results show a typical experiment from six separate experiments performed.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Identification of 59Fe- and 67Ga-containing bands in R and S HL60 cells using antibody supershift experiments. Supershift experiments were performed using lysates from cells incubated for 4 h with 59Fe-Tf ([Tf] = 1 µM; [Fe] = 2 µM) (A and B) or 24 h with 67Ga-Tf ([Tf] = 1 µM; [Ga] = 2 µM) (C and D). To the cytosolic fraction, BSA (protein control) or antibodies against Ftn, Tf, TfR1, cyclin D1 (CD1), HFE, or DMT1 were added and incubated for 1 h at 25°C before native PAGE 59Fe or 67Ga autoradiography (see Materials and Methods for details). D, in addition to the effect of antibodies and BSA on 67Ga distribution in S cells, the result of adding 1 mM DFO to the lysate and migration of 67Ga-citrate in the gel is illustrated. The results show typical experiments from three to four separate experiments performed.

The band migrating immediately below band A, designated as "B", was only clearly apparent in R cells and only discernible between 2 and 6 h (Fig. 2A). Band C comigrated with horse spleen Ftn and ⁵⁹Fe uptake into this protein was similar between R and S cells (Fig. 2, A and B). We adopted the hypothesis that band C represented Ftn, and the identity of this band is confirmed below (see Fig. 4). Densitometric analysis revealed that the percentage of total cellular ⁵⁹Fe uptake into Ftn was not significantly (p > 0.05) different between S and R cells. This was equal to 36 ± 3 and 39 ± 4% (4 experiments) of total ⁵⁹Fe in S and R cells, respectively, after a 24-h incubation with ⁵⁹Fe-Tf. A faint band migrating between band C and D was observed in the S cell type (Fig. 2A). However, it was not a consistent or prominent finding in all experiments and will not be discussed further.

Band D, which migrated below C (circled; Fig. 2A), was only found in S cells. This band tended to be above a diffuse lower band that was removed by the addition of the chelator desferrioxamine (DFO; 1 mM) to the lysate (Fig. 2C). In fact, DFO caused a pronounced and significant (p < 0.0001) ablation in the intensity of band D after six experiments (Fig. 2C). Therefore, we termed this latter band the labile iron pool (LIP). However, we are not definitively stating that this entity corresponds to the LIP observed by others (Breuer et al., 1996). Band D was not significantly decreased (p > 0.05) after incubation with DFO, suggesting that it was different from the LIP (Fig. 2C). Band B was not evident in the gel shown in Fig. 2C, since the lysates were from cells incubated with ⁵⁹Fe-Tf for 6 h, at which time this band disappears into the background (Fig. 2A). Band D was only clearly apparent after a 4-h incubation with ⁵⁹Fe-Tf, whereas LIP was already evident at 2 h (Fig. 2A). In addition, band D did not comigrate with purified ⁵⁹Fe-Tf (Fig. 2C).

Figure 2D illustrates ⁵⁹Fe uptake into the SMM, which was similar to that observed in the cytosol (Fig. 2A), although there are some differences. The most notable difference was that band B is not clearly discernible in the SMM fraction (Fig. 2D).

Resistance to Gallium Results in Marked Alterations in ⁶⁷Ga Uptake from Transferrin. Because we were interested in understanding the mechanism of gallium resistance, uptake and intracellular distribution of ⁶⁷Ga were also assessed in R and S cells (Fig. 3). As observed for ⁵⁹Fe uptake, gallium resistance resulted in marked changes in ⁶⁷Ga distribution. Similarly to experiments examining ⁵⁹Fe distribution (Fig. 2), ⁶⁷Ga-Tf (1 µM) uptake was performed for 2 to 24 h at 37°C, and the cytosolic and SMM fractions were prepared (Fig. 3). Examining ⁶⁷Ga uptake from Tf, we found linear incorporation into the cytosol and SMM as a function of time (Fig. 3A). The uptake of ⁶⁷Ga into the cytosol was significantly (p < 0.0005) greater than that in the SMM in both R and S cells. However, in the cytosol, ⁶⁷Ga uptake was significantly (p < 0.005) lower in R cells than S cells. This was most apparent after 24 h, where R cell ⁶⁷Ga incorporation was 75% (n = 6) that of S (Fig. 3A). In contrast, in the SMM fraction, the reverse was true; ⁶⁷Ga uptake was 28% (6) greater (p < 0.05) in R cells than S cells after 24 h (Fig. 3A). Therefore, there was evidence of preferential ⁶⁷Ga trafficking into the SMM compartment of R cells relative to S cells. The SMM protein fraction represented 15 ± 5% (6) of the total protein pool in the cell and incorporated approximately 10 ± 3% (6) of the total ⁶⁷Ga uptake after 24 h. When total cellular ⁶⁷Ga uptake from Tf (i.e., cytosol and SMM uptake) into the R cells was assessed after 24 h, it was 81 ± 6% (6) of the uptake into S cells. This was similar (p > 0.05) to the corresponding value of 85 ± 6% (n = 4) for total cellular ⁵⁹Fe uptake in R cells relative to S cells. Potentially, this may be significant in terms of gallium resistance.

View larger version (39K): [in this window] [in a new window]   Fig. 3. R and S cells display differences in 67Ga trafficking following incubation with 67Ga-Tf. R and S HL60 cells were incubated with 67Ga-Tf ([Tf] = 1 µM; [Ga] = 2 µM) for 2 to 24 h at 37°C and then washed and the cytosolic and SMM fractions isolated. A, 67Ga uptake (cpm/100 µg of protein) as a function of incubation time in the cytosolic and SMM fractions; B and D, R and S HL60 cells were labeled as described above and native PAGE 67Ga autoradiography performed (see Materials and Methods for details). Densitometric analysis of band C and the LGP is also shown for S cells ( and ) and R cells ( and ) for the cytosolic fraction (C) and SMM fraction (E). The results show a typical experiment from six separate experiments performed.

Strikingly, in comparison to ⁵⁹Fe-Tf uptake (Fig. 2), the majority of the ⁶⁷Ga was present in a low mol. wt. form in the cytosolic and SMM fractions, and we have termed this the "labile gallium pool" (LGP; Fig. 3, B and D). This was the case in both R and S cells. However, a significantly (p < 0.01) greater proportion of the total cytosolic ⁶⁷Ga incorporated was present in the LGP in R cells (81 ± 3%; 6) compared with S cells (67 ± 5%; 6) after 24 h (Fig. 3B).

The SMM fraction (Fig. 3, D and E) displayed similar ⁶⁷Ga distribution to that found in the cytosol (Fig. 3, B and C). However, neither band A (the putative Tf-TfR1 complex) nor band B was clearly discernible (Fig. 3D). As observed for the cytosol, most ⁶⁷Ga in the SMM was incorporated into the LGP. Interestingly, more ⁶⁷Ga was incorporated into the LGP of the SMM in R cells compared with S cells (Fig. 3, D and E). In fact, in three experiments the ⁶⁷Ga uptake into the LGP compartment of the SMM fraction of R cells after an incubation of 2, 4, 6, and 24 h was 12 ± 1-, 3 ± 0.3-, 2 ± 0.1-, and 1.5 ± 0.1-fold greater (p < 0.05), respectively, than that found for S cells.

Antibody Supershifts Identify Ftn, TfR1, and HFE among the ⁶⁷Ga- and ⁵⁹Fe-Containing Protein Bands. To aid the identification of ⁵⁹Fe- and ⁶⁷Ga-containing bands observed in Figs. 2 and 3, supershift experiments were performed using antibodies specific for Ftn, Tf, TfR1, HFE, and DMT1 (Fig. 4). As a relevant negative control, the same concentration of another antibody against a protein not known to be directly involved in iron metabolism, namely cyclin D1 (CD1), was also used. In addition, bovine serum albumin (BSA) was used as an additional protein control. Neither CD1 nor BSA resulted in any significant (p > 0.05) supershifts or perturbations of ⁵⁹Fe or ⁶⁷Ga distribution.

Lysates from cells that had been labeled for 4 or 24 h with ⁵⁹Fe-Tf (1 µM) or ⁶⁷Ga-Tf (1 µM), respectively, were incubated for 1 h at 25°C with antibodies, and the samples then electrophoresed on a 5% native gel and subjected to autoradiography. The addition of an anti-Ftn antibody (Fig. 4A) prevented the entrance of band C into the gel in R and S cells, confirming it represented Ftn. The addition of the anti-TfR1 antibody to a lysate derived from cells incubated for 4 h with ⁵⁹Fe-Tf resulted in a supershift (denoted by an arrow) of band A compared with the relevant control in R and S cells (Fig. 4, A and B). The shift in this band upon the addition of antibody was found to be significant (p < 0.0005) over four experiments. These results indicated that band A was consistent with the Tf-TfR1 complex. It is of interest to note that band A was significantly (p < 0.001) more pronounced in S cells than R cells (Fig. 4, A and B), and this could be accounted for by the greater expression of the TfR1 in S cells that binds ⁵⁹Fe-Tf (Fig. 1, B–D). Surprisingly, the anti-Tf antibody had no effect on band A (Tf-TfR1 complex; Fig. 4A), even though this complex would be expected to contain ⁵⁹Fe-Tf as well as TfR1. This may indicate that Tf within the complex was not accessible to the antibody. Band D that was exclusive to S cells and which migrated below Ftn, was not affected by any of the antibodies (Fig. 4, A and B).

The HFE antiserum was used at two dilutions (1/3 and 1/13) and in R and S cells resulted in a titer-dependent shift of band A, which as described above, is consistent with the TfR1 (Fig. 4B). This indicates that the Tf-TfR1 complex in R and S cells also contains HFE (i.e., it is consistent with the Tf-TfR1-HFE complex), as documented in other systems (Parkkila et al., 1997; Roy et al., 1999). Instead of HFE antiserum resulting in a shift of band A toward the origin, the addition of the antiserum caused a significant (p < 0.0001) shift resulting in two bands migrating below Ftn (denoted by arrows; Fig. 4B). This may be due to perturbation of the Tf-TfR1-HFE complex caused by binding of the HFE antiserum, resulting in disruption of this complex or conformational or electronic changes, causing it to migrate more rapidly. Previous studies using various antibodies have also shown that interaction of these proteins with other types of molecular complexes can lead to their disruption (Crossley et al., 1996; Davies et al., 2004).

The addition of the HFE antiserum also resulted in a decrease of the intensity of band B in R cells (Fig. 4B). Although the intensity of this band is not strong in the control, assessment of the density of this band over three separate experiments demonstrated that it was significantly (p < 0.001) decreased by 85 ± 5% upon the addition of the HFE antiserum. This may potentially explain in R cells why two bands (see arrows in Fig. 4B) are observed migrating below Ftn following the addition of HFE antiserum; that is, bands A and B are shifted down the gel (Fig. 4B). Band B is not apparent in S cells. However, two bands are also observed in S cells below Ftn after the addition of HFE antiserum (Fig. 4B). Band B may be the source of this second band in R cells, whereas in S cells, the origin of this second very faint band (see arrow) is not apparent. These results suggest HFE is present in two complexes with ⁵⁹Fe-Tf and presumably the TfR1 in R and S cells, i.e., those in bands A and B. However, anti-TfR1 antibody only supershifted band A but not band B, suggesting the different nature of these bands (Fig. 4B). The addition of an antibody specific for the iron-responsive element- and non-iron-responsive element-containing forms of DMT1 (Fig. 4B) had no significant (p > 0.05) effect on any bands.

As found for ⁵⁹Fe uptake (Fig. 4, A and B), the identity of the ⁶⁷Ga bands was explored using antibodies added to lysates derived from a 24-h incubation with ⁶⁷Ga-Tf (Fig. 4, C and D). In all studies, the ⁶⁷Ga-containing bands were not as well defined as those found for ⁵⁹Fe. However, the Ftn antibody resulted in loss of band C in R cells (Fig. 4C) and S cells (Fig. 4D), identifying it as Ftn. The use of TfR1 antibody resulted in a significant (p < 0.005) supershift of band A in R and S cells (see arrow in Fig. 4C), suggesting it is a TfR1 complex. The addition of 1 mM DFO to the lysate markedly and significantly (p < 0.0001) decreased the LGP in S cells (Fig. 4D) and R cells (data not shown). This LGP comigrated with free ⁶⁷Ga-citrate (Fig. 4D). Similarly to cells labeled with ⁵⁹Fe, bands A and B were significantly (p < 0.01) supershifted down the gel upon the addition of anti-HFE to the ⁶⁷Ga-labeled lysates (data not shown).

Redistribution of ⁵⁹Fe and ⁶⁷Ga in Prelabeled R and S Cells Upon Reincubation. Gallium resistance may also be mediated by release of gallium and, hence, possibly iron from the cell. To investigate this, cells were incubated with ⁵⁹Fe-Tf for 4 h, followed by washing and reincubation for up to 24 h. Cellular ⁵⁹Fe redistribution and its release into the overlying media was then assessed. Total cellular ⁵⁹Fe decreased similarly in R and S cells as a function of time; there was a significant (p < 0.0005) difference between time 0 and after a reincubation of 18 and 24 h (Fig. 5A). A concomitant increase of ⁵⁹Fe into the media was also found with no difference being evident between R and S cells (data not shown). The decrease in SMM-⁵⁹Fe levels was shown to be similar to that in the cytosol (data not shown). In R and S cell cytosol, band A (the Tf-TfR1-HFE complex) displayed kinetic properties of an intermediate not discernible after 1 h of reincubation (Fig. 5, B and C); there was a pronounced and significant (p < 0.0001) decrease in the intensity of this band after the 0-h time point. This suggests ⁵⁹Fe uptake from ⁵⁹Fe-Tf via the TfR1 followed by its cycling through the cell and its subsequent release (Richardson and Ponka, 1997). During reincubation, Ftn-⁵⁹Fe (band C) was significantly (p < 0.01) increased until 6 h and then slightly decreased up to 24 h (Fig. 5, B and C). This was consistent with initial ⁵⁹Fe uptake into Ftn followed by gradual ⁵⁹Fe release until 24 h. The LIP and band D essentially disappeared after an 18- and 24-h reincubation, indicating their roles as intermediates or molecules with short half-lives (Fig. 5B).

View larger version (40K): [in this window] [in a new window]   Fig. 5. Kinetics of the decrease in cellular 59Fe levels after reincubation of prelabeled R and S cells in media alone and the identification of intermediates involved in iron trafficking. A, decrease of cellular 59Fe levels (cpm/100 µg of protein) as a function of time in R and S HL60 cells that were prelabeled for 4 h with 59Fe-Tf ([Tf] = 1 µM; [Fe] = 2 µM), washed, and then reincubated in media alone for up to 24 h. B, lysates derived from the cells in A were then used for native PAGE 59Fe autoradiography (see Materials and Methods for details). C, densitometric analysis of the Ftn (band C) and Tf-TfR1 complex (band A) in B. The results shown are a typical experiment from five separate experiments performed.

View larger version (33K): [in this window] [in a new window]   Fig. 6. A to D, kinetics of the decrease in cellular 67Ga levels after reincubation of prelabeled R and S cells in media alone. E, the intracellular distribution of 59Fe, but not 67Ga, is similar between R and S cells. A, decrease of cellular 67Ga in R and S HL60 cells that were prelabeled for 24 h with 67Ga-Tf ([Tf] = 1 µM; [Ga] = 2 µM), washed, and then reincubated in media alone for up to 24 h. B, lysates derived from the cells in A were then used for native PAGE 67Ga autoradiography (see Materials and Methods for details) and exposed for 24 (Bi) or 72 h (Bii). Bi was used for the densitometric analysis of the LGP (C), and Bii was used for densitometric analysis of the Ftn band (D). The results in A–D show a typical experiment from four performed. E, R and S HL60 cells were incubated with 59Fe-Tf ([Tf] = 1 µM; [Fe] = 2 µM) or 67Ga-Tf ([Tf] = 1 µM; [Ga] = 2 µM) for 24 h at 37°C. The cytosolic fraction was then prepared and used for native PAGE 59Fe autoradiography, which was followed by densitometric analysis of the Ftn band as well as the LGP and LIP. The Ftn, LGP, and LIP bands were expressed as a percentage of the total 59Fe or 67Ga incorporated in the R or S cells after a 24-h incubation, as recorded by densitometric analysis. The results show the mean ± S.D. from four to six separate experiments.

For the reincubation experiments in Figs. 5, A to C, and 6, A to D, the levels of ⁵⁹Fe and ⁶⁷Ga steadily declined in R and S cells in both cytosol and SMM. Examination of reincubation media showed that ⁶⁷Ga and ⁵⁹Fe levels significantly (p < 0.01) increased over time (data not shown). Hence, the decline in cellular ⁵⁹Fe and ⁶⁷Ga may be at least partially due to their efflux.

Intracellular Distribution of ⁶⁷Ga Differs Between R and S Cells. Figure 6E shows the densitometric analysis of Ftn, the LIP, and LGP for cells incubated with ⁵⁹Fe-Tf or ⁶⁷Ga-Tf for 24 h. There was no significant (p > 0.05) difference in ⁵⁹Fe distribution between these compartments in S and R cells after 24 h. In contrast, R and S cells displayed significant differences in ⁶⁷Ga distribution after 24 h (Fig. 6E). In fact, S cells displayed significantly (p < 0.02) greater (4.5 ± 1.6%; 6) incorporation of total ⁶⁷Ga into Ftn, compared with 1.6 ± 0.5% (6) for R cells. Furthermore, S cells incorporated significantly (p < 0.01) less (67 ± 5%; 6) of total ⁶⁷Ga into the LGP, compared with 81 ± 3% (6) for R cells (Fig. 6E). Analysis of ⁵⁹Fe and ⁶⁷Ga uptake in the SMM produced similar results (data not shown).

These results highlight the marked differences in distribution of ⁶⁷Ga relative to ⁵⁹Fe (Fig. 6E). For instance, in the labile pool, ⁶⁷Ga levels are 5-fold greater (p < 0.0001) than those found for ⁵⁹Fe, whereas for Ftn, ⁵⁹Fe levels were 8- and 24-fold greater (p < 0.0001) than that of ⁶⁷Ga in the S and R cells, respectively (Fig. 6E).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
For many years, the trafficking of iron and gallium was thought to be very similar (Chitambar and Zivkovic, 1987). This view was derived from research examining the binding of iron and gallium to Tf and their delivery to the cell via the TfR1 (Chitambar and Zivkovic, 1987). However, apart from this, the subsequent metabolism and intracellular trafficking of gallium have remained uncertain. Despite rapidly advancing knowledge regarding specific molecules involved in iron metabolism, little is known concerning the trafficking pathways involved in ⁵⁹Fe or ⁶⁷Ga transport. The recent interest in the use of gallium as an antineoplastic agent (Chitambar, 2004) has underlined the importance of understanding pathways involved in its metabolism. In particular, the development of tumor cell lines that are gallium-resistant (Chitambar et al., 1990) could suggest altered uptake mechanisms that provide an opportunity for delineating gallium and iron transport. Furthermore, understanding these pathways may lead to strategies to prevent gallium resistance in tumor cells and lead to improved ⁶⁷Ga radio-imaging techniques for tumors (Warrell et al., 1983; Chitambar et al., 1997).

In the present study, we have shown that although there are some common aspects in iron and gallium uptake, there are clear differences in their intracellular trafficking and distribution. For the first time, we show that ⁵⁹Fe and ⁶⁷Ga trafficking are distinct in R and S cells. One of the most striking differences between ⁵⁹Fe and ⁶⁷Ga incorporation was that a large proportion of ⁵⁹Fe was incorporated into Ftn with comparatively less in the LIP, whereas ⁶⁷Ga was poorly incorporated into Ftn, with most present as the LGP (Fig. 6E). Intriguingly, we also identified novel intermediates and alterations in the ⁵⁹Fe and ⁶⁷Ga trafficking pathways in R cells compared with their sensitive counterparts.

Although previous studies suggest that gallium may be incorporated into Ftn (Hegge et al., 1977; Nakamura et al., 1984; Chitambar and Zivkovic, 1987), the extent of this in comparison to other cellular compartments has remained unclear. Our results showed that Ftn cannot incorporate ⁶⁷Ga efficiently compared with ⁵⁹Fe (compare Figs. 2 and 3). In view of the mechanism of iron uptake (Richardson and Ponka, 1997), the observation that ⁶⁷Ga is taken up into Ftn at all is surprising. Indeed, it is not even known how Tf-bound gallium is transported from the endosome. A possible candidate could be DMT1, since Fe(III) released from Tf is reduced to Fe(II) and transported through the endosomal membrane by this molecule (Richardson and Ponka, 1997). However, unlike Fe(III), Ga(III) cannot be reduced to Ga(II). Interestingly, DMT1 may transport Fe(III) (Thomas and Oates, 2004) and, thus, could potentially mediate Ga(III) uptake. Once Fe(II) is transported through the endosome, it is incorporated into Ftn and oxidized to Fe(III), leading to its deposition (Richardson and Ponka, 1997). Because Ga(III) cannot be reduced to Ga(II), other mechanisms must result in limited gallium being incorporated into Ftn.

Apart from cytosolic Ftn, an immunologically identical band to Ftn was found in the SMM (Fig. 2D). Because the SMM can contain mitochondria and other organelles (Patel and Rickwood, 1995), it may represent mitochondrial Ftn, although levels of this molecule are exceedingly low in many cell lines (Levi et al., 2001; Corsi et al., 2002). However, a membrane- and nuclear-associated Ftn has been identified (Iancu et al., 1976; Surguladze et al., 2005), and we favor this corresponds to the SMM-associated Ftn.

The inefficient uptake of ⁶⁷Ga by Ftn could contribute to the marked accumulation of ⁶⁷Ga in the LGP (Fig. 3). The LGP comigrates with ⁶⁷Ga-citrate and is labile because it is bound by the chelator DFO (Fig. 4D). A similar observation was also seen for the LIP (Fig. 2C). However, it is unclear whether these compartments exist in cells as low mol. wt. complexes, and it may be that the buffers (e.g., Tris) used for native PAGE could chelate ⁵⁹Fe or ⁶⁷Ga from labile binding proteins. Considering this, there is little direct evidence of large quantities of low mol. wt. iron within cells (Richardson and Ponka, 1997). In fact, the participation of chaperones and direct protein-protein transfer are possible alternative mechanisms that achieve iron trafficking (Richardson et al., 1996).

Another important finding in the current investigation was that two ⁵⁹Fe-containing bands (A and B) were identified in R cells that were consistent with two forms of the Tf-TfR1-HFE complex (Figs. 2 and 4). In contrast, in S cells, only one Tf-TfR1-HFE complex (band A) was observed, and the intensity of this band was greater than that in R cells (Fig. 2). Both bands A and B reacted with HFE antiserum, whereas only band A reacted with TfR1 antibody, suggesting its different nature. Considering this, it may be suggested that band B represented a Tf-HFE complex with TfR2, although previous studies by others have shown that HFE-binding to the TfR2 is not detectable (West et al., 2000). However, it is well known that HFE regulates Tf-binding to the TfR1, as it competes for the Tf-binding site on this receptor (Parkkila et al., 1997; Corsi et al., 1999; Roy et al., 1999; West et al., 2000). HFE has also been shown to decrease ⁶⁷Ga uptake from ⁶⁷Ga-Tf (Chitambar and Wereley, 2003), and we demonstrated decreased ⁶⁷Ga and ⁵⁹Fe uptake in R cells compared with their sensitive counterparts. Together with the lower TfR1 expression in R cells (Chitambar and Wereley, 1997), the two Tf-TfR1-HFE complexes may be involved in reducing ⁶⁷Ga and ⁵⁹Fe uptake from Tf compared with S cells (Figs. 1, D and E, and 3A). Hence, this alteration could potentially be important in the gallium-resistance observed. Indeed, previous investigations suggested that the interaction of HFE and TfR1 in intracellular vesicles may be important for the function of HFE in iron trafficking (Waheed et al., 2002; Davies et al., 2003).

The two Tf-TfR1-HFE bands observed in our studies may arise from the different compositions of these complexes with other proteins, such as DMT1 or the ferrireductase, involved in iron release from Tf (Richardson and Ponka, 1997). The changed ratios of these molecules in the complex may result in the two bands and contribute to altered gallium metabolism. Moreover, the dissimilar compositions of these complexes could explain the differential reactivity of bands A and B toward the TfR1 antibody, since different complexes may block TfR1-antigenic sites.

A novel band labeled D was identified in this investigation that was located just above the LIP (Fig. 2A). However, unlike the LIP, band D was not removed upon adding DFO to the lysate (Fig. 2C), suggesting its different character. Band D had the properties of a long-lived iron-containing intermediate or short-lived iron-containing protein, only disappearing after an 18-h reincubation (Fig. 6B). Interestingly, band D was only found in S cells; it was absent from R cells. Whether this molecule plays a role in the sensitivity to gallium remains a subject for further investigation.

It is of interest to discuss that although the differences presented in gallium distribution in Ftn and labile pool are statistically different between R and S cells (Fig. 6), this difference is rather modest. Hence, why is there a 6-fold difference in gallium toxicity between R and S cells? Considering this, and the alterations in iron and gallium trafficking that potentially confer gallium resistance, a number of differences between R and S cells were identified in this study. These findings include 1) the fact that R cells incorporated less gallium than S cells because of the lower expression of the TfR1 than S cells; 2) the identification of an altered Tf-TfR1-HFE complex, which was more intense in S cells compared with R cells; 3) the presence of an additional and novel Tf-TfR1-HFE complex that was more pronounced in R cells; 4) the absence of band D in R cells, which may act as a potential "sensitivity factor"; and 5) relative to S cells, there was less gallium uptake into the Ftn of R cells and more incorporation into the SMM. This latter observation suggests that the altered compartmentalization between Ftn and the SMM may prevent the deleterious effects of gallium in R cells. Hence, all of these changes may contribute to gallium resistance.

In conclusion, as well as providing clues to the mechanism of gallium resistance, this investigation results in considerable insight into the differences between iron and gallium trafficking in cells, which should lead to a greater understanding of the critical processes of tumor cell iron and gallium metabolism. Such knowledge may eventually lead to the development of more effective treatment strategies with gallium. Specifically, this study demonstrates for the first time that in contrast to ⁵⁹Fe, little ⁶⁷Ga is incorporated into Ftn, with most present as a labile ⁶⁷Ga pool. We also identified unique alterations in gallium and iron trafficking between R and S cells. In fact, in R cells, there was a distinct Tf-TfR1-HFE complex (band B) not observed in S cells. Moreover, the two Tf-TfR1-HFE complexes in R cells may be involved in reduced ⁶⁷Ga and ⁵⁹Fe uptake compared with S cells. Finally, in S cells, a novel iron-binding intermediate (band D) was identified that was not present in R cells and may be a sensitivity factor to gallium.

	Acknowledgements

 
We gratefully acknowledge the tremendous assistance of Dr. Ralph Watts in the preparation of the figures. The assistance of Dr. Jon Howard is also very kindly acknowledged in studies assessing the effects of iron on gallium cytotoxicity. Dr. David Lovejoy and Danuta Kalinowski are thanked for kind help in reviewing the article before submission.

	Footnotes

 
Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.

This project was supported by a National Health and Medical Research Council fellowship and a grant from the Leukemia Foundation of New South Wales (to D.R.R.).

doi:10.1124/jpet.105.099044.

ABBREVIATIONS: Tf, transferrin; TfR1, transferrin receptor 1; PAGE, polyacrylamide gel electrophoresis; R, resistant; S, sensitive; Ftn, ferritin; DMT1, divalent metal transporter 1; HFE, hemochromatosis protein; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; SMM, stromal-mitochondrial membrane; DFO, desferrioxamine; LIP, labile iron pool; LGP, labile gallium pool; CD1, cyclin D1; BSA, bovine serum albumin.

Address correspondence to: Prof. Des R. Richardson, Iron Metabolism and Chelation Program, Department of Pathology, Blackburn Building (DO6), University of Sydney, Sydney, NSW 2006, Australia. E-mail: d.richardson{at}pathology.usyd.edu.au

	References

Top Abstract Materials and Methods Results Discussion References

 

Breuer W, Epsztejn S, and Cabantchik ZI (1996) Dynamics of the cytosolic chelatable iron pool of K562 cells. FEBS Lett 382: 304–308.[CrossRef][Medline]

Chitambar CR (2004) Gallium nitrate for the treatment of non-Hodgkin's lymphoma. Expert Opin Investig Drugs 13: 531–541.[Medline]

Chitambar CR, Matthaeus WG, Antholine WE, Graff K, and O'Brien WJ (1988) Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. Blood 72: 1930–1936.[Abstract/Free Full Text]

Chitambar CR and Seligman PA (1986) Effects of different transferrin forms on transferrin receptor expression, iron uptake and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium. J Clin Investig 78: 1538–1546.[Medline]

Chitambar CR and Wereley JP (1997) Resistance to the antitumor agent gallium nitrate in human leukemic cells is associated with decreased gallium/iron uptake, increased activity of iron regulatory protein-1 and decreased ferritin production. J Biol Chem 272: 12151–12157.[Abstract/Free Full Text]

Chitambar CR and Wereley JP (1998) Transferrin receptor-dependent and -independent iron transport in gallium-resistant human lymphoid leukemic cells. Blood 91: 4686–4693.[Abstract/Free Full Text]

Chitambar CR and Wereley JP (2003) Expression of the hemochromatosis (HFE) gene modulates the cellular uptake of 67Ga. J Nucl Med 44: 943–946.[Abstract/Free Full Text]

Chitambar CR, Zahir SA, Ritch PS, and Anderson T (1997) Evaluation of continuous-infusion gallium nitrate and hydroxyurea in combination for the treatment of refractory non-Hodgkin's lymphoma. Am J Clin Oncol 20: 173–178.[CrossRef][Medline]

Chitambar CR and Zivkovic Z (1987) Uptake of gallium-67 by human leukemic cells: demonstration of transferrin receptor-dependent and transferrin-independent mechanisms. Cancer Res 47: 3929–3934.[Abstract/Free Full Text]

Chitambar CR, Zivkovic-Gilgenbach Z, Narasimhan J, and Antholine WE (1990) Development of drug resistance to gallium nitrate through modulation of cellular iron uptake. Cancer Res 50: 4468–4472.[Abstract/Free Full Text]

Corsi B, Cozzi A, Arosio P, Drysdale J, Santambrogio P, Campanella A, Biasiotto G, Albertini A, and Levi S (2002) Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects cellular iron metabolism. J Biol Chem 277: 22430–22437.[Abstract/Free Full Text]

Corsi B, Levi S, Cozzi A, Corti A, Altimare D, Albertini A, and Arosio P (1999) Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron-deficient phenotype. FEBS Lett 460: 149–152.[CrossRef][Medline]

Crawford ED, Saiers JH, Baker LH, Costanzi JH, and Bukowski RM (1991) Gallium nitrate in advanced bladder carcinoma: Southwest Oncology Group study. Urology 38: 355–357.[CrossRef][Medline]

Crossley M, Whitelaw E, Perkins A, Williams G, Fujiwara Y, and Orkin SH (1996) Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells. Mol Cell Biol 16: 1695–1705.[Abstract]

Davies N, Freebody J, and Murray V (2004) Chromatin structure at the flanking regions of the human beta-globin locus control region DNase I hypersensitive site-2: proposed nucleosome positioning by DNA-binding proteins including GATA-1. Biochim Biophys Acta 1679: 201–213.[Medline]

Davies PS, Zhang AS, Anderson EL, Roy CN, Lampson MA, McGraw TE, and Enns CA (2003) Evidence for the interaction of the hereditary haemochromatosis protein, HFE, with the transferrin receptor in endocytic compartments. Biochem J 373: 145–153.[CrossRef][Medline]

Hegge FN, Mahler DJ, and Larson SM (1977) The incorporation of Ga-67 into the ferritin fraction of rabbit hepatocytes in vivo. J Nucl Med 18: 937–939.[Abstract/Free Full Text]

Iancu TC, Neustein HB, and Landing BH (1976) The liver in thalassaemia major: ultrastructural observations. Ciba Found Symp (51): 293–316.

Knorr GM and Chitambar CR (1998) Gallium-pyridoxal isonicotinoyl hydrazone (Ga-PIH), a novel cytotoxic gallium complex. A comparative study with gallium nitrate. Anticancer Res 18: 1733–1737.[Medline]

Larson SM, Rasey JS, Allen DR, Nelson NJ, Grunbaum Z, Harp GD, and Williams DL (1980) Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor. J Natl Cancer Inst 64: 41–53.[Medline]

Le NT and Richardson DR (2004) Iron chelators with high antiproliferative activity up-regulate the expression of a growth inhibitory and metastasis suppressor gene: a link between iron metabolism and proliferation. Blood 104: 2967–2975.[Abstract/Free Full Text]

Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, Arosio P, and Drysdale J (2001) A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem 276: 24437–24440.[Abstract/Free Full Text]

Nakamura K, Kawaguchi H, Shimizu K, and Orii H (1984) The role of ferritin in the intracellular distribution of gallium 67. Eur J Nucl Med 9: 237–240.[Medline]

Parkkila S, Waheed A, Britton RS, Bacon BR, Zhou XY, Tomatsu S, Fleming RE, and Sly WS (1997) Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 94: 13198–13202.[Abstract/Free Full Text]

Patel D and Rickwood D (1995) Optimization of conditions for specific binding of antibody-coated beads to cells. J Immunol Methods 184: 71–80.[Medline]

Richardson DR and Baker E (1990) The uptake of iron and transferrin by the human malignant melanoma cell. Biochim Biophys Acta 1: 1–12.

Richardson DR, Neumannova V, Nagy E, and Ponka P (1995a) The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells. Blood 86: 3211–3219.[Abstract/Free Full Text]

Richardson DR and Ponka P (1997) The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells. Biochim Biophys Acta 1331: 1–40.[Medline]

Richardson DR, Ponka P, and Vyoral D (1996) Distribution of iron in reticulocytes after inhibition of heme synthesis with succinylacetone: examination of the intermediates involved in iron metabolism. Blood 87: 3477–3488.[Abstract/Free Full Text]

Richardson DR, Tran EH, and Ponka P (1995b) The potential of iron chelators of the pyridoxal isonicotinoyl hydrazone class as effective antiproliferative agents. Blood 86: 4295–4306.[Abstract/Free Full Text]

Roy CN, Carlson EJ, Anderson EL, Basava A, Starnes SM, Feder JN, and Enns CA (2000) Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells. FEBS Lett 484: 271–274.[CrossRef][Medline]

Roy CN, Penny DM, Feder JN, and Enns CA (1999) The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J Biol Chem 274: 9022–9028.[Abstract/Free Full Text]

Seidman AD, Scher HI, Heinemann MH, Bajorin DF, Sternberg CN, Dershaw DD, Silverberg M, and Bosl GJ (1991) Continuous infusion gallium nitrate for patients with advanced refractory urothelial tract tumors. Cancer 68: 2561–2565.[CrossRef][Medline]

Seligman PA and Crawford ED (1991) Treatment of advanced transitional cell carcinoma of the bladder with continuous-infusion gallium nitrate. J Natl Cancer Inst 83: 1582–1584.[Free Full Text]

Surguladze N, Patton S, Cozzi A, Fried MG, and Connor JR (2005) Characterization of nuclear ferritin and mechanism of translocation. Biochem J 388: 731–740.[CrossRef][Medline]

Thomas C and Oates PS (2004) Differences in the uptake of iron from Fe(II) ascorbate and Fe(III) citrate by IEC-6 cells and the involvement of ferroportin/IREG-1/MTP-1/SLC40A1. Pflueg Arch Eur J Physiol 448: 431–437.[Medline]

Vyoral D and Petrak J (1998) Iron transport in K562 cells: a kinetic study using native gel electrophoresis and 59Fe autoradiography. Biochim Biophys Acta 2: 179–188.

Waheed A, Grubb JH, Zhou XY, Tomatsu S, Fleming RE, Costaldi ME, Britton RS, Bacon BR, and Sly WS (2002) Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci USA 99: 3117–3122.[Abstract/Free Full Text]

Warrell RP Jr, Coonley CJ, Straus DJ, and Young CW (1983) Treatment of patients with advanced malignant lymphoma using gallium nitrate administered as a seven-day continuous infusion. Cancer 51: 1982–1987.[CrossRef][Medline]

West AP Jr, Bennett MJ, Sellers VM, Andrews NC, Enns CA, and Bjorkman PJ (2000) Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE. J Biol Chem 275: 38135–38138.[Abstract/Free Full Text]

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