From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Transferrin Receptor-Dependent and -Independent Iron Transport
in Gallium-Resistant Human Lymphoid Leukemic Cells
By Christopher R. Chitambar and Janine P. Wereley
Recent studies showed that gallium and iron uptake are
decreased in gallium-resistant (R) CCRF-CEM cells; however,
the mechanisms involved were not fully elucidated. In the
present study, we compared the cellular uptake of 59Fetransferrin (Tf) and 59Fe-pyridoxal isonicotinoyl hydrazone
(PIH) to determine whether the decrease in iron uptake by R
cells is caused by changes in Tf receptor (TfR)-dependent or
TfR-independent iron uptake. We found that both 59Fe-Tf and
59Fe-PIH uptake were decreased in R cells. The uptake of
59Fe-Tf but not 59Fe-PIH could be blocked by an anti-TfR
monoclonal antibody. After 59Fe-Tf uptake, R cells released
greater amounts of 59Fe than gallium-sensitive (S) cells.
However, after 59Fe-PIH uptake 59Fe release from S and R
cells was similar. 125I-Tf exocytosis was greater in R cells. At
confluency, S and R cells expressed equivalent amounts of
TfR; however, at 24 and 48 hours in culture, TfR expression
was lower in R cells. Our study suggests that the decrease in
Tf-Fe uptake by R cells is caused by a combination of
enhanced iron efflux from cells and decreased TfR-mediated
iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells,
both uptake systems may be controlled at some level by
similar regulatory signal(s).
r 1998 by The American Society of Hematology.
G
out of the endosome. The receptor-apoTf (metal-free) complex
then recycles back to the cell surface where Tf is released to the
exterior.18-20 In addition to TfR-mediated uptake, certain cells
can acquire iron and gallium (as low molecular weight chelates)
through a Tf-independent uptake system.21-25
Our recent investigation showed that in addition to the
decrease in gallium uptake, R cells also have a decrease in their
uptake of iron.12 However, the mechanisms responsible for this
decrease in gallium/iron uptake remained to be determined. In
the present study, we have investigated the steps involved in the
transport of iron into R cells to determine whether the previously observed downregulation of iron uptake by these cells is
caused by changes in TfR-dependent or -independent iron
uptake pathways. We show that both Tf-Fe and non-Tf iron
uptake are downregulated in R cells and that this is associated
with changes in TfR synthesis and cycling and the egress of iron
from cells.
ALLIUM NITRATE IS A group-IIIa metal salt in clinical
use for the treatment of hypercalcemia and certain
malignancies.1,2 As an antineoplastic agent, gallium has significant activity against bladder cancer and lymphoma.3-8 Recent
investigations have shown that the mechanism of cytotoxicity
of gallium includes perturbation of iron-dependent cell proliferation, including inhibition of ribonucleotide reductase, an ironcontaining enzyme responsible for deoxyribonucleotide synthesis.9-11
Malignant lymphoid cells in vitro and in animal tumor
models are uniformly sensitive to growth inhibition by gallium.2 However, clinical studies have shown that 40% to 50% of
patients with relapsed lymphoma respond to treatment with
gallium nitrate whereas the remainder have disease that is
resistant to gallium.8 In an attempt to understand why certain
lymphomas and other malignancies are relatively resistant to
the cytotoxicity of gallium, investigation in our laboratory has
focused on elucidating the biological changes that tumor cells
undergo during the development of drug resistance to gallium.
Recently, we reported that human lymphoid leukemic CCRFCEM cells with acquired resistance to gallium (R cells) have a
decrease in their uptake of gallium, suggesting that drug
resistance to gallium involves a downregulation of gallium
transport into cells.12
Gallium binds avidly to the iron transport protein transferrin
(Tf)13 and the cellular uptake of gallium closely parallels that of
iron.14-17 The uptake of iron and gallium by cells occurs by Tf
receptor (TfR)-mediated endocytosis of Tf-Fe or Tf-Ga. Inside
the cell, the TfR-ligand complex translocates to an acidic
endosome where iron/gallium dissociates from Tf and trafficks
From the Division of Hematology/Oncology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI.
Submitted August 8, 1997; accepted February 3, 1998.
Supported by US Public Health Service Grant No. RO1 CA68028.
Address reprint requests to Christopher R. Chitambar, MD, Division
of Hematology/Oncology, Medical College of Wisconsin, 9200 W
Wisconsin Ave, Milwaukee, WI, 53226.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9112-0012$3.00/0
4686
MATERIALS AND METHODS
Gallium nitrate was obtained from Alpha Aesar (Ward Hill, MA).
Human Tf (substantially iron free), pronase and 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma Chemical Co (St Louis, MO). [35S]methionine was obtained
from Dupont (Wilmington, DE). 59FeCl3 and 125I-Na were obtained
from Amersham (Arlington Heights, IL). 59Fe-Tf was prepared as
described by Bates and Schlabach,26 whereas 125I-Tf was prepared by
the Chloramine T method.27 Pyridoxal isonicotinoyl hydrazone (PIH)
and 59Fe-PIH were prepared as described by Ponka.28 Monoclonal
antibody (MoAb) 42/6 and rabbit antiserum against the human TfR
were generously provided by Ian Trowbridge (The Salk Institute) and
Caroline Enns (Oregon Health Sciences University).
Cells. Human T lymphoblastic leukemic CCRF-CEM cells (galliumsensitive or S cells) were obtained from American Type Culture
Collection (Rockville, MD) and were grown in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS; complete medium) in an
atmosphere of 6% CO2. A gallium-resistant CCRF-CEM cell line (R
cells) was developed from the parent line through a process of
continuous exposure of cells to increasing concentrations of gallium
nitrate over a course of several months. R cells were grown either in
medium containing 150 µmol/L gallium nitrate (R1 cells) or in medium
without gallium nitrate (R2 cells). R2 cells displayed a stable galliumresistant phenotype even in the absence of gallium.
Cell growth experiments. Cell growth in the presence and absence
of gallium was determined by MTT assay as previously described by
Mosmann29 or by counting cells directly with a hemocytometer. For the
Blood, Vol 91, No 12 (June 15), 1998: pp 4686-4693
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IRON TRANSPORT IN GALLIUM RESISTANCE
MTT assay, cells grown to confluency were plated at an initial density of
2 3 105 cells/mL in 96-well microwell plates and incubated for 72 hours
in the presence of 0 to 1,000 µmol/L gallium nitrate. At the end of the
incubation, 10 µL of MTT (5 mg/mL stock solution) was added to each
well and the cells were incubated at 37°C for an additional 4 hours.
Cells were then solubilized by the addition of 100 µL of 0.04 N HCl in
isopropanol to each well, and the absorbance of each well was
determined spectrophotometrically at dual wave length 570/630 nm by
using an EL 310 microplate auto-reader (Biotech Instruments, Winooski,
VT). The absorbance of the wells containing gallium nitrate was
compared with that of wells in which the drug was omitted. The growth
rate of S, R1, and R2 cells in the absence of gallium nitrate was also
compared by counting cells after 24, 48, and 72 hours of growth.
Uptake of 59Fe by cells. 59Fe uptake studies were performed by
using either 59Fe-Tf or 59Fe-PIH. S, R1, and R2 cells in growth phase
(after 24 or 48 hours of incubation in fresh medium) or in confluent/
stationary phase (after 72 hours of incubation in medium) were washed
twice with medium and replated (0.5 3 106 cells/mL) in 1-mL 24-well
plates in complete medium or serum-free medium. 59Fe-Tf or 59Fe-PIH
was added to each well as specified in the figure legends and incubation
continued for 3 to 24 hours. Because of potential loss of cell viability in
serum-free medium, uptake times for studies in this medium did not
exceed 24 hours. In certain experiments, 59Fe uptake was performed in
the presence of 10 µg/mL of MoAb 42/6. At specified times, cells were
removed from the wells, washed twice by centrifugation with ice-cold
phosphate-buffered saline (PBS) and 59Fe cpm in the cell pellet was
determined using a Wallac Compugamma gamma counter (Wallac Inc,
Gaithersburg, MD).
Release of 59Fe from cells. S, R1, and R2 cells (106 cells/mL) were
incubated in complete medium with 59Fe-Tf (4 µg/mL Tf, 5.9 ng Fe/mL,
28,000 59Fe cpm/mL) for 1 or 3 hours at 37°C in a CO2 incubator. At the
end of the incubation, an aliquot of cell suspension was removed and
centrifuged, and the amount of 59Fe taken up by cells was determined.
The remaining cells were washed twice by centrifugation with ice-cold
PBS to remove unincorporated 59Fe-Tf and suspended in the original
volume of fresh complete medium without 59Fe-Tf (release medium).
These cells were then reincubated in tissue culture flasks at 37°C. At
specified times, aliquots of cell suspension were removed and centrifuged. The radioactivity in the cell pellet and supernatant (medium) was
counted to determine the fraction of 59Fe released from cells. In
additional experiments, 59Fe uptake and release conditions were similar
except that after the 59Fe uptake step, cells were incubated with pronase
(150 µg/mL) for 20 minutes at 4°C to remove surface-bound 59Fe before
reincubation in fresh medium. 59Fe release by cells after uptake of
59Fe-PIH was also examined. The experimental conditions were similar
to those described for 59Fe-Tf except that 59Fe-PIH uptake was
performed in serum-free medium over 3 hours, whereas the release
medium was supplemented with 1% FCS.
125I-Tf binding. Cellular TfR expression in cells was determined by
an 125I-Tf binding assay as previously described.30 S and R1 cells were
harvested after incubation in medium for 24, 48, and 72 hours in the
absence of gallium. Cells were washed with PBS containing 0.1%
bovine serum albumin and assayed for 125I-Tf binding at 4°C. Maximum Tf binding was determined according to the method of Scatchard.31
TfR synthesis. Cellular TfR synthesis was examined as described by
Rutledge.32 S, R1, and R2 cells (5 3 105/mL) were incubated for 3 or 20
hours with 10 µC/mL [35S]methionine in methionine-free RPMI 1640
medium supplemented with 5% FCS. Cells were washed with PBS and
lysed in 10 mmol/L Tris pH 7.4/150 mmol/L NaCl/5 mmol/L EDTA
buffer containing 1% Triton X-100. Cell lysates were preadsorbed with
50 µL Staphylococcus aureus cells (Pansorbin cells; Calbiochem, La
Jolla, CA) at 4°C for 1 hour. Pansorbin cells were then removed by
centrifugation and the supernatant containing the radiolabeled TfR was
immunoprecipitated by incubation with 1.4 µL of anti-TfR antiserum
and 25 µL fresh Pansorbin cells. Pansorbin cells with receptor-antibody
4687
complexes bound to it were washed extensively and finally resuspended
in 23 Laemmli sample buffer.33 The sample was heated in a boiling
waterbath, it was centrifuged to remove the Pansorbin cells, and the
supernatant was resolved by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing conditions. Autoradiography of the gel was performed by exposing the dried gel to XAR-5 film
(Eastman Kodak, Co, Rochester, NY) with intensifying screens at
270°C for 24 to 48 hours.
125I-Tf internalization and release. The kinetics of internalization
of cell surface TfR-bound 125I-Tf and the release of internalized 125I-Tf
was examined by using a modification of a previously described
method.18 For the Tf internalization experiments, 107 cells were
harvested at confluency, washed with PBS-BSA, and incubated at 4°C
for 60 minutes in 100 µL of the same buffer with 138 ng 125I-Tf
(approximately 6,200 cpm/ng Tf) to allow for ligand binding to cell
surface TfR. Cells were then washed by centrifugation with ice-cold
PBS-BSA and resuspended in 1 mL serum-free medium prewarmed to
37°C. The cell suspension was maintained at 37°C in a water bath. One
hundred-microliter aliquots were removed at 2.5-minute intervals,
added to 1 mL of ice-cold 10 mmol/L acetic acid/150 mmol/L NaCl pH
3 buffer (acid wash) to remove 125I-Tf on the cell surface, and
centrifuged in a microfuge centrifuge for 1 minute at full speed. The
supernatant was carefully removed and the radioactivity in the cell
pellet and supernatant counted to determine the fraction of 125I-Tf
internalized by cells (acid-resistant cpm). For the 125I-Tf release studies,
pulse-chase experiments were performed in which 107 cells were first
incubated at 37°C in 500 µL serum-free medium containing 0.1% BSA
with 125I-Tf to allow for uptake of the radiolabeled ligand (pulse). After
30 minutes of incubation, cells were washed twice with ice-cold
serum-free medium to remove unincorporated 125I-Tf and resuspended
in 1 mL of serum-free medium containing 100 µg/mL of nonradioactive
Tf-iron at 37°C (chase). The cell suspension was maintained at 37°C in
a water bath and 100-µL aliquots were removed at 2.5-minute intervals
and centrifuged. Radioactivity in the pellet and supernatant was counted
to determine the percent of 125I-Tf released from cells over time.
RESULTS
Gallium-resistant CCRF-CEM cells display a stable drugresistant phenotype. The effect of gallium nitrate on the
growth of S and R1 cells is shown in Fig 1A. To determine
Fig 1. Effect of gallium nitrate on the growth of gallium-sensitive
(S) and -resistant (R1 and R2) CCRF-CEM cells. Cells were plated at
2 3 105 cells/mL in the presence of increasing concentrations of
gallium nitrate, and growth was determined by MTT assay after a
72-hour incubation. (A) gallium-resistant cells (d) that had been
maintained continuously in medium containing 150 mmol/L gallium
nitrate (R1 cells). (B) gallium-resistant cells (d) that had been grown
without gallium in the medium for 10 weeks (R2 cells). (s) galliumsensitive cells.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4688
whether R cells would revert to a gallium-sensitive phenotype
in the absence of gallium, they were propogated in complete
medium without gallium for 10 weeks and then analyzed for
sensitivity to gallium. As shown in Fig 1B, these cells (R2 cells)
remained resistant to gallium, indicating that continued exposure to gallium was not necessary to maintain a stable galliumresistant phenotype. Subsequent experiments were performed
by using both R1 and R2 cells. S, R1, and R2 cells displayed
similar growth rates.
Iron uptake by cells. Recently, we showed that the development of drug resistance to gallium nitrate in CCRF-CEM cells is
related to a decrease in their uptake of gallium and that this is
accompanied by a parallel decrease in iron uptake.12 To confirm
that these differences in iron uptake between S and R cells were
consistent, 59Fe-Tf uptake was examined by using cells that
were actively proliferating (after 24 or 48 hours of growth in
culture) or were confluent (after 72/0 hours of growth in
culture). Cells that had been previously grown in medium for
the specified times were reincubated in fresh medium containing 59Fe-Tf and the amount of 59Fe taken up by cells was
measured after 3, 6, and 24 hours of incubation. As shown in Fig
2, 59Fe uptake by R1 cells was significantly less than S cells
regardless of whether they were initially confluent or actively
proliferating.
It is known that the cellular uptake of 59Fe-Tf is mediated by
the TfR, whereas the uptake of 59Fe-PIH occurs independent of
Tf and its receptor.28 Therefore, the uptakes of 59FeTf and
59Fe-PIH were compared to further define the pathway(s)
involved in the decrease of iron transport into R cells. As shown
in Fig 3A and B, 59Fe uptake by R1 and R2 cells was
significantly lower than S cells regardless of whether iron was
delivered to cells as 59Fe-Tf or 59Fe-PIH. After a 6-hour
incubation in complete medium, 59Fe-Tf uptake by R1 and R2
cells was 56% and 60% that of S cells, whereas 59Fe-PIH uptake
by R1 and R2 cells was 75% that of S cells (Fig 3A). After a
24-hour incubation, 59Fe-Tf uptake by R1 and R2 cells was 48%
and 60% that of S cells, whereas 59Fe-PIH uptake was 51% and
Fig 2. 59Fe-Tf uptake by S and R1 CCRF-CEM cells at different
times of proliferation. CCRF-CEM cells were grown for 0 to 72 hours in
fresh medium and then used for 59Fe uptake studies. Cells were
plated at 2 3 105 cells/mL in complete medium containing 59Fe-Tf (228
pmole 59Fe/mL), and 59Fe uptake by cells was determined at the times
shown. (A) 59Fe uptake by confluent, 0/72-hour cells; (B) 59Fe uptake
by cells previously grown for 24 hours in fresh medium; and (C) 59Fe
uptake by cells previously grown for 48 hours in fresh medium. (d) S
cells; (s) R cells. Values are means 6 standard error (SE) of a
representative experiment performed in triplicate.
CHITAMBAR AND WERELEY
56% that of S cells (Fig 3B). Figure 3A and B also illustrate that
the cellular uptake of radioiron from 59Fe-Tf was several times
greater than from 59Fe-PIH.
Because the 59Fe-PIH uptake experiments were performed in
serum-supplemented medium, the possibility existed that Tf
(present in bovine serum) may have influenced iron uptake. To
confirm that cells incorporated 59Fe from 59Fe-PIH independent
of the TfR pathway, cellular 59Fe-PIH uptake studies were also
performed in serum-free, Tf-free medium. Under these conditions, 59Fe uptake by R1 and R2 cells was 44% and 66% of S
cells, respectively (Fig 3D), and was comparable with 59Fe
uptake in serum-supplemented medium (Fig 3C). To exclude
the possibility that the decreased 59Fe-PIH uptake by R cells
was not a function of the amount of 59Fe-PIH in the medium,
59Fe-PIH uptake by S and R cells was measured over a fivefold
range of 59Fe-PIH concentrations. As shown in Fig 4, 59Fe
uptake by S and R cells increased progressively with increasing
concentrations of 59Fe-PIH; however, the amount of 59Fe uptake
by R cells was markedly less than that of S cells at all
concentrations of Fe-PIH examined and reached approximately
80% of saturation levels with 500 pmole of Fe-PIH in the
medium.
To further verify that the decrease in 59Fe-PIH uptake by R1
and R2 cells was truly independent of the TfR, additional 59Fe
uptake studies were performed in the presence of 42/6, a MoAb
that blocks internalization of the TfR.34 As expected, 42/6
blocked the uptake of 59Fe-Tf by S, R1, and R2 cells (Fig 5A);
however, it had no effect on the uptake of 59Fe-PIH (Fig 5B).
Collectively, these experiments indicate that 59Fe-PIH delivers
iron to CCRF-CEM cells independent of the TfR and that
TfR-independent iron uptake is decreased in R1 and R2 cells.
Iron release from cells. To determine whether the efflux of
iron from cells could play a role in the decrease in iron uptake
by R1 and R2 cells, cells that had incorporated 59Fe-Tf were
reincubated in fresh medium to determine whether they would
release 59Fe to the external environment. These studies showed
that after their initial uptake of 59Fe-Tf, R1 and R2 cells released
significantly greater amounts of 59Fe than S cells. As shown in
Fig 6A, after a 1-hour uptake of 59Fe-Tf, R1 and R2 cells
released approximately 1.7-fold and 2.1-fold more 59Fe to the
medium than S cells over the subsequent 75 minutes of
reincubation. A similar pattern of 59Fe release was seen when
cells were allowed to incorporate 59Fe-Tf over 3 hours and then
were reincubated in fresh medium for 20 hours (Fig 6B). After 3
hours of reincubation, R1 and R2 cells released 2.9-fold and
2.3-fold more 59Fe to the medium than S cells. Even after 20
hours of reincubation, both R1 and R2 cells continued to release
significantly greater amounts of 59Fe than S cells (Fig 6B). To
determine whether 59Fe released from cells after the 1- or 3-hour
uptake represented 59Fe released from the cell surface (external)
or 59Fe released from inside the cell, cells were treated with
Pronase (to remove 59Fe on the cell) before reincubation in fresh
medium. Under these conditions, results similar to those shown
in Fig 6 were obtained, thus indicating that the 59Fe released
from cells represented the efflux of intracellular 59Fe.
In contrast to 59Fe-Tf, significant differences in the release of
59Fe from S, R1, and R2 cells were not seen after they had
incorporated 59Fe-PIH (Fig 7).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IRON TRANSPORT IN GALLIUM RESISTANCE
4689
Fig 3. 59Fe-Tf and 59Fe-PIH uptake by S, R1, and R2
cells. Cells were plated in medium containing equivalent amounts of 59Fe (106 pmole Fe/mL) as either
59Fe-Tf or 59Fe-PIH and incubated for 6 to 24 hours. (A)
59Fe uptake by cells over 6 hours in complete medium; (B) 59Fe uptake by cells over 24 hours in
complete medium; (C) 59Fe-PIH uptake over 24 hours
in complete medium; (D) 59Fe-PIH uptake over 24
hours in serum-free medium. Values shown represent means 6 SE of an experiment performed in
triplicate. Similar results were obtained in two additional experiments.
TfR expression and synthesis. Because the TfR plays a
central role in the uptake of Tf-Fe, 125I-Tf binding and TfR
synthesis were examined. At confluency (0 and 72 hours) in
culture, R1 and S cells displayed equivalent cell surface 125I-Tf
binding. In contrast, after 24 and 48 hours of growth in fresh
medium, maximal 125I-Tf binding to R1 cells was lower than
that to S cells by approximately 29% and 23%, respectively (Fig
8A). Measurement of TfR synthesis at the 24-hour time point by
using a 3-hour [35S]methionine pulse-label showed that the
synthesis of new TfR at this time point was decreased in R1 and
R2 cells (Fig 8B). Interestingly, [35S]methionine pulse-labeling
of cells over a longer period (20 hours) during the first 20 hours
of incubation in fresh medium did not show differences in TfR
synthesis. With the 3-hour [35S]methionine pulse, the reduced
TfR was identified on SDS-PAGE analysis as two bands
corresponding to 86 kD and 90 kD, consistent with different
glycosylation states of the TfR.35 However, with the 20-hour
pulse, the major band was 90 kD, consistent with the size of the
mature TfR.
Tf cycling. To determine whether S, R1, and R2 cells differ
with regard to TfR function, the kinetics of internalization and
release of receptor-bound Tf were examined by using cells that
were in the same growth phase as those used for the iron release
studies. Cells were allowed to internalize surface receptorbound 125I-Tf at 37°C and the amount of 125I-Tf within the cell
(acid-resistant cpm) determined. Figure 9A shows that the rates
of 125I-Tf internalization by S, R1, and R2 cells were similar
during the first 10 to 15 minutes. However, the amount of
acid-resistant 125I-Tf in R1 and R2 cells peaked at the 20-minute
time point and decreased thereafter, indicating that after this
time a fraction of 125I-Tf had cycled out of these cells. In
contrast, the amount of acid-resistant 125I-Tf in S cells peaked at
the 30-minute time point and decreased only slightly thereafter.
Because these results suggested that the exocytosis of
internalized Tf was greater in R cells than in S cells, an
additional experiment was performed in which cells were
pulsed with 125I-Tf for 30 minutes and then chased with
nonradioactive Tf. As shown in Fig 9B, these studies showed
the egress of 125I-Tf from R1 cells to be significantly greater
than from S cells.
DISCUSSION
Fig 4. Uptake of 59Fe-PIH by cells. Cells were incubated in serumfree medium with increasing concentrations of 59Fe-PIH. 59Fe uptake
was determined after 20 hours of incubation. (d) S cells; (s) R cells.
Values represent the means of a duplicate experiment.
In an earlier investigation, we showed that gallium-resistant
CCRF-CEM cells have a decrease in their uptake of both
gallium and iron.12 Whereas the decrease in gallium uptake
protects these cells from the cytotoxicity of gallium, the
decrease in iron uptake could potentially threaten cellular
iron-dependent processes. However, because the growth of R
cells is similar to S cells, it is clear that R cells still acquire a
critical amount of iron needed for viability and proliferation.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4690
CHITAMBAR AND WERELEY
The finding that both TfR-dependent and -independent iron
uptake was decreased in R cells was unexpected because these
two iron uptake pathways are generally perceived as separate,
independent systems. One possible explanation for the parallel
decrease in TfR-dependent and -independent iron uptake is that
both iron uptake pathways may be controlled at some level by
the same mechanism and that this regulatory mechanism is
affected during the development of drug resistance to gallium.
TfR-mediated uptake of iron is generally in balance with the
Fig 5. Effect of anti-TfR MoAb 42/6 on 59FeTf and 59Fe-PIH uptake.
Cells were plated in serum-free medium containing either 59Fe-Tf (109
pmole Fe/mL) or 59Fe-PIH (245 pmole Fe/mL) with (1) or without (2)
10 mg/mL 42/6. 59Fe uptake by cells was determined after a 24-hour
incubation. (A) 59Fe-Tf uptake; (B) 59Fe-PIH uptake. Values represent
means 6 SE (n 5 3).
The decrease in iron uptake by R cells raised important
questions regarding the relative roles of TfR-dependent and
-independent iron transport into these cells, and therefore, we
sought to determine whether this decrease was caused by
changes in TfR-dependent or TfR-independent iron transport.
TfR expression and the uptake of Tf-Fe by nonerythroid cells is
closely linked to the need for iron to maintain cell viability and
DNA synthesis.30,36,37 The TfR-independent iron uptake system
in contrast, may serve to remove potentially toxic low molecular weight iron complexes from the circulation and may
represent a transport system shared by several metals.23,38-40
Because Fe-PIH has been shown to support the growth of cells
independent of Tf,41 this iron complex was used to examine
TfR-independent iron uptake.
Comparison of 59Fe uptake by using 59Fe-Tf or 59Fe-PIH as
the source of iron showed that, whereas TfR-dependent iron
uptake was always greater than TfR-independent iron uptake,
both pathways were significantly downregulated in R cells.
Evidence that the PIH-mediated uptake of iron was independent
of the TfR was provided by performing the uptake studies in
serum-free, Tf-free medium and by showing that 59Fe-PIH
uptake was unaffected by blockade of the TfR with MoAb 42/6.
Fig 6. 59Fe release after 59Fe-Tf uptake. S, R1, and R2 cells were
allowed to incorporate 59Fe-Tf over 1 or 3 hours and then washed and
reincubated in fresh medium. At the specified times, aliquots of cell
suspension were harvested and the 59Fe in the medium and cells was
counted to determine the percent of 59Fe released from cells to the
medium. (A) 59Fe released by cells after a 1-hour uptake. Insert figure
shows the amount of 59Fe (pmole Fe/106 cells) taken up by cells over
the 1-hour incubation before release. Data represent means 6 SE (n 5
3). (B) Experimental conditions were similar to (A) except that 59Fe
release was examined after a 3-hour uptake of 59Fe-Tf and the percent
59Fe released was measured over 20 hours. Data represent means 6
SE (n 5 3). Insert figure shows the amount of 59Fe (pmole Fe/106 cells)
taken up by cells over the 3-hour incubation before release.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IRON TRANSPORT IN GALLIUM RESISTANCE
4691
Fig 7. 59Fe release after 59Fe-PIH uptake. Experimental conditions
were similar to that described in Fig 6 except that cells were
incubated in serum-free medium with 59Fe-PIH for 3 hours and then
washed and reincubated in fresh medium supplemented with 1%
FCS. Data represent means 6 SE (n 5 4).
amount of iron needed to support cellular iron-dependent
processes. When iron is incorporated in excess of cellular
requirements, it is sequestered in ferritin, and hence, very little
of it effluxes from cells under normal conditions. However, R
cells released significantly greater amounts of incorporated iron
to the exterior than S cells, indicating that, with respect to Tf-Fe,
Fig 9. (A) Internalization of 125I-Tf by cells. Cells were incubated
with 125I-Tf at 4°C to allow for ligand binding to cell surface TfRs. Cells
were washed to remove unbound 125I-Tf and incubated at 37°C to
allow for internalization of 125I-Tf. At the specified times, aliquots of
cells were removed and centrifuged through an acidic buffer to
determine the fraction of 125I-Tf internalized. Data shown are representative of three separate experiments. (•) S; (300) R1; (S) R2 cells. (B)
Release of 125I-Tf from cells. Cells were allowed to incorporate 125I-Tf
at 37°C, washed to remove unincorporated radioactivity, and then
incubated in serum-free medium containing 100 mg/mL Tf-Fe (nonradioactive). The amount of 125I-Tf released by cells at the specified
times was determined as described the text. (•) S; (300) R1 cells. Data
represent means 6 SE (n 5 3). Differences between S and R1 cells
after the 5-minute time point are significant (P F .004).
Fig 8. (A) 125I-Tf binding studies. 125I-Tf binding to cells was
measured after growth of cells in fresh medium for the times shown.
Open columns, S cells; hatched columns, R1 cells. Data shown
represent the means 6 SE (n 5 3). (B) TfR synthesis. Newly synthesized TfRs were labeled with [35S]methionine over a 3-hour or 20-hour
pulse as described in the text. The 3-hour pulse was performed on
cells after they had been incubated in fresh medium for 24 hours. The
20-hour pulse was performed immediately after the initial plating of
confluent cells in fresh medium (0 to 20 hours). The autoradiograph
shown is representative of three separate experiments.
an increased efflux of iron contributes to the decrease in iron
uptake. The basis for the increase in iron release from R cells
remains to be determined. Under normal conditions, the intracellular release of iron from Tf occurs through a process of
endosomal acidification involving an adenosine triphosphate
(ATP)-dependent proton pump.20 The movement of iron out of
the endosome is poorly understood and may include the activity
of low or high molecular weight intermediates or even direct
interaction between organelles.20 Perturbation of any of these
processes could lead to a block in the unloading of iron from Tf
and a subsequent release of iron from cells.
The decrease in Tf-Fe uptake and the increase in iron release
from R cells could be caused by changes in the synthesis,
posttranslational modification, or cycling of the TfR. Earlier
studies did not show differences in the affinity of the TfR for Tf
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
4692
CHITAMBAR AND WERELEY
in R cells.12 In the present study, differences in the size of the
TfR were not seen on SDS-PAGE analysis, making it unlikely
that the TfR in R cells is structurally altered. Although TfR
expression in R cells was comparable with S cells when
measured at confluency (0 and 72 hours), TfR expression was
lower in R cells only after 24 and 48 hours of subculture. Hence,
the decrease in Tf-Fe uptake by R cells over the 24- and 48-hour
period of incubation can be explained in part by the decrease in
TfR number. In contrast, experiments showing an increase in
the release of iron from R cells were performed over the initial 1
and 3 hours of subculture of cells in fresh medium, time points
at which TfR expression in S and R cells was similar.
Pulse-chase experiments performed at these time points showed
that the cycling of 125I-Tf out of R cells was also increased.
Based on these results, we conclude that the decrease in Tf-Fe
uptake by R cells is caused by a combination of enhanced efflux
of iron from cells and decreased TfR-mediated iron transport
into cells. The former mechanism appears to dominate during
the initial period of subculture of cells in fresh medium,
whereas the latter mechanism comes into play later on.
Although our studies shed light on the mechanisms involved
in the decrease in TfR-dependent iron uptake by R cells, the
basis for the decrease in TfR-independent iron uptake remains
to be determined. Differences in the release of iron from S and R
cells were not seen after the uptake of 59Fe-PIH, suggesting that
the primary mechanism for the decrease in TfR-independent
iron uptake involves a quantitative or qualitative decrease in a
non-Tf iron transport system. It remains to be determined
whether these differences in the handling of TfR-dependent and
-independent Fe by R cells are the result of changes in single or
multiple mechanisms involved in the regulation of iron uptake
during the development of gallium resistance.
To our knowledge, there have been no previously reported
examples in which the downregulation of uptake of one metal as
a protective adaptation by a tumor cell results in a decrease in
iron transport as well. Because of the interaction between
gallium and iron proteins, R cells may serve as a unique model
system to gain insights into adaptive changes in cellular iron
transport. It is hoped that further investigation of iron metabolism in these cells will yield new information regarding
regulatory mechanisms responsible for the uptake and intracellular trafficking of iron and iron proteins.
REFERENCES
1. Warrell RP, Jr: Clinical trials of gallium nitrate in patients with
cancer-related hypercalcemia. Semin Oncol 18:26, 1991
2. Foster BJ, Clagett-Carr K, Hoth D, Leyland-Jones B: Gallium
nitrate: The second metal with clinical activity. Cancer Treat Rep
70:1311, 1988
3. Seligman PA, Crawford ED: Treatment of advanced transitional
cell carcinoma of the bladder with continuous infusion gallium nitrate. J
Natl Cancer Inst 83:1582, 1991
4. Crawford ED, Saiers JH, Baker LH, Costanzi JH, Bukowski RM:
Gallium nitrate in advanced bladder carcinoma: Southwest Oncology
Group study. Urology 38:355, 1991
5. Seidman AD, Scher HI, Heinemann MH, Bajorin DF, Sternberg
CN, Dershaw DD, Silverberg M, Bosl GJ: Continuous infusion gallium
nitrate for patients with advanced refractory urothelial tumors. Cancer
68:2561, 1991
6. Keller J, Bartolucci A, Carpenter JT, Jr, Feagler J: Phase II
evaluation of bolus gallium nitrate in lymphoproliferative disorders: A
Southeastern Cancer Study Group trial. Cancer Treat Rep 70:1221,
1986
7. Weick JK, Stephens RL, Baker LH, Jones SE: Gallium nitrate in
malignant lymphoma: A Southwest Oncology Group study. Cancer
Treat Rep 67:823, 1983
8. Warrell RP, Jr, Coonley CJ, Straus DJ, Young CW: Treatment of
patients with advanced malignant lymphoma using gallium nitrate
administered as a seven-day continuous infusion. Cancer 51:1982, 1983
9. Chitambar CR, Seligman PA: 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 Invest 78:1538, 1986
10. Chitambar CR, Matthaeus WG, Antholine WE, Graff K, O’Brien
WJ: Inhibition of leukemic HL60 cell growth by transferrin-gallium:
Effects on ribonucleotide reductase and demonstration of drug synergy
with hydroxyurea. Blood 72:1930, 1988
11. Chitambar CR, Narasimhan J, Guy J, Sem DS, O’Brien WJ:
Inhibition of ribonucleotide reductase by gallium in murine leukemic
L1210 cells. Cancer Res 51:6199, 1991
12. Chitambar CR, Wereley JP: 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, 1997
13. Harris WR, Pecoraro VL: Thermodynamic binding constants for
gallium transferrin. Biochemistry 22:292, 1983
14. Harris AW, Sephton RG: Transferrin promotion of 67Ga and 59Fe
uptake by cultured mouse myeloma cells. Cancer Res 37:3634, 1977
15. Larson SM, Rasey JS, Allen DR, Nelson NJ, Grunbaum Z, Harp
GD, Williams DL: Common pathway for tumor cell uptake of
Gallium-67 and Iron-59 via a transferrin receptor. J Natl Cancer Inst
64:41, 1980
16. Chitambar CR, Zivkovic Z: Uptake of gallium-67 by human
leukemic cells: Demonstration of transferrin receptor-dependent and
transferrin-independent mechanisms. Cancer Res 47:3929, 1987
17. Chitambar CR, Zivkovic-Gilgenbach Z: Role of the acidic
receptosome in the uptake and retention of 67Ga by human leukemic
HL60 cells. Cancer Res 50:1484, 1990
18. Klausner RD, van Renswoude J, Ashwell G, Kempf C, Schecter
AN, Dean A, Bridges KR: Receptor-mediated endocytosis of transferrin
in K562 cells. J Biol Chem 258:4715, 1983
19. Klausner RD, Ashwell G, van Renswoude J, Harford J, Bridges
KR: Binding of apotransferrin to K562 cells: Explanation of the
transferrin cycle. Proc Natl Acad Sci USA 80:2263, 1983
20. Richardson DR, Ponka P: The molecular mechanisms of the
metabolism and transport of iron in normal and neoplastic cells.
Biochim Biophys Acta 1331:1, 1997
21. Basset P, Quesneau Y, Zwiller J: Iron-induced L1210 cell
growth: Evidence of a transferrin-independent iron transport. Cancer
Res 46:1644, 1986
22. Sturrock A, Alexander J, Lamb J, Craven CM, Kaplan J:
Characterization of a transferrin-independent uptake system for iron in
HeLa cells. J Biol Chem 265:3139, 1990
23. Inman RS, Wessling-Resnick M: Characterization of transferrinindependent iron transport in K562 cells. J Biol Chem 268:8521, 1993
24. Seligman PA, Kovar J, Schleicher RB, Gelfand EW: Transferrinindependent iron uptake supports B lymphocyte growth. Blood 78:
1526, 1991
25. Chitambar CR, Sax D: Regulatory effects of gallium on transferrin-independent iron uptake by human leukemic HL60 cells. Blood
80:505, 1992
26. Bates GW, Schlabach MR: The reaction of ferric salts with
transferrin. J Biol Chem 248:3228, 1973
27. Hunter HM, Greenwood FC: Preparation of iodine-131 labelled
human growth hormone of high specific activity. Nature 194:495, 1962
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
IRON TRANSPORT IN GALLIUM RESISTANCE
28. Ponka P, Schulman HM, Wilczynska A: Ferric pyridoxal isonicotinoyl hydrazone can provide iron for heme synthesis in reticulocytes.
Biochim Biophys Acta 718:151, 1982
29. Mosmann T: Rapid colorimetric assay for cellular growth and
survival: Application to proliferation and cytoxicity assays. J Immmunol Methods 65:55, 1983
30. Chitambar CR, Massey EJ, Seligman PA: Regulation of transferrin receptor expression on human leukemic cells during proliferation
and induction of differentiation. Effects of gallium and dimethylsulfoxide. J Clin Invest 72:1314, 1983
31. Scatchard G: The attractions of proteins for small molecules and
ions. Ann NY Acad Sci 51:660, 1949
32. Rutledge EA, Root BJ, Lucas JJ, Enns CA: Elimination of the
O-linked glycosylation site at Thr 104 results in the generation of a
soluble human-transferrin receptor. Blood 83:580, 1994
33. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970
34. Taetle R, Castognola J, Mendelsohn J: Mechanisms of growth
inhibition by anti-transferrin receptor monoclonal antibodies. Cancer
Res 46:1759, 1986
4693
35. Omary BM, Trowbridge IS: Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem 256:12888, 1981
36. Larrick JW, Cresswell P: Modulation of cell surface iron
transferrin receptors by cellular density and state of activation. J
Supramol Struct 11:579, 1979
37. Trowbridge IS, Omary MB: Human cell surface glycoprotein
related to cell proliferation is the receptor for transferrin. Proc Natl Acad
Sci USA 78:3039, 1981
38. Brissot P, Wright TL, Ma W, Weisiger RA: Efficient clearance of
non-transferrin–bound iron by rat liver. J Clin Invest 76:1463, 1985
39. Kaplan J, Jordan I, Sturrock A: Regulation of the transferrinindependent iron transport system in cultured cells. J Biol Chem
266:2997, 1991
40. Olakanmi O, Stokes JB, Pathan S, Britigan BE: Polyvalent
cationic metals induce the rate of transferrin-independent iron acquisition by HL-60 cells. J Biol Chem 272:2599, 1997
41. Landschulz W, Thesleff I, Ekblom P: A lipophilic iron chelator
can replace transferrin as a stimulator of cell proliferation and differentiation. J Cell Biol 98:596, 1984
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1998 91: 4686-4693
Transferrin Receptor-Dependent and -Independent Iron Transport in
Gallium-Resistant Human Lymphoid Leukemic Cells
Christopher R. Chitambar and Janine P. Wereley
Updated information and services can be found at:
http://www.bloodjournal.org/content/91/12/4686.full.html
Articles on similar topics can be found in the following Blood collections
Neoplasia (4182 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.