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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2569-2577
Optimal Proliferation of a Hematopoietic Progenitor Cell Line
Requires Either Costimulation With Stem Cell Factor or Increase of
Receptor Expression That Can Be Replaced by Overexpression of Bcl-2
By
Huei-Mei Huang,
Jian-Chiuan Li,
Yueh-Chun Hsieh,
Hsin-Fang Yang-Yen, and
Jeffrey Jong-Young Yen
From the Institute of Biomedical Sciences and Institute of Molecular
Biology, Academia Sinica, Taipei, Taiwan; and Graduate Institute of
Life Sciences, National Defense Medical Center, Taipei, Taiwan.
 |
ABSTRACT |
In vitro proliferation of hematopoietic stem cells requires
costimulation by multiple regulatory factors whereas expansion of
lineage-committed progenitor cells generated by stem cells usually
requires only a single factor. The distinct requirement of factors for
proliferation coincides with the differential temporal expression of
the subunits of cytokine receptors during early stem cell
differentiation. In this study, we explored the underlying mechanism of
the requirement of costimulation in a hematopoietic progenitor cell
line TF-1. We found that granulocyte-macrophage colony-stimulating
factor (GM-CSF) optimally activated proliferation of TF-1 cells
regardless of the presence or absence of stem cell factor (SCF).
However, interleukin-5 (IL-5) alone sustained survival of TF-1 cells
and required costimulation of SCF for optimal proliferation. The
synergistic effect of SCF was partly due to its anti-apoptosis activity. Overexpression of the IL-5 receptor  subunit (IL5R) in
TF-1 cells by genetic selection or retroviral infection also resumed
optimal proliferation due to correction of the defect in apoptosis
suppression. Exogenous expression of an oncogenic anti-apoptosis
protein, Bcl-2, conferred on TF-1 cells an IL-5-dependent phenotype.
In summary, our data suggested SCF costimulation is only necessary when
the expression level of IL5R is low and apoptosis suppression is
defective in the signal transduction of IL-5. Expression of Bcl-2
proteins released the growth restriction of the progenitor cells and
may be implicated in leukemia formation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS IS A complex process in
which a small population of stem cells is needed to generate
continuously large populations of mature cells in eight major lineages.
These diverse proliferative, differentiative, and maturation events are
achieved by a network of multiple hematopoietic regulatory molecules,
including stem cell factor (SCF), interleukins (IL), and
colony-stimulating factors (CSF). Early studies using in vitro colony
formation assay discovered that a combination of two or more regulatory
factors was required to stimulate the proliferation of highly purified stem cells whereas lineage-committed progenitor cells can be stimulated to proliferate by single regulators.1-5 Notably, when cells
were treated with granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-3, or IL-6, they formed small colonies of committed progenitor cells in various hematopoietic lineages. However, when the
cells were cultured in combination with SCF there was a consistent 10-fold to 20-fold increase of cell numbers per colony. The lineage spectrum of these committed progenitors did not change when the fractionated stem cells were incubated in the presence of CSF with or
without SCF.1-5
It has been suggested that hematopoiesis is regulated by the sequential
interaction of regulators present in the microenvironment with the
corresponding receptors present on the cellular surface (see
reviews6,7). Changes in growth factor requirements during the progression of differentiation may reflect the hierarchy of cytokine receptor expression and function. To clarify how the activation of lineage-restricted growth factor receptor genes is
coupled with the activation of the corresponding differentiation program, the pattern of expression of several hematopoietic growth factor receptor genes during the differentiation has been studied. In
the most undifferentiated stem cell population, the  subunit of the
IL-3 receptor was clearly expressed and the expression remained
throughout the differentiation process.6 The common  subunit of IL-3, IL-5, and GM-CSF receptor (c) and the subunit of the GM-CSF receptor (GMR) were expressed in low levels in stem
cells, but their expression increased in the differentiated progenitor
population.8 The differential temporal expression of
receptor subunit genes in vitro was consistent with the flow cytometric
study in hematopoietic stem and progenitor cell populations purified
from bone marrow.9 However, the underlying molecular mechanism of the hierarchical response of the cells to cytokine during
the differentiation program is not yet clear.
TF-1 is an immortalized cytokine-dependent cell line established from
the bone marrow of an erythroleukemic patient.10 In many
aspects TF-1 cells represent the multiple-lineage progenitor cells that
acquire an ability of unlimited self-renewal and lose the ability of
differentiation commitment. Our study and other studies showed that
TF-1 cells not only expressed CD34 surface marker10 and
were responsive to SCF,11 but also differentially expressed
the subunits of the IL-5 receptor and the GM-CSF
receptor.12 Interestingly, TF-1 cells also manifested
distinct proliferation response toward IL-5 and GM-CSF. GM-CSF alone
efficiently stimulated the optimal proliferation of TF-1 cells, but
IL-5 could only sustain the viable cell number.12
In the present study, we explored the molecular mechanism of the
necessity of multifactor costimulation in TF-1 progenitor cells for
optimal proliferation. Our results suggest that SCF costimulation is
required only when the expression level of IL5R is low and apoptosis
suppression is defective in the signal transduction of IL-5.
Furthermore, increased expression of IL5R or Bcl-2 protein released
the requirement of SCF for maximum proliferation of TF-1 in medium
containing IL-5.
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MATERIALS AND METHODS |
Cell lines, culture conditions, and cytokines.
The TF-1 cell line10 and its variant, JYTF-1,12
were both maintained in RPMI1640 supplemented with 10% fetal bovine
serum (FBS), 50 µmol/L -mercaptoethanol, 2 mmol/L L-glutamine, 100 u/mL penicillin G, 100 µg/mL streptomycin, and 2 ng/mL GM-CSF. TF1
and TF8 were two hIL5R overexpressing subclones of the TF-1 cell
line and were maintained in the same medium as described for TF-1
except 200 µg/mL of G418 was supplemented. TF/Bcl-2 was a pool of
mixed Bcl-2 overexpressing TF-1 subclones. This pool of cells was
established by Chao et al13 and was maintained in the same
medium as that for TF1 cells. Recombinant human GM-CSF, IL-3, IL-5,
and SCF were purchased from R & D Systems (Minneapolis, MN).
Cell proliferation and DNA synthesis assay.
For analyzing proliferation activity, cells were cultured in the
indicated growth medium at a density of 2 × 105
cells/mL. The numbers of viable cells were monitored daily by trypan
blue exclusion assay. DNA synthesis rate (cpm/10,000 cells/h or
cpm/20,000 cells/h as indicated) was measured by the
[3H]thymidine pulse-labeling assay as previously
described12 with slight modification. In brief, 2 × 105 cells were cultured in the indicated medium for 48 hours, then 1 × 104 or 2 × 104
viable cells were seeded in a 96-well plate and 1 µCi of
[3H]thymidine was added. After 1 hour of incubation, the
labeled cells were harvested and lysed with FilterMate cell harvester (Parkard Instrument Co, Canberra, Australia). Samples were counted in a
microplate scintillation counter (Parkard Instrument Co).
Surface IL-5R staining for flow cytometric analysis.
For analyzing surface expression of IL-5R, one million rapidly
growing cells were washed twice with ice-cold phosphate-buffered saline
(PBS) and resuspended in 1 mL staining buffer (2% FBS and 0.1% sodium
azide in PBS) and kept on ice for 30 minutes. The cells were pelleted,
mixed with 0.5 µg of primary antibody (antihuman IL-5R antibody
A14 was a gift of Dr Charles Shih of PharMingen, San Diego, CA), and
incubated on ice for 1 hour. After washing twice with staining buffer,
the cells were incubated with fluorescein isothiocyanate
(FITC)-conjugated goat antimouse antibody (Zymed, Carlton Court, South
San Francisco, CA) for 45 minutes on ice. Fluorescence intensity of
surface bound antibody was analyzed by FACScan (Becton Dickinson,
Mountain View, CA) after removal of unbound secondary antibody.
Bromodeoxyuridine (BrdU) labeling and propidium iodide (PI) staining
for cell cycle analysis.
Cell cycle analysis of BrdU-labeled and PI-stained cells was performed
according to the manufacturer's instruction (Becton Dickinson
Immunocytometry System, San Jose, CA) with slight modifications. Three
to five million cells were cultured in the indicated medium for 24 hours before labeling with 10 µmol/L BrdU for 1 hour. Cells were then
washed and fixed overnight in ice-cold methanol. After denaturation as
described by the vender's manual, cells were stained in 5 µg/50 µL
of FITC-conjugated anti-BrdU antibody at 37°C for 30 minutes.
Before flow cytometric analysis, cells were stained with 1 µg/mL of
PI (Sigma, St Louis, MO) and treated with 100 U/mL of RNaseA
(Worthington Biochemical Corp, Freehold, NJ) in 0.1% glucose at room
temperature for 30 minutes.
Histone releasing assay and DNA fragmentation analysis.
Two methods were employed to monitor the relative activity of
apoptosis. Histone releasing assay (Boehringer Mannheim) is an
enzyme-linked immunosorbent assay (ELISA) for the detection of
cytoplasmic histone-associated-DNA-fragments after induction of
apoptosis. It was performed according to the instructions of the
manufacturer. Briefly, 1 × 104 cells were seeded in a
96-well plate in the indicated medium. After 24 hours of incubation,
cells were pelleted in the bottom and lysed to release the cytosolic
contents. An aliquot of lysate was placed into a streptavidin precoated
96-well plate. Subsequently, a mixture of biotin conjugated antihistone
antibody and peroxidase conjugated anti-DNA antibody were added and
incubated for 2 hours. After removal of unbound antibodies, the amount
of the bound nucleosomes was photometrically determined with
2,2'-Azino-di[3-ethylbenz-thiazolin-sulfonat] (ABTS) as a
substrate. After reaction, the absorption optical density at 405 nm was
measured by a THERMOmax microplate reader (Molecular
Devices, Menlo Park, CA). The DNA fragmentation was analyzed as
previously described.12 In brief, 1 million cells were
cultured for 24 hours in the indicated medium, then washed, centrifuged, and resuspended in 50 µL of Williams lysis buffer (50 mmol/L Tris HCl, pH 8.0, 10 mmol/L EDTA, 0.5% Sarkosyl, and 500 µg/mL proteinase K) and incubated at 50°C for 3 hours. The samples were incubated for 1 hour at 37°C after addition of 10 µL
of RNase A (2 mg/mL). After addition of 1 µL of ethidium bromide (10 mg/mL), the samples were extracted with an equal volume of phenol/chloroform (1:1), and stored at 4°C after the addition of 10 µL of 1% low melting agarose solution containing 10 mmol/L EDTA (pH
8.0). Samples were melted at 70°C and allowed to solidify inside
the well before electrophoresis was initiated.
Phase contrast microscopy.
Phase contrast micrography was conducted as previously
described.12 The photos were taken from three different
fields for each sample, and a total of 300 to 500 cells were counted
for each sample.
Retroviral construct and viral infection.
The entire coding region of hIL5R complementary DNA (cDNA) was
released from pSG5IL5R by EcoRI digestion and cloned into the EcoRI site of pBabeNeo14 to yield
pBabeNeohIL5R. This plasmid was then introduced into PA317
amphotropic packaging cells obtained from American Type Culture
Collection (Rockville, MD). Stable packaging subclones were selected in
the presence of 400 µg/mL of G418 by the standard procedure.
Infectious virus was collected as a 24-hour conditioned supernatant
from the virus-producing PA317 subclones and was used to infect TF-1
cells in the presence of 8 µg/mL of polybrene. Many independent
stable infected subclones were established under the selection of G418
for two weeks.
Northern blot analysis.
Total RNA was isolated from cultured cells by the modified acidic
phenol method described by Wilkinson.15 Twenty micrograms of each RNA sample was separated in 1% formaldehyde agarose gel and
transferred to a Hybond N+ membrane (Amersham, Little
Chalfont, Buckinghamshire, UK). The RNA blot was probed with
[-32P]-dCTP labeled cDNA for the hIL5R and G3PDH genes.
Western blot analysis.
Two hundred microgram lysates were subjected to standard Western blot
analysis as previously described.16 After binding with
horseradish peroxidase conjugated secondary antibodies, blots were
visualized with an ECL detection system (Amersham). The anti-Bcl-2 antibody (N-19) and anti-Bax antibody (N-20) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti- tubulin antibody was purchased from Amersham.
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RESULTS |
SCF enhances the proliferation effect of IL-5 but not GM-CSF in TF-1
cells.
Proliferation is defined in this report as the phenomenon of an
increase of viable cell number in a given cell population. A successful
proliferation under certain culture conditions is usually governed by
the capabilities of cells that undergo mitogenesis, which is defined as
an ability of cells to enter S phase and replicate DNA, and prevent
apoptosis in individual cells. With the lack of either one of these
capabilities, cytokines may only support survival or suboptimal
proliferation of the target cells.
In a previous study, SCF in the GM-CSF-dependent cell lines was shown
to act more as a survival factor and not a proliferation factor due to
its ability to sustain viable cell numbers.11 In another
study, a synergistic proliferation effect of SCF and IL-3 was only
observed when SCF was cocultivated with low concentrations of IL-3 in
an IL-3-dependent cell line.17 To extend the findings of
the synergistic ability of SCF, we explored the possibility for SCF to
complement the defective antiapoptotic effect of IL-5 in a
GM-CSF-dependent TF-1 cell line. As shown in
Fig 1, TF-1 cells only survived in
IL-5-containing medium, but partially proliferated in SCF-containing
medium (Fig 1). A synergistic proliferative response between IL-5 and
SCF was clearly observed when TF-1 cells were costimulated with both
factors for more than 3 days, and this phenomenon became even more
obvious thereafter (Fig 1). SCF enhanced the growth of TF-1 cells
cultured in all concentrations of IL-5 tested (data not shown), which
is in contrast to previous observations using IL-317 (see
below). Interestingly, GM-CSF, in its optimal concentration, stimulated
the proliferation of TF-1 cells maximally regardless of the presence or
absence of SCF (Fig 1). Therefore, TF-1 cells manifested a differential
requirement of SCF costimulation in media containing IL-5 or GM-CSF for
optimal proliferation.
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| Fig 1.
Proliferation synergism of SCF with IL-5 in TF-1 cells.
Rapid growing TF-1 cells were washed and cultured separately in
cytokine-free medium ( ), SCF (50 ng/mL,  ), GM-CSF (2 ng/mL,  ),
GM-CSF+SCF ( ), IL-5 (5 ng/mL,  ), or IL-5 + SCF
( )-containing medium. The viable cell numbers were determined daily
by trypan blue staining and are shown as the percentage of the initial
viable cell number. Each number represents the average from four
independent duplicated experiments. The standard deviations are
indicated as error bars.
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The synergy of SCF is partly due to its antiapoptosis activity.
We next explored whether the synergistic proliferation of TF-1 cells in
medium containing SCF and IL-5 was due to increased mitogenesis,
decreased apoptosis, or both. Because TF-1 cells manifested
differential growth rates in various culture media, we first examined
the possibility of alteration of cell cycle distribution among viable
cells in these culture conditions. Using BrdU-labeling and PI staining,
we clearly showed that there was no obvious alteration of cell cycle
distribution under all culture conditions examined
(Fig 2A). The percentages of cells in the G1, S, and G2/M phases were generally about
37% ± 2%, 52% ± 2%, and 9% ± 1%, respectively,
regardless of whether the cells were cultured in GM-CSF-, IL-5-,
SCF-, or IL-5 + SCF-containing medium, or cytokine-free medium (Fig
2A). We next investigated the DNA synthesis rate in an equal number of
viable cells grown under various culture conditions. As shown in Fig
2B, [3H]thymidine pulse-labeling assay showed that
although SCF promoted mitogenic activity (Fig 2B, column SCF) at a
level comparable to that achieved by IL-5 (Fig 2B, column IL5), neither
synergism nor addition of mitogenic activity was observed in TF-1 cells costimulated with SCF and IL-5 (Fig 2B, column IL5 + SCF). Therefore, the synergism of proliferation was not due to the increase of mitogenic
activity of TF-1 cells. On the other hand, SCF showed a dose-dependent
suppression of apoptosis in TF-1 cells in a DNA fragmentation assay
(data not shown). Interestingly, this suppression effect was never
complete when TF-1 cells were treated with SCF at concentration up to
50 ng/mL (Fig 3A, lane 6). IL-5 was
partially defective in suppression of apoptosis and a clear DNA ladder
could be observed in the DNA fragmentation assay (Fig 3A, lane 4). This defect was rescued by SCF (Fig 3A, lane 5). Consistent with these observations, a quantitative measurement of apoptosis by histone release assay further showed a synergistic antiapoptosis effect between
SCF and IL-5. As shown in Fig 3B, the apoptosis suppression activity of
IL-5 was about half of that of GM-CSF, and this suppression activity
was further suppressed when IL-5 was combined with SCF. When the
efficiency of apoptosis suppression by GM-CSF was set as 100% and that
under cytokine-free condition as 0%, IL-5 suppressed apoptosis with
about 52% efficiency (Fig 3B, column IL5). SCF suppressed apoptosis
with about 77% efficiency (Fig 3B, column SCF). When TF-1 cells were
costimulated with optimal concentrations of both SCF and IL-5,
apoptosis was further suppressed, up to 92% (Fig 3B, column IL5 + SCF). This synergism was highly reproducible and significant (P <<
.001). Although other factors cannot be excluded, our data suggested
that the synergism in proliferation could partially be contributed by
the synergism in antiapoptosis activity.
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| Fig 2.
IL-5 in combination with SCF had no additional mitogenic
activity. (A) Cell cycle distribution of TF-1 cells cultured in various
combinations of cytokines. TF-1 cells were cultured in the indicated
media as described in Fig 1 for 24 hours before BrdU-labeling and PI
staining as described in Materials and Methods. Similar numbers of
viable cells were analyzed for the percentages of cells in the G1, S,
and G2/M phases and results are shown within the panel. The cytokines
present in each culture medium are also indicated inside the panels.
The x-axis represents the intensity of PI fluorescence and the y-axis
represents the intensity of FITC-conjugated anti-BrdU antibody
fluorescence. These plots are a set of representative results and the
percentages of cells in various cell cycle stages are the averages from
four independent determinations. (B) DNA synthesis rates of TF-1 cells
cultured in various combinations of cytokines. TF-1 cells were cultured
in the indicated media for 48 hours and ten thousand viable cells were
subjected to [3H]thymidine incorporation assay to
determine the DNA synthesis rate. The data shown are the averages of
four independent triplicated experiments.
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| Fig 3.
Synergistic antiapoptotic activity of SCF and IL-5 in
TF-1 cells. (A) DNA fragmentation assay. The genomic DNA was prepared
from TF-1 cells treated with various combinations of cytokines as
indicated in the figure and was subjected to agarose gel
electrophoresis. The dose of cytokines used in these experiments was
identical to those used in Fig 1. (B) Histone releasing assay. TF-1
cells were cultivated in the indicated cytokines and the released
cytosolic contents were subjected to histone measurement according to
the manufacturer's instruction. Data shown are the averages of two
independent duplicated experiments. The difference between the values
in columns SCF and IL5 + SCF was statistically significant
(P<< .001). The relative antiapoptotic activities for cells
cultured in various conditions are referred to that of cells in
cytokine-free medium (set as 0%) and in GM-CSF-containing medium (set
as 100%) and are shown underneath the bar graph.
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High level expression of IL5R also results in apoptosis
suppression.
In our previous study we successfully isolated a variant of the TF-1
cell line, JYTF-1, which proliferated well in IL-5 and had an increased
expression of IL5R.12 One possible explanation for why
IL-5 cannot support the long-term growth of parental TF-1 cells is that
there may be a mutation in the IL5R gene that renders its gene
product nonresponsive to IL-5. In the present study, we ruled out this
possibility by sequencing the PCR-amplified cDNA from TF-1 cells. The
result showed that the sequence of IL5R in parental TF-1 cells was
identical to the published sequence of IL-5R (data not shown). Because
JYTF-1 cells had an increased expression of the IL5R gene, we
performed gene transfer experiments to determine whether the high-level
expression of IL5R rescues the antiapoptosis defect and confers the
phenotype of IL-5-dependent growth in parental TF-1 cells. A
retroviral expression plasmid of IL5R was constructed and
transferred into TF-1 cells by virus infection. Several stable
transfectants of IL5R were established after retroviral infection
and G418 selection. Surface overexpression of hIL5R protein was
further confirmed (Fig 4A) by indirect
immunofluorescence staining and flow cytometric analysis with a mouse
monoclonal anti-hIL5R antibody A14. TF-1 cells expressed the lowest
level of hIL5R on individual cells, JYTF-1 expressed about three
times more hIL5R, and both TF1 and TF8 cells expressed the
highest amount of hIL5R (about 10 times more). The expression levels of c were also checked with specific mouse monoclonal antihuman c
antibody and the results confirmed similar expression levels of c in
all cell lines (data not shown). The expression of virus-derived hIL5R messenger RNA (mRNA) was confirmed by Northern blot analysis (Fig 4B). A hybridization signal (~5 kb) that moved slightly faster than that of the endogenous full-length hIL5R mRNA (~6 kb) was observed in two representative transfectants, TF1 and TF8 (Fig 4B, vIL5R v IL5R). Quantitatively, TF-1 cells expressed
the lowest level of IL5R. JYTF-1 cells expressed about six times more IL5R than TF-1 cells, and TF1 and TF8 expressed 13 and 16 times more than TF-1 cells, respectively (densitometric analysis, data
not shown). The growth property of transfectants were then compared
with that of TF-1 and JYTF-1 cells in the presence or absence of IL-5.
In the presence of IL-5, TF1, and TF8 grew very well and the
growth rate was comparable to the same cells cultivated in medium
containing GM-CSF, whereas TF-1 cells in IL-5 only survived and JYTF-1
grew at a suboptimal rate (Fig 4C). The growth rates of TF8 cells
were not further stimulated when cells were cultured in a combination
of IL-5 and SCF (see below). These results strongly suggested that
high-level expression of IL5R ensured optimal proliferation of the
hematopoitic progenitor cell line in response to IL-5 in an
SCF-independent manner.
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| Fig 4.
Establishment of hIL5R overexpressing TF-1 cells. (A)
Surface expression of hIL5R protein in TF-1, JYTF-1, and hIL5R
transfectants TF1 and TF8 cells. The target cells were cultured
in IL-5 for 24 hours and then subjected to flow cytometric analysis as
described in Materials and Methods. The primary antibody was either a
control mouse IgG (dashed line) or a mouse monoclonal anti-hIL5R
antibody (solid line). The X-axis indicates the viable cell number and
the Y-axis shows the fluorescence intensity. (B) Expression of the
virus-derived IL5R mRNA in TF1 and TF8 cells. Total RNA was
isolated from the indicated cell lines and 20 µg of RNA was analyzed
by Northern blot hybridization. Probes used were cDNA-specific for the
IL5R gene (upper panel) or for the glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) gene (lower panel), which were used as internal
controls for the loading control. The positions of the endogenous
full-length IL5R mRNA (IL5R) and the virus-encoded IL5R mRNA
(v IL5R) are indicated at the right-hand side of the figure. (C)
Distinct growth curve of TF-1, JYTF-1, TF1, and TF8 cells in
IL-5. Cells were grown in the absence of cytokine () or in the
presence of GM-CSF () or IL-5 () at densities of 5 × 105 cells/mL and 1 × 105 cells/mL (GM-CSF and
IL-5), respectively. Viable cell number was counted at 0 hours, 24 hours (day 1), and 48 hours (day 2) after seeding and was converted
into the percentage of initial cell number (at day 0) as shown on the
left-hand side of each plot. Each value is the average of four
independent measurements.
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Pulse labeling of TF1 and TF8 cells with
[3H]thymidine showed that levels of DNA synthesis
activity were similar to levels in TF-1 and JYTF-1 cells in the
presence of IL-5 (Fig 5). These numbers
were about twofolds of the levels of DNA synthesis activity in the
absence of cytokine. These results further suggested that the mechanism
leading to optimal growth effect of IL-5 was not due to enhanced
mitogenesis. We then examined if the enhanced growth of TF1 and
TF8 in IL-5 was due to decreased apoptosis. To address this issue,
three distinct methods were employed to assess the degree of the
apoptosis, including (1) micrographic analysis
(Fig 6A), (2) histone-releasing analysis
(Fig 6B), and (3) DNA fragmentation analysis (Fig 6C). As shown in Fig
6, all three assays showed that TF-1 cells were more prone to undergo apoptosis in IL-5-containing media (21% in Fig 6A) than cells overexpressing IL5R, including JYTF-1 (6% in Fig 6A), TF1 (2%, Fig 6A), and TF8 cells (2%, Fig 6A). Therefore, we concluded that
overexpression of IL5R correlated very well with the improvement of
antiapoptotic activity of TF-1 cells in IL-5-containing medium, rather
than with the increase of mitogenic activity.
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| Fig 5.
Overexpression of IL5R does not increase the mitogenic
activity of IL-5. Rapid growing TF-1, JYTF-1, TF1, and TF8 cells
were cultured in IL-5-containing ( ) or cytokine-free () media for
24 hours and then 1 × 104 viable cells were subjected to
[3H]thymidine pulse-labeling assay as described in
Materials and Methods. For each sample, data shown is the average of 13 to 17 independent measurements (n).
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| Fig 6.
Suppression of apoptosis by overexpression of IL5R in
TF-1 cells. (A) Microscopic measurement. Cells were grown in indicated
culture media for 24 hours and the percentage of apoptotic cells in the
whole population was calculated from the photomicrographs (see
Materials and Methods). Each number represents the average of two
independent measurements. For each measurement, 300 cells were counted
in cytokine-free cultures (); 500 cells were counted in GM-CSF-
() and IL-5-containing ( ) medium. (B) Histone-releasing assay.
Cells were treated as described in (A), and were lysed to release the
cytoplasmic contents. The bound nucleosomes were detected with ELISA as
described in Materials and Methods. A representative set of data of
three independent duplicated experiments is shown and each number is
the average of two measurements. (C) DNA fragmentation analysis. Cells
grown in the cultures as described in (A) were harvested and the
genomic DNA was prepared and analyzed as described in Fig 3A and
Materials and Methods. A molecular weight marker (1 kb ladder from Life
Technology, Gaithersburg, MD) is loaded at the left-hand side of the
figure.
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Oncogenic protein Bcl-2 also supports the optimal proliferation of
progenitor cells.
The Bcl-2 oncogene, first identified in human follicular
lymphomas18,19 and implicated in the formation and
therapeutic resistance of numerous human tumors, is a powerful
antiapoptosis gene.20-22 We were interested in determining
whether the role of SCF in antiapoptosis could be replaced by the
overexpression of Bcl-2 oncoprotein. A pool of mixed TF-1 stable clones
overexpressing human Bcl-2 oncoprotein was established by retroviral
infection as previously described13 and was further
analyzed in the present study. As shown in
Fig 7, panel A, Bcl-2 protein was
dramatically overexpressed in TF/Bcl-2 cells compared with both TF-1
and JYTF-1 cells (lanes 1, 2,and 3). Cytokine-withdrawal-induced
apoptosis was dramatically blocked by Bcl-2 oncoprotein in TF/Bcl-2
cells according to DNA fragmentation,19 morphological
observation (data not shown), and viable cell counting (Fig 7B). These
cells showed a significant proliferation response to IL-5 that was
comparable to the proliferation activity obtained by SCF costimulation
or IL5R overexpression (Fig 1 and Fig 4C). In contrast, the
proliferation response of TF/Bcl-2 cells in the GM-CSF-containing
medium remained unchanged compared with that of TF-1 cells (Fig 7B
v Fig 1). Interestingly, SCF did not show synergistic
proliferation when costimulated with IL-5 in either TF8 (Fig 7C) or
TF/Bcl-2 cells (Fig 7C). These data further suggested that the
mechanism of SCF costimulation with IL-5 in TF-1 cells and the effect
of IL5R overexpression were mainly due to suppression of apoptosis.
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| Fig 7.
Overexpression of Bcl-2 oncoprotein rescues the
proliferation of TF-1 cells in IL-5. (A) Overexpression of Bcl-2
protein in TF/Bcl-2 cells. Two hundred and fifty micrograms of protein
lysates from each indicated cell line were analyzed by Western blot for
the expression of Bcl-2, Bax, and -tubulin proteins (indicated at
the right-hand side of the figure). (B) The growth curves of TF/Bcl-2
cells in the indicated culture media. TF/Bcl-2 cells were grown in
media as indicated and the viable cell numbers were determined at each
time point by trypan blue staining. (C) TF/Bcl-2 and TF8 cells were
refractory to SCF stimulation in IL-5-containing medium. TF-1, TF8,
and TF/Bcl-2 cells were treated with IL-5 alone (), SCF alone ( ),
or IL-5 + SCF () and the numbers of viable cells were determined
by trypan blue staining at different time points. The dose of cytokines
was the same as described in Fig 1. The results were the averages from
three duplicated experiments. The error bars represent the standard
deviations.
|
|
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DISCUSSION |
In this study, by taking advantage of a human leukemic cell line, we
explored the relationship between the differential expression of
cytokine receptors and the differential requirement of costimulation for proliferation of cells in the early differentiation stage of
hematopoiesis. The TF-1 cell line we used may represent a clonal cell
population at the committed multilineage progenitor stage. It expressed
the markers and receptors specific for stem and progenitor cells at
high levels, including CD34,10 c-kit,11
erythropoietin receptor,23 IL-3 receptor subunit,
common receptor subunit, and GM-CSF receptor subunit.12 However, the IL-5 receptor subunit was
expressed only at a low level.12 Low-level expression of
IL5R may be a characteristic of these multilineage progenitor cells
wherein costimulation of SCF and IL-5 is usually necessary for
eosinophil colony formation in vitro.
We showed in this study that differential expression levels of the IL-5
receptor have a tight relationship with the antiapoptosis ability of
IL-5. Cells like TF-1 having low numbers of IL5R are intrinsically
defective in antiapoptosis ability and the underlying mechanism remains
to be investigated. Although SCF itself had some mitogenic activity in
TF-1 cells, it did not exert any additive or synergistic mitogenic
activity with IL-5 in TF-1 cells (Fig 2). Instead, SCF played a
significant role in synergizing with IL-5 in the antiapoptotic activity
(Fig 3C). However, SCF was not always required for IL-5 to achieve its
optimal proliferation. When IL5R was overexpressed, SCF was no
longer effective in synergizing with IL-5 and was not required for
optimal proliferation (Fig 7C). Therefore, according to our results,
the requirement of SCF for optimal proliferation is largely dependent
on the quality of IL-5 signals.
These results are consistent with recent observations in other studies
of the function of cytokine receptors.6,7 In early studies
many cytokines were shown to require the SCF costimulation to achieve
maximum efficacy for blast cell growth in in vitro colony formation
assays. However, when receptors of these cytokines were molecularly
cloned and highly expressed in surrogate factor-dependent cell lines,
most receptors achieved maximal growth effect by their cognate cytokine
alone.6,7,24 There was no evidence in previous studies that
costimulation with SCF was absolutely required for the function of any
specific cytokine receptor. Whether or not the expression levels of
these cytokine receptors were low and hence there was a deficiency in
the activity of cell death suppression in the purified stem cell
population remains to be investigated.
It has been shown that in the presence of SCF, IL-3 at concentrations
below ED50 could stimulate growth similar to that of IL-3
alone at the saturation dose in an IL-3-dependent cell line that
highly expressed IL-3 receptors. The mitogenic and antiapoptotic effects of IL-5 were uncoupled in TF-1 cells, and it was not clear whether this uncoupling also occurred when IL-3 concentration went
below ED50. Nevertheless, according to our and others data, SCF was effective in complementing the defects caused by either low
levels of receptors or low levels of cytokines. These observations may
have significance in suggesting the functions of SCF in vivo. Previous
studies showed that mice with Steel or Wv mutations that
lack the ability either to produce SCF or SCF receptors have much less
response to G-CSF in stimulating granulopoiesis and in elevating
progenitor cells.25 Therefore, the role of SCF in
preventing the apoptosis of the early hematopoietic progenitor cells
may be an important function of hematopoiesis.
Predisposition of Bcl-2 overexpression in transgenic animals has been
shown to increase the chance of occurrence of lymphoproliferative disorders due to its potent antiapoptosis activity.26-28
Recently, high level expression of Bcl-2 proteins was also observed in
many human tumors, including cholangiocarcinoma,29
endometrial carcinoma,30 acute myeloid leukemia
(AML),31 solitary fibrous tumor,32 and
medullary thyroid tumor.33 Our demonstration of replacing SCF with Bcl-2 expression in Fig 7 had two major implications. First,
it re-emphasized the notion that antiapoptosis is one of the major
contributions of SCF costimulation. Second, it indicated that the
Bcl-2-transduced cells became immortal and had growth advantages over
normal progenitor cells in the absence of cytokines. These Bcl-2
overexpressing cells were not factor-independent, however, they
survived for a longer period of time and re-entered proliferation
rapidly in the presence of cytokines. It is known that some primary
leukemia cells from patients with AML are dependent on exogenous growth
factors.34 The mechanism involved in this phenomenon
remains to be investigated, and it will be interesting to explore the
correlation between survival response and the expression of Bcl-2
protein in these leukemia cells.
Strong suppression of apoptosis by exogenous overexpression of Bcl-2 in
TF-1 cells may suggest an important role for Bcl-2 in the regulation of
cytokine-withdrawal induced apoptosis. However, the regulation of
endogenous Bcl-2 cannot explain the mechanism of
cytokine-withdrawal-induced apoptosis or apoptosis in IL-5-containing medium (unpublished data). Instead, another Bcl-2 member, Mcl-1, showed
a nice correlation between expression level and the occurrence of
apoptosis (unpublished data and Chao et al13) in several cytokine-dependent cell lines. Further study on the regulation of the
Mcl-1 gene and the signal transduction of IL-5 in TF-1 cells should
shed light on the antiapoptosis signaling pathways of cytokines in
hematopoietic cells.
| |
ACKNOWLEDGMENT |
The authors thank Dr Charles Shih (PharMingen) for the gift of
antihuman IL-5R antibody, and Dr Mi-Hua Tao for his careful reading of
this manuscript and helpful suggestions.
| |
FOOTNOTES |
Submitted August 21, 1998; accepted December 10, 1998.
Supported by grants from both Academia Sinica and the National Science
Council of Taiwan, NSC85-2331-B-001-007 to J.J.Y.Y.
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.
Address correspondence to Dr Jeffrey Jong-Young Yen, Institute of
Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan; e-mail:
bmjyen{at}novell.ibms.sinica.edu.tw.
| |
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ABSTRACT
INTRODUCTION