[CANCER RESEARCH 48, 3425-3429, June 15, 1988]
Transferrin-like Autocrine Growth Factor, Derived from T-Lymphoma Cells, That
Inhibits Normal T-Cell Proliferation1
Giovanni Morrone,2 Laura Corbo, Maria Caterina Turco, Rosa Pizzano, M. De Felice, Sandy Bridges, and
Salvatore Venuta
Institute of Experimental and Clinical Oncology, Medical School, University of Reggio Calabria, Calamaro [G. M., L. C., M. C. T., M. D. F., S. V.] and Institute of
Biochemical Sciences, II Medical School, Naples [R. P., S. B.J, Italy.
ABSTRACT
Transfer rin, the major iron-binding protein in the plasma of vertebrate
species, is an essential growth factor for cells in serum free medium. We
have established a cell line, Fr, from peripheral blood mononuclear cells
of a patient affected by Sézarysyndrome. Fr cells show a very immature
antigenic phenotype, while constitutively bearing transfeirin receptor on
their surface. Furthermore the Fr line does not produce or respond to
interleukin 2. Finally its conditioned medium contains both a growth
stimulating activity for the Fr cell line and a factor which inhibits I lymphocyte proliferation. We have identified a protein, produced in large
amounts by Fr cells, which shares the immunological properties of human
transferrin. Our data suggest that this transfemn-like factor can act as
an autocrine growth factor for the producer cells and as an inhibitory
factor for normal lymphocytes.
INTRODUCTION
GFs3 play a central role in cell transformation
(1, 2). In
several cell lines it has been reported that the production of
autocrine growth mediators does occur (3-7). It has been shown
that tumor cell growth can be regulated by "normal" growth
factors (4, 6-7, 8), and the products of many viral and cellular
oncogenes have been found to be related to GFs (9-12) or G F
receptors (l3-15).
Another growth advantage for malignant cells could derive
from the production of inhibitory factors which interfere with
the host defence mechanism and the proliferation of normal
cells. In particular, hemopoietic inhibitory factors such as acidic
isoferritins are produced by leukemic cells (16), and suppressive
immunoregulatory factors have been detected in extracts of
fresh as well as cultured human tumor cells (17).
The iron carrier protein Tf acts as a growth factor: its
presence is required for the proliferation of almost all cell types
in vitro (18) and it appears to be involved in the regulation of
growth and differentiation of human tissues (18-19). Further
more, transferrin receptor expression seems to be related to the
proliferative status of normal (18-21) and tumor (18) cells and
monoclonal antibodies to Tf receptor inhibit cell growth (2223).
In this paper we report the establishment of a cell line, Fr,
from peripheral blood cells of a patient with Sézarysyndrome,
which exhibits the following features: (a) the cells strongly
express Tf receptor while lacking other surface membrane
molecules present on mature lymphocytes and Sézarycells; (b)
Fr cells do not produce nor respond to IL-2. FrCM contains
both a growth stimulating activity for Fr cells and a factor
which inhibits T-lymphocyte proliferation; (c) in Fr supernatant
a protein has been identified which shares the biochemical and
immunological properties of human transferrin. Such a transferrin-like molecule appears to be responsible for both of the
above mentioned effects on cell growth.
MATERIALS AND METHODS
Establishment of Cell Line and Culture Conditions. Mononuclear cells
were isolated from peripheral blood of the patient G. F. (female, 67,
affected by Sézary
Syndrome diagnosed by clinical, morphological, and
immunological criteria) by centrifugation on Ficoll-Hypaque (Phar
macia, Uppsala, Sweden). Cells were plated at 5 x I05/ml in RPMIClick's (Flow Laboratories, Rockville, MD), supplemented with 15%
FCS (Flow Laboratories) and 5 units/ml human purified IL-2 (kindly
provided by Dr. K. Weite, Sloan-Kettering Cancer Center, New York,
NY), and incubated at 37°Cin 5% CO2, 90% humidity. Cultures were
fed every third day by replacing 50% of the spent medium with fresh
medium.
After 20 passages, attempts were made to grow Fr cells without
exogenous IL-2. Thereafter, IL-2 was not added to the culture medium
anymore, because cells showed the same growth rate in either the
presence or absence of the factor.
The medium used in routine cultures of Fr cells was RPMI-Click's
supplemented with penicillin and streptomicin (Flow Laboratories),
glutamine (Flow Laboratories), and 15% FCS. Where indicated, FCS
concentration has been changed, or the medium has been supplemented
with 10 ¿ig/mlhuman transferrin (Sigma, St. Louis, MO) or with a
pool of growth factors containing Tf (10 ng/ml), glycyl-histidyl-lysine
(20 /jg/ml). hydrocortisone (3.6 Mg/ml), insulin (10 fig/ml), thyroidstimulating hormone (1 milliunit/ml), and somatostatin (10 ng/ml).
TPA, used in some experiments, was kindly provided by Dr. A. Pinto
(CRO, Aviano, Italy).
Fr Conditioned Medium (FrCM) Preparation. Cells (3.5 x nr/nil)
were plated in RPMI-Click's 0.5% FCS and incubated at 37'C in 5%
CO2 and 90% humidity. Forty-eight h later, cultures were harvested
and centrifugea for 10 min at 400 x g, and the supernatant was
collected, filter sterilized, and stored at 4'C. FrCM was collected only
when cell viability, as assessed by trypan blue exclusion, was higher
than 80%.
Antibodies. MoAbs OKT3, OKT4, OKT6, OKT8, OKT9, OKT10,
OKT11, OKM1, OKIa were purchased from Ortho Diagnostic System,
Milan, Italy. BAI was purchased from Hybritech, San Diego, USA.
The MoAb anti-Tac, which recognizes the IL-2 receptor, was kindly
provided by Dr. T. Waldmann (NIH, Bethesda, MD).
MoAbs S4-7 and R IB-19 were kindly provided by Dr. G. Rovera
(Wistar Institute, Philadelphia, PA).
Fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoReceived 8/10/87; revised 2/26/88; accepted 3/7/88.
globulins were from Ortho Diagnostic Systems, Milan, Italy.
The costs of publication of this article were defrayed in part by the payment
Rabbit anti-human Tf immunoglobulins were from Miles, Elkhart,
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
IN.
1This work was supported by Consiglio Nazionale delle Ricerche projects
Immunofluorescence. One hundred n\ of PBMNC or Fr cell suspen
"Oncologia" and "Ingegneria Genetica e Basi Molecolari delle Malattie Eredi
sion (4 x 106/ml) were incubated 30 min at 4'C with 100 ti\ of a MoAb
tarie" and by grants from Associazione Italiana per la Ricerca sul Cancro.
solution. Cells were washed three times and incubated 30 min at 4'C
2To whom requests for reprints should be addressed.
3The abbreviations used are: GF, growth factor; Tf, transferrin; IL, interleukin;
with 100 f/1of fluorescein isothiocyanate-conjugated rabbit anti-mouse
IgG ant ¡scrum.Cells were washed twice, re-suspended in a drop of 50%
FrCM, Fr cell line conditioned medium; FCS, fetal calf serum; MoAb, mono
clonal antibody; PBMNC, peripheral blood mononuclear cells; SDS, sodium
glycerol-phosphate-buffered saline (140 mM NaCI-8 mM Na2PO4-1.5
dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PHA, phytohemagIHMKI l -PO.,, pH 7.4) solution, spread on a slide, and examined with
glutinin; FrGF, Fr-derived growth factor; TPA, 12-O-tetradecanoylphorbol-13acetate.
an immunofluorescence microscope.
3425
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AUTOCRINE AND IMMUNOSUPPRESSION
Thymidine Incorporation Assays. For IT cells, tritiated thymidine
incorporation was assessed by incubating cells in the presence of O.S
fiCi/well of [3H]thymidine (specific activity, 47 Ci/mM; Amersham
International pic, Amersham, England) for 4 h. Cells were subsequently
harvested by an automatic device, and the thymidine incorporation
measured in a beta counter.
For human PBMNC proliferation assays were performed as previ
ously described (8).
11-2 Production Assay. Ir supernatants were assayed for the presence
of IL-2 by a biological assay with the IL-2-dependent murine line (111
(24).
SDS-PAGE of Proteins Produced by Fr Cells. Fr cells (5 x 10s) were
plated in RPMI supplemented with 0.5 or 10% PCS; 48 h later, cells
were collected, washed twice in phosphate buffered saline solution, and
incubated at 2 x 10"/ml in methionine-free RPMI containing 0.5%
FCS and 50 ¿iCi/mlof ["SJmethionine (Amersham, International pic,
Amersham, England). After 24 h, the medium was harvested and an
aliquot was loaded on a SDS-polyacrylamide gel. As a control, the
medium of resting or PHA-activated human T-lymphocytes from
healthy donors was analyzed on the same gel. A sample of the media
was used for immunoprecipitation with rabbit anti-human Tf inuminoglobulins.
Immunoprecipitation of the Fr-derived Transferrin-like Factor. One
hundred n\ of the above media to be tested for the presence of Tf were
incubated with 100 /«I
of immunoprecipitation buffer and 5 M'of rabbit
anti-Tf immunoglobulins. After 6 h, 50 ^1 of 50% Sepharose-Protein
A were added. After 1 additional h, the mixture was centrifuged for 10
min at 5000 x g; the pellet was resuspended in 50 M'of reducing buffer,
then centrifuged again. The supernatant was collected and loaded on
the SDS-PAGE gel.
The precipitate was resuspended in the same volume of medium as
the original sample and used for SDS-PAGE or tested in functional
assays.
Purification of the Fr-derived Transferrin-like Factor. Two ml of
I r( \l were applied on a 1 nil column of Sepharose 4B (Pharmacia)
coupled with mouse antibodies against human transferrin. The column
was washed with 0.1 M phosphate buffer and eluted with 2 ml of citrate
buffer, pH 3.5. The eluate was dialyzed against RPMI-Click's medium
and analyzed on 12% SDS-PAGE or tested in functional assays.
RESULTS
Surface Membrane Phenotype of Fr Cells. The antigenic
profile of mononuclear cells from fresh peripheral blood of the
patient and of Fr cells after 30 culture passages was studied.
Results are shown in Table 1. The most striking difference
between fresh and cultured cells is that the latter shows no
reactivity with the MoAbs OKT3 and OKT4 while they strongly
bind the anti-la antibody and OKT9, which recognizes the Tf
receptor. The Tac antigen (IL-2 receptor) is not present on Fr
FUNCTIONS OF A Tf-LIKE MOLECULE
membrane. This is in agreement with the IL-2 independence of
Fr cells (see below). Fr cells were negative for the myeloid
antigens recognized by the MoAbs OKM1, S4-7, and R IB- 19
(Table 1).
Cells were treated with TPA (10 ng/ml) for 3 days; they
stopped proliferating, regardless of the presence of Tf in the
medium. Surface phenotype was determined. The OKT9- and
OKTIO-positive cells were reduced to 18 and 38%, respectively,
while 10% of OKT1 1-positive cells could be detected (Table 2).
Lack of IL-2 Production in Fr Cells. Fr supernatant was tested
on the IL-2 dependent murine cell line, CTLL, for the presence
of interleukin 2. Table 3 shows that Fr conditioned medium
was not able to support CTLL growth, even at concentrations
up to 50%. This indicates that the cells do not produce IL-2.
Autocrine Growth-stimulating Activity in FrCM. Growth
curves were made under different conditions in order to assess
the effect of cell density, FCS concentration, Fr conditioned
medium, and growth factors on Fr proliferation.
Fig. 1 shows the growth curve of Fr cells cultured at 5,000
(A) or 40,000 (B) cells/ml, in the presence of different concen
trations of FCS. Cells growing in high serum concentration
(15%) show the same proliferation rate regardless of the cell
Table 2 Effect of TPA on Fr cell surface phenotype
Fr cells were cultured with or without TPA (10 ng/ml) for 3 days, washed, and
analyzed in immunofluorescence (see "Materials and Methods") with the indi
cated MoAbs.
of positive cells
Without TPA
MoAb
With TPA
OKT3
<1OKT4
<1OKT6
<1OKT8
<1OKT9
76OKT10
74OKT11
<1Anti-Tac
< 1<1<1<1<118381034
Table 3 IL-2 detection in FrCM
Samples of FrCM were harvested and tested at the indicated concentrations
for their content of IL-2 in CTLL assay (see "Materials and Methods"). Recom
binant IL-2 was used as a standard.
[JH]Thymidine
Sample
incorporation (cpm ±SD)
NoneIL-2,
707,620
±
5 units/ml
IL-2, 2.5 units/ml
IL-2, 1.25 units/ml
unit/mlFrCM,
IL-2, 0.3
±980
4,709 ±550
1,904
350444
±
±75465
±65
419 ±76
50%
FrCM, 25%372
Table 1 Surface phenotype of Fr cells
Fr cells were analyzed in immunofluorescence (see "Materials and Methods")
with the indicated MoAbs. Results are compared with those obtained with
noncultured PBMC.
B
cellsNoncultured
positive
100
Fr after 30
MoAbOKT3OKT4OKT6OKT8OKT9OKT10OKT11OKM1OKIaAnti-TacBAIS4-7RIBPBMNC
passages9898NO"1NDNDND1<1NDNDNDND<1<1<1<18075<1<170<1<1<1<1
90
_ go
Ó 70
« 60
Õ50
=:
«°
u
4
48
72
Timi Ihrsl
48
72
96
Time (hrs)
Fig. 1. Growth curves of Fr cells. Fr cells, SxlO3 (A) and 4x10" (B), were
plated in RPMI-Click's supplemented with 0.5 (O) and 15% (•)FCS. After 24,
48, 72, and 96 h the viable cells were counted. Points, average of triplicate
determinations.
19%of
1ND, not done.
3426
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AUTOCRINE AND 1MMUNOSUPPRESSION FUNCTIONS OF A Tf-LIKE MOLECULE
concentration, the doubling time being about 48 h. When serum
concentration was 0.5%, however, only cells plated at 40,000/
ml were still able to proliferate after 24 h lag period.
Fig. 2 shows the effect of FrCM on Fr cell growing at low
density (9000/ml) in a low serum concentration. The addition
of 50% Fr supernatant to the culture medium strongly stimu
lates cell proliferation.
Since the cell line was OKT9+, we tested the possibility that
survival (60 h) compared with the immediate decline of cells
cultured in serum- and Tf-free medium (data not shown). A
pool of six growth factors, including Tf, showed almost the
same effect of Fr proliferation as transferrin alone (Fig. 3).
Presence of T-Lymphocyte Inhibitory Activity in FrCM. The
effect of Fr supernatant on the growth of PHA-stimulated
human PBMNC was studied. Fig. 4. / shows that the addition
of 25% of FrCM to the cultures inhibited cell growth by about
90%. In order to confirm that T-cells were the target of such
an inhibitory activity, the same experiment was performed using
the monoclonal antibody OKT3, mitogenic for T-lymphocytes.
The effect of FrCM in these experiments was the same as that
on PHA-stimulated cells (Fig. 4B).
Detection of a Tf-like Protein in FrCM. In order to identify
the factor(s) responsible for the autogenous stimulation of Fr
growth, the proteins secreted by the cell line were endogenously
labeled with [35S]methionine and then analyzed on a SDS-
transferrin was able to support Fr cell growth. Fig. 3 shows
that the addition of 10 ng/m\ of human Tf stimulates the
proliferation of Fr growing in 0.5% FCS. A growth curve with
5000 cells/ml in the presence of Tf showed that the cells were
Tf-dependent for growth even at low density in the presence of
0.5% PCS; in serum-free medium the addition of Tf was not
sufficient to allow Fr proliferation but resulted in only a longer
polyacrylamide gel in comparison with the proteins produced
by resting and PHA-stimulated human PBMNC. Anti-Tf immunoprecipitates from the same supernatants were run as well.
As shown in Fig. 5, a major band is present in Fr supernatant
(lane 1) that is absent or barely detectable in the conditioned
medium from PHA-stimulated human peripheral blood leuko
cytes (lane 2) and that is immunoprecipitated by antibodies
against human transferrin (lane 6) while no bands were detected
50
40
30
f)
o
20
X
Ia>
»
O 10
9
8
7
6
48
72
96
120
144
Time Ihrs)
Fig. 2. Self-stimulating activity in Fr supernatant. Cells ('»,l(l') were plated
in RPMI-Click's supplemented with 0.5% FCS (O), 15% FCS (•),and 50%
FrCM (A). At the indicated times the viable cells were counted.
I
200
6
1:100
1:500
1:200
1:1000
dilution
1:10«
48
72
96
1:10*
1:10»
1:10'
1:10"
1:10'
120
Time (hrs)
Fig. 3. Effect of transferrin on Fr proliferation. Fr cells (30x103) were plated
in RPMI-Click's supplemented with 0.5% FCS (O), 15% FCS (•),10 jig/ml
transferrin alone (A), and together with a pool of 5 growth factors (A), as described
in "Materials and Methods."
dilution
Fig. 4. Dose-dependent inhibition by FrCM of mitogen-stimulated PBMNC
proliferation. PBMNC (4xl05) were incubated at 37'C in 96-well microtiter
plates in a final volume of 0.2 ml of RPMI plus 5% FCS with PHA (A) and
OKT3 (B) at the indicated concentrations in the absence (•)and presence of 25%
FrCM (O). After 68 h [3H]thymidine incorporation was measured.
3427
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AUTOCRINE AND IMMUNOSUPPRESS1ON
FUNCTIONS OF A Tf-LIKE MOLECULE
shown in Fig. 6, the electrophoretical mobility of FrGF was
different from that of transferrin.
We conclude that FrGF is a Tf-like protein, somehow differ
ent from normal transferrin. Its concentration in FrCM was
estimated to be approximately 100 ng/m\.
90K-
DISCUSSION
The role of Gfs in the process leading to malignant transfor
mation of the cell has not yet been well defined. However, many
data have drawn a close relationship between these molecules
and tumor cell growth (3-15, 25-27). Transferrin appears to
be centrally involved, at least as a cofactor, in the regulation of
growth of both normal and malignant cells. Tf receptor is
expressed in the majority of tissues with a high proliferation
rate. Blocking the interaction of Tf with its receptor results in
growth inhibition of normal and transformed cells.
We have obtained a cell line, Fr, from peripheral blood cells
of a patient with Sézary
syndrome, studied its surface phenotype
and the growth requirement, and analyzed its protein secretion.
The Fr antigenic profile is dramatically different from that of
normal lymphocytes and of fresh peripheral blood cells from
the same patient. In particular, the antigens recognized by
MoAbs OKT3 and OKT4 present on the patient's circulating
69K-
46K-
30K-
14KFig. 5. SDS-PAGE analysis of |"S]melhionine-labeled proteins secreted from
Fr cells before and after immunoprecipitation with anti-transferrin antibody.
Samples were run on 12% polyacrylamide gel under reducing conditions. Lanes
1 and 2, 10 pi of "S-labeled supernatant from FrCM and 1% PHA-stimulated
PBMNC, respectively; lane 3, 0.5-*tl labeled Fr supernatant containing as many
cpms as contained in 10 *ilof the supernatant from PHA-stimulated PBMNC. In
parallel antitransferrin immunoprecipitates from resting and PHA-stimulated
PBMNC supernatants (lanes 4 and 5, respectively) and from FrCM (lane 6) were
run. A, thousands.
mononuclear cells are not detectable on the surface of Fr cells.
Furthermore the line shows a very strong expression of Tf
receptor and TÕOand HLA Class II antigens. Fr cells do not
express the myeloid antigens recognized by the MoAbs OKM1,
S4-7, and R IB-19. After treatment with TP A, expression of
IL-2 receptor (35% of the cells) and to a lesser extent, of Tl 1
1
Table 4 Effect of FrGF on Fr and PBMNC proliferation
Fr cells were cultured at 3 x lO^/ml in RPMI supplemented with 0.5% PCS
in the absence or presence of 10 fig/ml of FrGF. [3H]thymidine incorporation
was measured at the indicated times. Human PBMNC (4 x 10') were stimulated
with 1% PHA and 5 ng/ml OKT3 in the absence or presence of 10 ng/ml of
FrGF. [JH]Thymidine incorporation was measured after 72 h. Values are mean
±SD of triplicate determinations.
['HJThymidine
incorporation (cpm)
FrGF18,614
CellsFr
(48 h)
Fr (72 h)
h)PBMNC
Fr (96
FrGF8,929
±850
12,005 ±1,300
17,738
1,820866
±
±2,500
29,552 ±3,400
32,770
4,0002,300
±
±120
±300
PBMNC + PHA
102,365 ±8.000
4,441 ±500
PBMNC + OKT3Without
66,461 ±3,000With 818 ±120inhibition95.7
98.8
in human PBMNC supernatants (lanes 4 and 5).
The Tf-like molecule was purified from serum free FrCM by
affinity chromatography on a Sepharose-conjugated
anti-Tf
column and its biological activity was tested. This protein was
able to support Fr growth while strongly inhibiting the prolif
eration of PHA- or OKT3-stimulated PBMNC (Table 4). Sim
ilar results were obtained with protein immunoprecipitated with
different anti-Tf antisera (data not shown). Hence it appeared
that both Fr-stimulatory and PBMNC-inhibitory activities de
tected in FrCM were displayed by this factor. Since the latter
activity is unusual for transferrin, it was conceivable that Fr
derived factor was not identical to this protein. To compare
FrGF to human transferrin, a SDS-PAGE was performed. As
14K
Fig. 6. Electrophoretical mobility of the Fr-derived Tf-like factor. Ten ¿/I
of
Tf-like factor eluted by Sepharose 4B conjugated with anti-transferrin antibodies
(lane /) were run in parallel with 10 pg of human transferrin (lane 2). K,
thousands.
3428
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AUTOCRINE AND IMMUNOSUPPRESSION
antigen (10% of the cells) could be detected by immunofluorescence. We suggest that our line derives from the clone of an
immature leukemic progenitor cell, which represents a very low
percentage of the peripheral blood cell population.
IL-2 is the growth factor responsible for T-cell proliferation
(8) and it also induces proliferation of B-lymphocytes (26, 28).
Cutaneous T-lymphoma cells have been shown to express the
IL-2 receptor (4). We studied whether Fr secretes or responds
to this lymphokine, and found that the cell line does not produce
the factor, nor does it express its receptor; Fr cells are not IL2-dependent for growth. The analysis of Fr supernatant also
failed to reveal the presence of IL-1 and of colony-stimulating
activities (data not shown). The only growth factor-like activity,
identified in FrCM, is the protein immunoprecipitated by antiTf antiserum. It cannot be excluded, however, that the Tf-like
FrGF acts only as a cofactor, since serum is required, even
though in very low concentration, for Fr proliferation.
Transferrin has been shown to be essential for growth of
most normal and malignant cells; this protein is probably
involved in other regulatory functions. Tf receptor is expressed
on the membrane of peripheral blood monocytes as a late
differentiation antigen, and suppressor T-cell derived Tf has
been reported to down-regulate the production of hematopoietins by T4 cells (29).
Furthermore, Tf production by T-lymphoma cell lines has
been recently described (30).
Fr supernatant contains a growth inhibitory activity for hu
man T-lymphocytes. We have tested the effect of the purified
Tf-like protein on stimulated PBMNC and observed that this
molecule was as active in inhibiting their proliferation as the
whole FrCM. It has to be cleared whether the FrGF immunosuppressive effect relies on a direct inhibition of T-lymphocytes
or is mediated by accessory cells and if stimulation of suppressor
T-cells is involved in its mechanism. Since FrGF is similar but
not identical to normal transferrin, it could be hypothesized to
act as a competitive analogue ineffective in T-lymphocyte pro
liferation. Preliminary experiments indicate that addition of
normal transferrin cannot bypass the inhibitory effect of FrGF.
Whatever the mechanism may be, it is noteworthy that a unique
protein synthesized by a transformed cell line can function as
an autocrine growth factor for the producer cells and as an
inhibitory factor for normal lymphocytes. The availability of a
cell line producing a factor with both autocrine growth stimu
lating properties and suppressor activity provides an interesting
model to study the mechanism responsible for triggering and
controlling cell proliferation.
ACKNOWLEDGMENTS
We thank Dr. G. Rovera for providing us with MoAbs S4-7 and
RIB-19, Dr. A. Pinto for his cooperation in performing fluorescence
experiments, and R. Bisogni and A. Visconti for technical help and E.
Tenore for editing the manuscript.
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3429
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1988 American Association for Cancer Research.
Transferrin-like Autocrine Growth Factor, Derived from
T-Lymphoma Cells, That Inhibits Normal T-Cell Proliferation
Giovanni Morrone, Laura Corbo, Maria Caterina Turco, et al.
Cancer Res 1988;48:3425-3429.
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