(CANCER RESEARCH 49, 1941-1947. April 15, 1989|
Transformation-induced Changes in Transferrin and Iron Metabolism in Myogenic
Cells1
Lydia M. Sorokin, Evan H. Morgan,2 and George C. T. Yeoh
Physiology Department, University of Western Australia, Nedlands, Perth 6009, Western Australia, Australia
ABSTRACT
The upta <e of transferrin and iron by cultured myogenic cells trans
formed with a temperature-sensitive strain of the Rous sarcoma virus
(tsLA24) »;s compared with that of normal developing myogenic cells
which were proliferating at the same rate as the transformed cells. The
mechanism »ftransferrin and iron uptake was the same in the transformed
cells as in normal myogenic cells and involved receptor-mediated endocytosis of transferrin. However, there were differences in transferrin
receptor numbers and receptor function. The number of receptors in
transformed cells was more than twice as great as in the normal cells
largely due :o increased surface receptor numbers. Despite this, the rate
of iron upta <e increased by only 20% in the transformed cells due to less
efficient cyi ling of the transferrin receptors and less efficient release of
iron from transferrin to intracellular sites. Some internalized iron was
released from the transformed cells still bound to transferrin. A fast and
a slow rate if transferrin exocytosis were identified in transformed cells,
as in norm: 1 cells, indicating that there were at least two intracellular
pathways for transferrin. The fast pathway predominated in the trans
formed cell;, compared with an equal importance of the two pathways in
the normal :ells.
INTRODUCTION
High le /els of expression of transferrin receptors have been
demonstrated in many types of transformed cells (1-6). It has
been suggested that this may provide a means of identifying
malignantly transformed cells (4, 7). However, large transferrin
receptor r umbers need not be a marker for malignant cells per
se since receptor numbers have also been shown to be elevated
on normal dividing cells in vitro (1, 3, 5, 8-10). This increase
is associa :ed with an increased iron demand by the cells for
DNA synthesis (1, 11, 12). The precise nature of the iron
requirement for cell proliferation is not known, but iron is an
essential :omponent of ribonucleotide reducÃ-ase, an enzyme
required or DNA synthesis (13, 14). As transformed cells
actively divide, it is possible that the increased expression of
transferrin receptors reflects an increase in iron demand asso
ciated with DNA synthesis.
Transfc rmation is not just a problem in cell proliferation;
rather, it is viewed by many to involve changes in both prolif
eration ar d differentiation (15). Transferrin receptor expression
varies wii h the state of differentiation of cells. Cells of the
erythroid series, normal erythroid cells, Friend erythroleukemic
cells, and K562 cells, have been the best studied in this respect
(16-20). In general, transferrin receptor numbers are signifi
cantly hif.her on differentiating cells with few or no receptors
expressed on the terminally differentiated cell. However, in the
case of III. 60 and Daudi leukemic cells, the receptors are
expressed in the undifferentiated, proliferating cells and disap
pear when the cells are induced to differentiate and mature (21,
22). Hence the increased expression of receptors on many types
Received 9/26/88; revised 12/5/88; accepted 12/14/88.
The cost; of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance 'vith 18 U.S.C. Section 1734 solely to indicate this fact.
1This wt rk was supported by grants from the Australian Research Grants
Committee ind the Cancer Foundation of Western Australia.
2To whom requests for reprints should be addressed.
of differentiating cells may also be related to proliferation.
In view of the changes in transferrin receptor expression
which occur with different states of growth and differentiation
of cells and the potential significance of the receptor on malig
nant cells, it would be of value to identify characteristics of the
transferrin receptor that are specific to the transformed state.
In order to do this it is necessary to characterize the changes in
receptor numbers and function in response to proliferation,
differentiation, and transformation in one cell type. The present
study presents the results of such an investigation.
Primary culture of chick embryo breast muscle is the model
studied inasmuch as these cells have been shown to grow and
develop normally in culture and there are numerous morpho
logical and biochemical markers for their differentiation (23).
Furthermore, these cells can be transformed to malignancy with
Rous sarcoma virus (24, 25). A temperature-sensitive variant
of the Rous sarcoma virus (tsLA24)3 is used to infect the
myogenic cultures in the present investigation. After infection
the cells behave like transformed cells when maintained at the
permissive temperature (35°C)for virus activity but undergo
normal myogenic cell development and ultimately form myotubes when shifted to restrictive conditions (41°C)for virus
activity (25). As myogenic cultures inevitably contain some
fibroblasts and as transformed myogenic cells and transformed
fibroblasts are not easily distinguishable either morphologically
or biochemically, the use of a temperature-sensitive virus pro
vided a means of checking that transformed cultures contain
significant proportions of myogenic cells. Transformed my
ogenic cells were maintained at 35°Cand transferrin and iron
uptake as well as release experiments were performed at 37°C
and 4°Cusing Tf-Fe2 labeled with 125Iand 59Fe. The results
were compared to nontransformed differentiating myoblast cul
tures in which the cells were growing exponentially (26).
MATERIALS AND METHODS
Reagents. "Fe (as 59FeCl3in 0.1 myogenic HC1) and '"I (as sodium
iodide) were purchased from Amersham International, Inc., England.
Eagle's MEM and horse serum were supplied by Flow Laboratories,
Annadale, New South Wales, Australia. Amphotericin B (Fungizone),
penicillin-streptomycin, and glutamine were obtained from Gibco,
Grand Island, NY. Bovine serum albumin and Polybrene were obtained
from Sigma Chemical Co., St. Louis. MO. Pronase was purchased
from Boehringer Mannheim, Mannheim, West Germany.
Cell Culture. Primary myogenic cultures were prepared as previously
described (26). Cells to be transformed with tsLA24 were also subjected
to a number of preplating steps as described by O'Neill and Stockdale
(27) in order to reduce fibroblast contamination of the cultures. These
cells were then plated at a density of 5 x IO5 cells/ml onto collagencoated culture dishes, as described for normal myogenic cultures (26)
and incubated at 37°Cfor 5-6 h to allow cells to attach to the culture
dish. Following this step, the cultures were infected with the Rous
sarcoma virus (tsLA24) according to a modified method of Tato et al.
(25). Briefly, this involved the addition of approximately 5 focusforming units of virus/cell in MEM (supplemented with 2.4 x 10~3M
3The abbreviations used are: Tf-Fe2, diferric transferrin; tsLA24, temperaturesensitive Rous sarcoma virus; Tf, transferrin; MEM, Eagle's minimum essential
medium.
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TRANSFERRIN
METABOLISM IN TRANSFORMED
glutamine; amphotericin B, 2.8 Mg/ml; and penicillin-streptomycin, 570
units/ml and 570 j/g/ml, respectively) plus 1% embryo extract, 1%
horse serum, and 8 ng/m\ of Polybrene. Polybrene is a nontoxic
amorphous mixture of polycations used to enhance the absorption of
virus by the myoblasts (28). The cultures were incubated overnight at
35*C (the permissive temperature for virus activity) with the virus
mixture in a water-saturated atmosphere of 95% air:5% CO2. The virus
mixture was then replaced with MEM plus 3% embryo extract and
10% horse serum which was the medium in which transformed cells
were maintained. Infected cultures were passaged twice in order to
ensure infection of all cells in the culture, and infected cultures were
maintained at 35'C until transferrin and iron uptake studies were
performed.
Because there is some evidence that transferrin receptor numbers
vary with the rate of growth, even in transformed cells (29-31), the
transformed cells used in transferrin and iron uptake studies were plated
at a density of 2.5 x 10s cells/ml in a total volume of 3 ml/60-mm
culture dish so that the rate of cell proliferation was the same as for
the normal myogenic cells. Viability of the cultures of transformed cells
was estimated by trypan blue exclusion (32) and daily monitoring of
cultures by phase-contrast microscopy. Growth of cultures was assessed
by daily cell counts, measurements of total DNA content per culture
dish, and DNA synthesis (as determined by [3H]thymidine incorpora
tion).
For every experiment, culture dishes were shifted to 41"C and 8:1:1
medium (MEM plus 10% embryo extract and 10% horse serum),
conditions restrictive for tsLA24 activity, in order to check for myotube
formation. In all cases, extensive myotube formation was found in these
control cultures, indicating that the cultures used for transferrin and
iron experiments contained predominantly transformed myogenic cells.
Transferrin Purification and Labeling. Ovotransferrin was isolated
from egg white, as described by Woodworth and Schade (33), and was
shown to be free of other proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein was labeled with '"I
and "Fe as described by Hemmaplardh and Morgan (34).
Transferrin and Iron Uptake and Release Experiments. The trans
formed myogenic cells used for transferrin and iron studies were grown
at 35*C. All uptake experiments, however, were performed at 37"C and
4'C to enable comparisons to be made with the results obtained with
normal myogenic cultures. Total uptake represents both binding and
internalization when experiments were performed at 37°C;in contrast,
uptake at 4'C only measures the binding component of the uptake
process. Both normal and transformed cells were used on days 2-3 of
culture when their rates of proliferation were the same.
In all experiments, cells were preincubated in MEM for at least l h
in order to minimize the extracellular concentration of transferrin in
contact with cells. The medium was discarded, and incubations with
labeled Tf-Fe2 were performed in MEM plus 1% bovine serum albumin
MYOGENIC CELLS
in a 95% air:5% CO: atmosphere. Nonspecific binding of the label was
determined by incubating cells with label plus a 100-fold molar excess
of unlabeled Tf-Fe2. Specific uptake or binding of labeled Tf-Fc2 was
defined as the uptake in the absence of unlabeled Tf-Fe2 minus the
uptake in the presence of unlabeled Tf-Fe2. All binding reactions were
stopped by washing cells five times with ice-cold buffered salt solution
(35).
Experiments involved incubating monolayers of cells at 37'C and
4°Ceither (a) at concentrations of labeled Tf-Fc2 ranging from 0.01 to
1 fiM for 90 min or (b) at a saturation concentration of labeled Tf-Fei
(0.25 ^M) for varying lengths of time. In order to measure both surfacebound and internalized radioactivity, cells were treated with 0.1%
Pronase in balanced salt solution for 30 min at 4"( ' as described
previously (26). For measurement of rates of endocytosis, cells were
incubated at 37°Cand 4°Cwith 0.25 ^M labeled Tf-Fe2 without and
with excess unlabeled Tf-Fej. Surface-bound and endocytosed transfer
rin were measured at 30-s intervals for the first 3 min and then at 4, 5,
7.5, 10, 15, 20, 30, 40, and 60 min of incubation.
Measurement of exocytosis of transferrin from cells involved incu
bating the cells for 16 h with 0.25 nM labeled Tf-Fe2 at 37°Cfor the
normal cells and 35°Cfor the transformed cells. This was followed by
washing and reincubation at 37°Cin MEM containing excess unlabeled
Tf-Fe2 (30 MM)for varying periods of time. Radioactivity in the reincubation medium was measured to estimate transferrin exocytosis and
the cells were treated with Pronase in order to measure surface-bound
and internalized radioactivity.
Expression of Results. To enable comparisons to be made between
normal and transformed cells all results were expressed per ¿¿g
cellular
DNA and, where appropriate, per cell values were also calculated.
Results presented are mean values ±SEM. The Student i test was used
to compare different groups of data which were obtained from multiple
experiments.
RESULTS
Growth of Transformed Myogenic Cells. Cultures of myogenic
cells transformed with tsLA24 and maintained at 35°Cunder
went an initial decline in cell numbers the day after subculturing
(Fig. 1/4). This was followed by a period of proliferation, as
revealed by increased cell number (Fig. IA) and increased DNA
synthesis (Fig. IB). The phase of rapid cell proliferation was
maintained for up to 6 days of culture. Greater than 98% of the
cells at all stages of culture were judged to be viable by the
criteria of trypan blue dye exclusion and cell morphology.
Transferrin and iron uptake studies were performed on trans
formed cells after 2-3 days in culture at 35°Cwhen the cells
were in rapid growth phase. At this stage, the rate of cell
30
Fig. 1. Growth of transformed myogenic
cells expressed as number of cells ill and
|'H|thymidine incorporation into DNA (B).
Cells were maintained in 60-mm-diameter
dishes. Results are expressed as means ±SEM
(bars).
4
6
DAYS IN CULTURE
4
6
DAYS IN CULTURE
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TRANSFERRIN
METABOLISM IN TRANSFORMED
proliferation was almost identical to that of cultured nontransformed muscle cells (26).
The extent of contamination of transformed myogenic cul
tures by fibioblasts is difficult to assess because of similarities
in cell morphology between transformed myogenic cells and
transforméefibroblasts. It was therefore necessary to adopt a
number of Drocedures to minimize fibroblast contamination,
including p-eplating to enrich for myoblasts and minimizing
the number of passages prior to transformation with virus.
These steps have been shown to significantly reduce overgrowth
by fibroblasts (25, 27) and in the present study all myogenic
cultures infected with virus contained less than 20% contami
nation by fibroblasts as judged by the extent of myogenesis with
occurred upon shift to permissive temperature.
Receptor-mediated Endocytosis of Transferrin. The time
course of ' !5I-Tf and 59Fe uptake by transformed myogenic
MYOGENIC CELLS
uptake was specific since it was inhibited by a 100-fold molar
excess of unlabeled transferrin.
The above results indicate that more than 90% of the trans
ferrin internalized by the cells is derived from that which is
bound to specific receptors. In order to quantitate these recep
tors the cells were incubated with varying concentrations of
radiolabeled Tf-Fe2. Specific transferrin uptake and specific
iron uptake showed saturation at 0.1-0.2 UMlabeled transferrin
at both 37°C(Fig. 3) and 4°C(Fig. 4). At labeled Tf-Fe2
concentrations above 0.2 pM an increasing proportion of the
transferrin and iron uptake was nonspecific. This can be largely
attribute to binding of transferrin to collagen used to coat the
culture dishes as this is observed in the absence of cells. Specific
20
cultures was similar to that previously described for normal
muscle cell cultures (26). At 37°Cthe total specific transferrin
TOTAL
15
uptake reached a maximum level after 10 min incubation with
label while total specific iron uptake continued to increase
£tÃ-o
linearly with time (data not shown). Internalization of both
transferrin ind iron as determined by Pronase-resistant uptake
Sì
of 125I-Tfand 59Fe was shown to occur at 37°Cbut not at 4°C < S
(Fig. 2). At 37°Cmore than 90% of the total internalized 125ITf uptake, and more than 95% of the total internalized 59Fe
i|
5
0.1
37°C
0.2
0.3
0.4
TRANSFERRIN CONCENTRATION
(pM)
2 90
o
o.
TOTAL
SPECIFIC
60
o
o>
S
n
n
n
NON-SPECIFIC
S 30
(O
0.1
TRANSFERRIN
0.2
0.3
CONCENTRATION
0.4
(/iM)
is
LU O
37°C
LL
*-
CO x
Z
< -z.
<
a:
\- o
Q
O)
Z °
§ £
LU O
20
40
0.1
TIME (min)
Fig. 2. Internalization of transferrin and iron at 37°Cand 4'C in transformed
myogenic celli. Results are the means of three experiments. Myogenic cultures
were transformed with a temperature-sensitive Rous sarcoma virus (tsLA24) and
cultures were naintained at 35°Cuntil the experiments were performed. Cells
were incubatec with 0.25 UMlabeled Tf-Fe2 in the presence and absence of excess
unlabeled Tf-Fe2. Surface-bound and internalized radioactivity was measured.
Total internalization (•);specific internalization (O).
0.2
0.3
0.4
TRANSFERRIN CONCENTRATION
(|iM)
Fig. 3. Effect of increasing concentration of labeled Tf-Fe2 on transferrin
uptake (A), iron uptake (A), and internalized transferrin (C) by transformed
myogenic cells. Cells were transformed with a temperature-sensitive Rous sar
coma virus and maintained at 35'C until the experiments were performed. Cells
were incubated with labeled Tf-Fe2 in the presence and absence of excess unlabeled
Tf-Fe2 at 37'C for 90 min.
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TRANSFERRIN
METABOLISM IN TRANSFORMED
formed cells were observed. The total number of transferrin
receptors was estimated to be 7.84 ±0.67 x 10'°molecule///}!
DNA (n = 5) in transformed cells, which is considerably more
than the figure of 3.78 ±0.24 x IO10molecules/^g DNA (n =
10
10
o
!¡
Ëa
£•¿=
s
w
SPECIFIC
TF.
n
X
0.1
0.2
0.3
*
m
0.4
TRANSFERRIN CONCENTRATION
Fig. 4. Effect of increasing concentration of labeled Tf-Fe2 on transferrin and
iron uptake at 4'C by transformed myogenic cells. Cells were transformed with a
temperature-sensitive Rous sarcoma virus and maintained at 35'C until experi
ments were performed. Cells were incubated with labeled Tf-Fez in the presence
and absence of unlabeled Tf-Fe¡for 90 min at 4'C. Results shown are specific
uptake values.
Table 1 Transferrin receptor numbers and affinity and iron uptake in
transformed myogenic cells
The A, values and surface receptor numbers were determined from transferrin
uptake data obtained by incubating transformed cells with increasing concentra
tions of labeled transferrin at 4'C for 90 min. The other results were calculated
from data obtained from similar experiments in which incubations were performed
at 37'C for 90 min. Each value is the mean ±SEM
Association constant (K,) (x IO7 M"')
13) in normal muscle. Since the number of internalized recep
tors was not significantly different, being 1.99 ±0.14 x 10'°
molecules/jig DNA (n = 10) in normal cells and 1.91 ±0.24 x
10'°molecules/Mg DNA (n = 5) in transformed cells, it is
reasonable to conclude that there has been a substantial increase
in the number of receptors on the surface of transformed cells.
Accordingly, the proportion of total receptors which were sit
uated intracellularly in transformed cells (range, 22-27%;
mean, 24%) was shown to be much lower than in their nontrans
formed counterparts (range, 50-56%; mean, 53%).
Rates of Endocytosis of Transferrin and Iron Accumulation.
To investigate whether the low intracellular transferrin receptor
number and comparatively low Vm^ value for iron uptake in
transformed cells was due to a slow rate of internalization of
the receptors, the rates of transferrin endocytosis were meas
ured at a Tf-Fe2 concentration of 0.25 P.M.The rate of transfer
rin endocytosis was found to be significantly higher (P < 0.01)
in transformed cells, being 0.42 ±0.04 molecule///g DNA/min
(n = 4), than in their nontransformed counterparts which gave
a value of 0.28 ±0.04 (n = 6). However, the maximal rate of
iron uptake in transformed cells, 0.73 ±0.12 x 10'°(n = 7)
iron atoms/¿/gDNA/min, was not significantly different (P >
0.05) when compared with the values for nontransformed cells,
0.62 ±0.26 x 10'°(n = 11) iron atoms/Mg DNA/min (26). The
±0.207.84
Receptor numbers (molecules/fig DNA x
10-'°)
Total
Internal
Surface rate of iron uptake ( 1',„.,,)
Maximal
(atoms/
„¿g
DNA/min x IO'10)n65
MYOGENIC CELLS
±0.67
1.91 ±0.24
567Value1.09
5.90 + 0.19
0.73 ±0.12
Tf and iron uptakes were analyzed by the methods of Scatchard
(36) and Eadie-Hofstee (37), respectively. The 37"C uptake data
were used to calculate the total and the internalized number of
transferrin receptors on the cells (Bmtx)and the maximum rate
of iron uptake ( Fmax).The 4°Cresults provided estimates of the
affinity of the receptors for transferrin (Kt) and the number
present on the cell surface. At 4°C,with saturating concentra
tions of Tf-Fc2, the number of atoms of iron taken up was twice
the number of molecules of transferrin. This indicates that iron
was not released from transferrin accumulated by the cells at
this temperature at which transferrin remained localized at the
cell surface.
The K. for transformed cells, 1.09 ±0.2 x IO7M'1 (Table 1),
rates of transferrin and iron internalization in individual cul
tures were closely correlated in transformed myogenic cells (r
= 0.992, P < 0.001), as reported previously for normal muscle
cultures (26). However, for every transferrin molecule endocytosed by transformed cells an average of only 1.6 iron atoms
were accumulated by the cells (Fig. 5). This value was signifi
cantly less (P< 0.001) than the 2.0 iron atoms per endocytosed
transferrin molecule which was found with normal muscle cells
and suggests a less efficient delivery of iron by transferrin in
transformed myogenic cells than in normal cells.
Transferrin Exocytosis. The release of transferrin and iron
from myogenic cells was studied after a 16-h incubation with
2.0
did not differ significantly (P > 0.05) from the values found in
normal chick myogenic cells in culture, 1.72 ±0.16 x IO7 M~'
N
<
5!i1-0
(26). The results for receptor numbers indicated that approxi
mately 75% of the receptors were situated on the cell surface,
25% being intracellular. These results also serve to validate the
methods used in their determination, since almost the same
number of surface receptors is obtained from the difference
between total and internal receptors estimated directly from
37°Cuptake data and surface-bound receptors estimated di
rectly from the 4°Cincubation results. The mean cycling time
for transferrin on the cells calculated from the total receptor
numbers and maximal rate of iron uptake, assuming that each
transferrin molecule donates two iron atoms to the cell per
cycle, is 21.5 min. This is much greater than the values calcu
lated previously for nontransformed cells which ranged from
6.7 to 11.4 min (26).
Several other differences between transformed and nontrans
NORMAL
z o
O x
rr
—¿c
o
E
LU -5
¡I
fi
/'"TRANSFORMED
/>
/
./L.
I
0.5
1.0
INTERNALIZED TRANSFERRIN
(molecules/pg DNA/min x1010)
Fig. 5. Relationship between the rates of transferrin endocytosis and iron
accumulation from 0.25 \M labeled Tf-Fe3 at 37'C in cultures of transformed and
normal myogenic cells. Data for myogenic cells transformed with Rous sarcoma
virus (A) and dividing presumptive myoblasts (O) are shown. The equation of the
line for the transformed cell data was>>= 1.6.x+ 0.011, r = 0.992, while that for
the normal cell data was y = 2.0* + 0.061, r = 0.988.
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TRANSFERRIN
METABOLISM IN TRANSFORMED
l25I-59Fe-labeled transferrin. Approximately 15% of the intracellular 59Fr was released from the transformed cells, mostly
during the first 10 min of reincubation (Fig. 6). In contrast, no
59Fe is released from normal cells (26). The release of intracellular 125I-tn.nsferrin from transformed cells was much greater
than that of 59Fe, 75% being exocytosed within 10 min reincu
bation (Fig. 6). As with 59Fe,the majority of the release occurred
within the f rst 10 min. The release curves for both normal and
transforméecells could be resolved into two components, fast
and slow. 11 the transformed cultures, the fast rate predomi
nated and n suited in the release of 75% of the total internalized
I25I-Tf within 10 min reincubation. After 90 min reincubation
less than 10% of the internalized I25l-Tf remained associated
MYOGENIC CELLS
different from the rate constant value for the fast transferrin
exocytosis pathway (P > 0.05). This result suggests that the
iron released from the transformed myogenic cells was transferrin-bound iron released by the fast exocytosis pathway of
transferrin. This conclusion was supported by the observation
that more than 95% of the I25l-transferrin released from the
cells was precipitable with 10% trichloroacetic acid.
DISCUSSION
The mechanism of transferrin and iron uptake by transformed
myogenic cells was similar to that described for most other
types of cells including normal myogenic cells (26). The results
with the cells. In normal cultures approximately 50% of the are consistent with those expected of receptor-mediated endointernalized I25l-Tf was released by each of the two pathways.
cytosis plus recycling of the transferrin to the extracellular
The rate constants for the fast and slow phases of transferrin
medium.
exocytosis from transformed cells were 0.39 ±0.04 and 0.0083
While the basic mode of transferrin and iron uptake by the
±0.002 miir1, respectively (n = 4). These values did not differ
transformed cells did not differ qualitatively from that which
significantly (P > 0.05) from those found with normal cells, occurs in normal myogenic cells, there were several quantitative
0.30 ±0.04 and 0.0051 ±0.0004 min"1, respectively.
differences. These could not be attributed to the rate of cell
To assess whether the iron released from transformed cells division since the cultures were studied when their proliferation
was associated with transferrin, rate constant values for iron rates were similar. The transformed cells were observed to have:
release weri calculated. Iron internalized by cells enters two (a) more total and cell surface transferrin receptors; (b) a higher
components. One consists of iron which is unavailable for rate of transferrin endocytosis; (c) a reduced efficiency of iron
release from the cells and represents iron utilized by the cells donation (Fig. 5); and (d) a greater proportion of transferrin
for growth md/or iron incorporated into iron-containing mol
molecules leaving the cell by the rapid efflux pathway (Fig. 6).
ecules. This is the 85% of the internalized 59Fe which remains
It therefore appears that the transformation process per se
unchanged with increasing time of reincubation in Fig. 6. The resulted in increased expression of transferrin receptors with
second component is iron which is available for release from their increased localization to the surface membrane of the cells
the cells. Internalized 59Fe at increasing time of reincubation
plus alterations in the intracellular cycling of the receptors and
was therefore expressed as a percentage of the latter fraction
transferrin which reduced the efficiency of iron delivery to the
and semilo¡;arithmic plots of this data were used to determine
cell.
the rate cor slant for iron release. A mean rate constant of 0.34
The large increase in total and surface transferrin receptor
±0.07 mir"1 was obtained. This value was not significantly
numbers in the transformed cells did not lead to a proportionate
increase in the Kmaxfor iron uptake and the rate of transferrin
endocytosis. This could be due to the presence of noncycling
100
plasmalemma receptors or the less frequent endocytosis of
receptors from this pool in transformed cells compared with
normal cells. It is not possible to distinguish between these
possibilities from the present data, but either would partly
explain the large mean cycling time, 21.5 min, calculated from
the measured total receptor numbers and Kmaxfor iron uptake.
Another contributor to the magnitude of this value is the less
efficient delivery of iron by transferrin in the transformed cells.
In these cells endocytosis of each transferrin molecule resulted
in the accumulation of an average of 1.6 iron atoms, instead of
the possible 2.0 which was used to calculate the mean cycling
time. Hence the true mean cycling time of transferrin molecules
and those receptors which are endocytosed is undoubtedly much
less than 21.5 min and is possibly close to the value of 11 min
found for untransformed myoblasts (26).
The presence of two rates of transferrin release from the
transformed myoblasts indicates that there are two intracellular
pathways for transferrin, as reported previously for normal
myoblasts (26); however, there is a greater proportion of the
fast release pathway in the transformed cells. Two pathways of
transferrin receptor cycling have also been described for K562
10
20
cells, one of which is monensin sensitive and may represent
TIME (min)
cycling of the receptors through the Golgi apparatus while the
Fig. 6. Kel' ase of internalized iron (O and •¿)
and transferrin (D and •¿)
from
other is resistant to monensin and is probably due to a nonnormal exponentially dividing myoblasts (open symbols) and myogenic cells
Golgi pathway (38). Whether similar pathways exist in muscle
transformed with Rous sarcoma virus (closed symbols). Results are means of six
experiments. Cells were incubated for 16 h with 0.25 /iM labeled Tf-Fe; at 35'C.
cells
remains to be established.
After washing, the cells were reincubated at 37°Cin fresh medium containing 30
There has not been a previous study of transferrin and iron
/iM unlabeled Tf-Fe2 and the intracellular 5'Fe and '"I-Tf were measured at the
metabolism in transformed myogenic cells. Boyd et al. (39)
times shown.
1945
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TRANSFERRIN
METABOLISM IN TRANSFORMED
have shown that a transformed mouse fibroblast cell line bound
more transferrin than the original, nontransformed line due to
altered receptor numbers. This was independent of the growth
rate, indicating that it was a transformation-related
change.
The findings of this study with myogenic cells are consistent
with those of Boyd et al. (39). In another investigation, the
distribution of transferrin receptors on normal human fibroblasts cultured in serum was found to be nonrandom, whereas
in malignant fibrosarcoma cells it was random (40). This dif
ference may be related to observations that a greater proportion
of the receptors were on the surface membrane and that they
showed a lower cycling rate in the transformed cells than in
their nontransformed counterparts. Recently, Vidal et al. (41)
reported a 5- to 6-fold increase in expression of surface trans
ferrin receptors in matched T-cell studies which compared
normal and human T-cell leukemia/lymphoma virus I-infected
cells. This study showed that the total number of receptors were
unaltered, but virus infection led to a redistribution in favor of
a cell surface localization.
In agreement with previous studies is the absence of any
difference between K, values for the receptor on normal and
transformed cells, suggesting similarities in receptor type (4244) and indicating that changes in transferrin binding and
uptake by the cells are due to alterations in receptor numbers,
distribution, and cycling rates. Also, there have been several
previous observations which suggest a low efficiency of receptor
function in transformed cells, i.e., high levels of receptors
associated with relatively low rates of iron uptake (11, 45-49).
In conclusion, this study has provided evidence for some
transformation-specific characteristics of transferrin and iron
metabolism in cells of the myogenic lineage. These include an
increase in transferrin receptor numbers on the cell membrane,
less efficient intracellular delivery of iron by transferrin, and
differences in the intracellular processing of transferrin and
iron. Whether similar transformation-specific characteristics
exist in other cell types besides muscle and lymphocytes requires
further investigation. The results raise the possibility that the
transferrin receptor may have an additional function as well as
that of iron donation in transformed cells and possibly, to a
lesser extent, in actively dividing myogenic cells. This alterna
tive function may involve growth promotion via a transmem
brane reducÃ-asesystem as has recently been reported to occur
in other cell types (50-52).
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
ACKNOWLEDGMENTS
The authors wish to thank D. Boettiger for providing the tempera
ture-sensitive Rous sarcoma virus and R. Coelen for helpful discussions
concerning viral transformation of cells.
30.
31.
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Transformation-induced Changes in Transferrin and Iron
Metabolism in Myogenic Cells
Lydia M. Sorokin, Evan H. Morgan and George C. T. Yeoh
Cancer Res 1989;49:1941-1947.
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