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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1548-1555
Both Stroma and Stem Cell Factor Maintain Long-Term Growth of ELM
Erythroleukemia Cells, but Only Stroma Prevents Erythroid
Differentiation in Response to Erythropoietin and Interleukin-3
By
Jim O'Prey,
Nick Leslie,
Katsukiko Itoh,
Wolfram Ostertag,
Chris Bartholomew, and
Paul R. Harrison
From the Beatson Institute for Cancer Research, CRC Beatson
Laboratories, Bearsden, Glasgow; the Department of Clinical Medical
Biology, Kyoto University, Kyoto, Japan; and Heinrich Pette Institut
for Experimental Virology and Immunology, Hamburg University, Hamburg,
Germany.
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ABSTRACT |
Defining how the stromal requirements of hematopoietic progenitors
change during leukemia progression is an important topic that is not
well understood at present. The murine ELM erythroleukemia is an
interesting model because the erythroid progenitors retain dependence
on bone marrow-derived stromal cells for long-term growth in vitro, and
they also undergo erythroid differentiation in the presence of
erythropoietin (EPO) and interleukin-3 (IL-3). In this report, we have
shown using neutralizing antibodies that stem cell factor (SCF),
insulin-like growth factor (IGF)-1, and integrin signaling
pathways are all involved. We then determined whether ELM cells can be
maintained long-term without stroma in various combinations of growth
factors produced by stroma cells or growth factors for which ELM cells
have receptors. This showed that ELM cells could be maintained with
high efficiency in SCF alone; furthermore, the cells remained
absolutely SCF-dependent and did not become more tumorigenic than cells
maintained on stroma. In contrast, ELM cells underwent clonal
extinction when serially cloned in IGF1; any cells that survived
long-term growth in IGF-1 were found to be IGF1-independent. One
important difference between maintaining ELM cells on stroma and growth
in SCF is that stroma reversibly inhibits their differentiation in
response to EPO and IL-3, whereas SCF does not.
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INTRODUCTION |
STROMAL CELL interactions are known to be
important for maintenance and growth of hematopoietic stem cells and
progenitors in vivo.1,2 Defining the stromal signals
involved is therefore important both for understanding the regulation
of hematopoietic cell homeostasis and, practically, for devising
methods for ex vivo amplification of the different hematopoietic cell
compartments for transplantation and protection against hematopoietic
failure after chemotherapy. Perhaps the best characterized system is
that of T-cell production, where activation of T cells by
antigen-presenting cells requires activation of the T-cell receptor in
conjunction with integrin signaling.3-5 In the erythroid
lineage, the importance of the stem cell factor (SCF) receptor/SCF
interaction has been demonstrated by the anemia-inducing effects of the
W and Sl mutations in mice, affecting SCF
receptor6 and SCF7 expression, respectively. In
contrast, many leukemic cells, in particular, most murine
retrovirus-induced erythroleukemias, have lost the stromal requirement
of their normal counterparts and grow readily in suspension culture,
often without added growth factors other than those present in serum;
they are also usually arrested in differentiation and no longer
responsive to erythropoietin (EPO) due to activation of the EPO
receptor by the retrovirus-encoded gp55 glycoprotein.8-10
The murine ELM erythroleukemia model is unusual in having a less
abnormal leukemic phenotype. We have shown, for example, that the cells
will not grow in the regular suspension culture medium suitable for
other erythroleukemia cells, but require direct contact with
marrow-derived stromal cells for long-term growth in
vitro.11-14 ELM cells also undergo erythroid
differentiation in response to EPO and interleukin-3
(IL-3).15 Moreover, stroma independent variants can be
obtained and we have shown that these have elevated expression of
members of the ets family of transcription factors/oncogenes.16 The aim of the present work was to
identify the stroma signaling processes that are critical for long-term ELM cell growth and determine whether these could be substituted by a
combination of soluble growth factors for which ELM cells have
receptors in conjunction with extracellular matrix (ECM) components.
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MATERIALS AND METHODS |
Cells.
ELM cells12 were grown on unirradiated or irradiated
(60 Gy) MS5 cells13 or the
Sl-/Sl- stromal cell line, UNC,17
kindly provided by Dr C. Eaves (Terry Fox Laboratory, British Columbia
Cancer Agency, Vancouver, Canada). Stroma and ELM cells were routinely
passaged in alpha minimal essential medium ( -MEM;
GIBCO, Paisley, Scotland) containing 8% horse serum
(Sigma, Poole, Dorset, UK) and 4 mmol/L glutamine. In some
cases, cells were grown in protein-free hybridoma medium (GIBCO; cat.
No. 12040-051).
Growth factors.
All murine (SCF, IL-3, granulocyte-macrophage colony-stimulating factor
[GM-CSF], colony-stimulating factor [CSF]-1, and IL-6) and human (IGF-1, IGF-2, and IL-11) recombinant growth factors were
obtained from R & D Systems (Abingdon, UK), except for
insulin, which was obtained from Sigma. Optimal concentrations of
growth factors were determined in short-term proliferation assays. A total of 104 ELM cells were freshly removed from stroma and
incubated in microtiter wells with increasing concentrations of growth
factor and 5 days later proliferation was assayed using the Promega MTT
kit (Promega, Madison, WI). The concentrations of growth
factors used in all other experiments were the minimum found to give
the optimal stimulation of growth in these assays (SCF, 10 ng/mL;
IGF-1, 100 ng/mL; insulin, 10 µg/mL; IGF-2, 100 ng/mL; IL-3, 120 U/mL). Other growth factors were used at the following
concentrations: GM-CSF, 1 ng/mL; CSF-1, 10 ng/mL; IL-6, 1 ng/mL; IL-11,
1 ng/mL.
Removal of ELM cells from MS5 stromal cells.
Fresh medium was added to flasks containing ELM cells growing on MS5
feeder layers and the flasks were shaken vigorously for a few seconds.
Most of the ELM cells became detached from the stromal layer and were
decanted into a fresh flask. To remove any contaminating MS5 cells, the
cells were allowed to settle for 30 to 60 minutes, when any residual
MS5 cells adhered to the plastic, whereas the ELM cells remained
unattached. To ensure removal of all MS5 cells, this procedure was
repeated two to three times. To check for contamination of ELM cells
with MS5 stromal cells, purified ELM cells were plated out in a flask
and the numbers of stromal cell colonies counted 2 weeks
later. Stromal cell colonies are unmistakably different from ELM cells
in morphology. Typically, 106 purified ELM cells gave 10 to
20 stromal cell colonies in this assay. In some experiments, ELM cells
were passaged on MS5 cells that had been lethally irradiated (60 Gy
Co60) to ensure that pure populations of ELM cells had been
obtained.
Clone-serial transfer experiments.
ELM cells were cloned on irradiated MS5 cells by end-point dilution in
96-well plates and after 2 to 3 weeks when the colonies contained about
105 cells, they were serially passaged 1:10 in microtiter
plates at weekly intervals in medium containing various growth factors.
Clone-clone transfer experiments.
ELM cells growing in culture were removed from stroma as described
above, centrifuged, and resuspended in fresh medium. The cells were
cloned by end-point dilution every 21 days on irradiated stroma or in medium containing growth factors.
Antibody inhibition experiments.
The anti-IGF-1 and anti-integrin antibodies were obtained from
Pharmingen (San Diego, CA). The anti-SCF-receptor
antibody (ACK2) has been described previously.18 ELM cells
were plated out on MS5 cells in wells in Terasaki plates (100 ELM and
200 MS5 in 20 µL medium) and incubated with antibody (30 µg/mL).
Plates were incubated for 5 days at 37°C, 5% CO2 in a
humidified incubator and the number of ELM cells counted under a light
microscope. Cell counts were averaged from at least eight wells for
each sample.
Preparation of extracellular matrix (ECM) in situ.
ECM was prepared using the method described by Scott-Burden et
al.19 Briefly, MS5 cells were plated out in
flasks or microtiter plates and allowed to grow for 3 to 5 days until
they became confluent. The cells were then washed twice with
phosphate-buffered saline (PBS), once with water, and the cells were
then lysed in 25 mmol/L NH4OH for 5 minutes at 4°C. The
plates were washed three times with PBS to remove any cell debris and
washed once with culture medium before plating of cells.
Tumorigenicity experiments.
ELM cells were injected via the tail vein into groups of 15 previously
irradiated (3 Gy Co60) C3H mice. Mice were checked twice
daily and the experiment terminated (usually after about 15 days) when
the first signs of ill health were apparent due to enlarged spleens.
Spleens were then dissected and individually weighed.
Detection of IGF-1 receptors by IGF-1 binding.
The IGF-1 binding assay was performed as described
previously,20 with modifications. Binding of IGF-1 to cells
was performed in suspension. A total of 5 × 105 cells
were incubated with 50,000 cpm 125I IGF-1 (Amersham, Little
Chalfont, UK) and increasing concentrations of unlabeled
IGF-1 (0 to 1,000 ng/mL) in a final volume of 0.25 mL of binding medium
(-MEM, 25 mmol/L [4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid] pH8.0, 0.1% bovine serum albumin) at 4°C for 18 hours.
Cells were washed twice in binding medium and cell-associated
125I IGF-1 counted in a gamma counter. Nonspecific binding
was assessed in the presence of unlabeled IGF-1 at 1,000 ng/mL.
Scatchard analysis was performed using the Cricket Graph computer
program (Computer Associates International, Islandia,
NY).
Northern analysis.
RNA was extracted using Trizol (GIBCO) and analyzed by Northern
transfer and hydridization with radioactive probes as described previously.16
Western blotting.
Activation of mitogen-activated protein (MAP) kinases was
assayed by Western blotting of whole cell lysates using a polyclonal antibody (Promega) with specificity for active, dually phosphorylated forms of MAP kinases (p44/p42 [ERK1/ERK2]). IGF-1-receptor levels were determined by Western blotting with a polyclonal antibody against
the IGF-1 receptor  subunit (Santa Cruz, Santa Cruz, CA). The manufacturer's instructions were followed for
preparation of lysates and Western blotting. Cells were lysed in 50 mmol/L Tris pH 6.8, 100 mmol/L dithiothreitol, 2% sodium dodecyl
sulfate (SDS), 0.1% bromophenol blue, 10% glycerol, fractionated by
SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto ECL
nitrocellulose membrane (Amersham). Immunodetection was performed by
enhanced chemiluminescence (ECL, Amersham).
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RESULTS |
Our previous studies have shown that the murine stromal cell line, MS5,
will support long-term growth of the stroma-dependent ELM cells,
whereas in the absence of stromal cells, ELM cells die within 7 to 14 days.14 To ensure that pure populations of ELM cells could
be obtained from the cocultures, the MS5 cells were normally lethally
irradiated before use. To determine which growth factor signaling
pathways might be involved in growth of ELM cells on stroma, we tried
to grow ELM cells without stroma in the presence of growth factors for
which ELM cells have receptors, or with growth factors produced by the
stromal cells, as judged by semiquantitative reverse
transcriptase-polymerase chain reaction (RT-PCR) experiments
(summarized in Table 1). Because the
interaction of ELM cells with extracellular matrix components may also
be involved, we determined by histocytochemistry which integrins are
expressed on the surface of the cells. Integrin 41 was present on
ELM cells and 51 was detected on MS5 stroma cells, whereas M, L, and 2 were absent
(data not shown).
Short-term growth of ELM cells in growth factors and extracellular
matrix.
We then measured the growth of ELM cells, after removal from stroma
cells, in each of the candidate growth factors (IL-6, CSF-1, IGF-1,
IGF-2, SCF, insulin, IL-3, GM-CSF, IL-11, and EPO), using a range of
growth factor concentrations in short-term proliferation (MTT) assays. In serum-containing medium, the only growth
factors that allowed ELM cells to proliferate over a 5-day period were SCF, IGF-1, and insulin (Fig 1A) (and to a
lesser extent IGF-2 and IL-3, data not shown). However, it should be
noted that the level of insulin required to support ELM growth was
100-fold higher than the level of IGF-1 required, suggesting that the
IGF-1 receptor is more important than the insulin receptor in
supporting ELM growth by IGF-1 or insulin. It is well established that
insulin can bind to the IGF-1 receptor with
low-affinity.20,21 The same growth factors also stimulated
ELM growth in serum-free medium for 5 days (Fig 1B, data for insulin
not shown). Addition of 4% serum without extra growth factors
stimulated ELM growth to a limited extent (Fig 1B); this is probably
mainly due to IGF-1 because neutralizing antibodies to IGF-1 (I1Ab)
reduced growth of ELM cells in serum alone, whereas antibodies against
the SCF-R (ACK2) had minimal effect (Fig 1B). The antibodies were
specific as shown by the fact that each antibody only reduced the
growth of ELM cells in serum-free medium stimulated by the growth
factor against which the antibody was raised (Fig 1B). The effect of ECM was tested by preparing ECM in situ from stroma cells growing in
the assay wells before adding the ELM cells. However, ECM did not
stimulate proliferation or enhance proliferation of ELM cells by any of
the growth factors (data not shown).
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| Fig 1.
Growth factor requirements for short-term growth of ELM
cells in the presence or absence of stroma. (A) Effect of growth
factors on the growth of ELM cells in serum-containing medium after
removal from MS5 stroma cells. A total of 104 ELM cells
were plated in 96-well plates in medium containing 8% horse serum in
the presence of increasing amounts of IGF-1, insulin, or SCF.
Proliferation under these conditions was assayed by the MTT method and
data shown are mean values from three independent experiments (12 cultures) with standard errors. The data shown are for the minimum
amounts of growth factors found to give the maximal stimulation (SCF,
10 ng/mL; IGF-1, 100 ng/mL; insulin, 10 µg/mL). (B) Effect of growth
factors and neutralizing antibodies on the growth of ELM cells in
serum-free medium or 4% serum. A total of 104 ELM cells
were plated in 96-well plates in serum-free medium in the presence of
100 ng/mL IGF-1 or 10 ng/mL SCF and 30 µg of neutralizing antibodies
to IGF-1 (I1Ab) or the SCF-R (ACK2). Proliferation under these
conditions was assayed by the MTT method and data shown are mean values
from three independent experiments (12 cultures) with standard errors.
(C) Effect of antibodies against IGF-1, SCF, or integrins on the growth
of ELM cells on MS5 stroma cells. A total of 100 ELM cells were plated
on 200 MS5 cells in wells in Terasaki plates and incubated for 5 days
in the presence of 30 µg/mL neutralizing antibodies to SCF-R (ACK-2),
IGF-1 (I1Ab), or 4 integrin (4Ab). The right-hand panel shows the
growth of ELM cells without stroma. The results are the number of cells in the wells relative to the number on stroma alone and represent the
mean of three independent experiments (24 cultures) with standard errors.
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Signaling pathways required for proliferation of ELM cells on stromal
cells.
To determine whether any of the growth factors shown to stimulate
short-term proliferation of ELM cells in the absence of stroma are
important for their growth on stroma, the effects of neutralizing
antibodies to the SCF-R, IGF-1, or integrin 41 were determined on
ELM growth in cocultures with stroma cells. Antibodies against IGF-1 or
4 integrin each reduced the growth of ELM cells after 5 days in
culture on stroma cells by about 50%. Antibodies against the SCF-R had
a slightly greater effect (about 60% reduction) (Fig 1C), as we have
previously shown.14 The use of antibodies to IGF-1 and
SCF-R together reduced the growth of ELM cells below the level found in
the absence of stroma cells (Fig 1C). These results imply that
integrin, SCF, and IGF-1 signaling pathways are required for optimal
growth of ELM cells on stromal cells.
Long-term growth of ELM cells in the absence of stromal cells.
To try to determine whether any combination of growth factors could
substitute for stroma in maintaining long-term growth of ELM cells, we
first grew ELM cells on extracellular matrix prepared from stroma cells
in a complex mixture of growth factors. To allow for any synergistic
interactions between growth factors, the nine growth factors selected
(IGF-1, IGF-2, insulin, SCF, GM-CSF, CSF-1, IL-3, IL-6, IL-11) were
those to which ELM cells had shown a proliferative response in
short-term assays plus any other growth factors for which ELM cells
have receptors, even if they did not respond to the growth factor on
its own. The concentrations of growth factors used were the minimum
amounts found to give the optimal stimulation of growth in short-term
assays when used individually (see Materials and Methods) or, in the
case of growth factors that did not stimulate ELM growth individually,
the concentrations shown to be optimal for growth of other cell types
(details in the Materials and Methods). Initially, 30 clones of ELM
cells were isolated on irradiated stroma cells by end-point dilution in
microtiter plates. When the clones had reached 104 to
105 cells in size, they were diluted and one tenth of each
clone was tested for growth under different conditions by weekly 1:10 serial passage, either on irradiated stromal cells or in the mixture of
the nine growth factors in wells containing extracellular matrix from
MS5 cells. We found that ELM cells could be maintained indefinitely (>200 days) under both of these conditions. During this period, the
cells remained dependent on the growth factor/ECM mix for long-term
growth, as removal of the growth factors at day 15, 50, and 70 resulted
in the rapid death of the cells (Fig 2A). We also attempted to grow ELM cells long-term in the mixture of growth
factors in serum-free medium (protein-free hybridoma medium), but the
cells died after about 5 weeks (data not shown).
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| Fig 2.
Long-term growth of ELM cells in growth factors. A total
of 30 clones of ELM cells were isolated as described in Materials and
Methods and transferred to wells in 96-well microtiter plates containing various growth factors and/or ECM and passaged 1:10 weekly. The viability of the clones was recorded before each passage. (A) ELM clones were transferred to wells preplated with MS5 ECM and a
cocktail of growth factors added (IGF-1, IGF-2, insulin, SCF, IL-3,
IL-6, IL-11, GM-CSF, and CSF1) ( ). At 15 days, 50 days, and 70 days
(arrows), growth factors and ECM were removed and the effect on
viability of the clones monitored ( ). (B) A total of 30 ELM clones
were passaged in a single growth factor (IGF-1 ( ), IGF-2 ( ),
insulin ( ), SCF (), and IL-3 (crosses)) and clone viability
monitored. (C) After clones had been maintained for 45 days in
IGF-1 (), insulin (), SCF (), and IGF-1+SCF () as
described in (B), they were either passaged 1:10 weekly in the same
growth factors until day 155 (solid lines) or in their absence (broken
lines).
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We then repeated the experiment using a less complex mixture of growth
factors comprising only the five growth factors to which ELM cells
respond in short-term proliferative assays (IGF-1, IGF-2, insulin, SCF,
and IL-3). In fact, the ELM clones maintained in the growth factors and
ECM grew almost as well as those maintained on stromal cells, and the
removal of ECM made little difference (data not shown).
To determine whether any of the growth factors used alone would permit
long-term growth of the cells in the absence of stroma, another set of
30 ELM clones was serially passaged for 155 days individually in IGF-1,
IGF-2, insulin, SCF, or IL-3 (Fig 2B). A total of 90% to 100% of the
clones passaged in SCF, insulin, or IGF-1 survived long-term (Fig 2B,
filled squares, open triangles, and open diamonds, respectively).
However, clones passaged in IL-3 (Fig 2B, crosses) or IGF-2 (Fig 2B,
filled triangles) died within 2 weeks, as did clones in medium
containing no growth factors (data not shown). To test whether the ELM
cells grown in SCF, IGF-1, or both remained dependent on these growth
factors, after ELM cell clones had been grown for 45 days in a
particular growth factor, the growth factor was removed and the cells
were serially passaged in its absence. This indicated that ELM cells
previously grown for 45 days in SCF (Fig 2C, filled squares) and
SCF+IGF-1 (Fig 2C, open squares) died rapidly upon removal of growth
factor(s) (broken lines). However, surprisingly, most of the clones
grown in IGF-1 (Fig 2C, open diamonds/broken line) or insulin (Fig 2C, open triangles/broken line) continued to proliferate after growth factor removal.
Cloning efficiency of ELM cells in SCF and IGF-1 compared with
stroma.
To evaluate more rigorously the ability of SCF to maintain ELM cells
and to elucidate how IGF-1-independent cells arise after long-term
growth in IGF1, we measured the cloning efficiencies of ELM cells were
serially recloned in either IGF-1 or SCF. ELM cells serially cloned in
SCF with high efficiency and remained SCF-dependent through three
rounds of cloning (Fig 3). However, ELM
cells from the third round of serial cloning in SCF were found to have
lost responsiveness to IGF-1, in contrast to ELM cells freshly removed
from stroma (Fig 3). The same change was also found in ELM cells that
were serially passaged in mass culture for several months in SCF or SCF
plus IGF-1 (data not shown). The reasons for this change in IGF-1
responsiveness are unknown, but it is not due to downregulation of
IGF-1-receptor levels or affinities (see below). As far as serial
cloning in IGF-1 is concerned, although ELM cells cloned with about
half the cloning efficiency as in SCF in the first round of cloning
(Fig 3), the cloning efficiency of the cells after two rounds of
cloning in IGF-1 declined dramatically (Fig 3). These results imply
that SCF is the only growth factor that, by itself, can support
indefinite growth or serial recloning of ELM cells.
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| Fig 3.
Cloning efficiency of ELM cells repeatedly cloned in SCF
or IGF-1. ELM cells cloned on stroma (30 clones) were cloned in 10 ng/mL SCF, 100 ng/mL IGF-1, or no growth factors and the cloning efficiency measured. Five clones surviving in each condition were then
picked and recloned again in the same growth factors. In the case of
cells cloned twice in SCF, the cloning in SCF or IGF-1 or no growth
factors was repeated a third time.
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The importance of SCF for long-term maintenance of ELM cells was
confirmed by cloning on SCF-deficient Sl/Sl stroma. Any clones that formed tended to be very small and overall cloning efficiency was
much lower than on normal stroma, unless SCF was added
(Table 2). Moreover, ELM cells surviving
long-term on Sl/Sl stroma became stroma-independent, and yet
they did not clone in SCF (Table 2). These altered
properties resemble those of ELM cells that survived long-term
selection in IGF-1. ELM cells also clone with only very low efficiency
on Sl/Sld stroma, which expresses only the soluble
form of SCF (data not shown). We presume this is because the cells do
not produce sufficient SCF to permit optimal growth/cloning of ELM
cells.
Mechanisms of IGF-1-independent growth.
To determine whether constitutive activation of a mitogenic signaling
pathway was responsible for the IGF-1-independence of ELM clones
obtained by continuous growth in IGF-1, we determined whether MAP
kinases were activated in serum-treated or serum-starved cells using
antibodies that recognize the activated forms of ERK1 and ERK2. The
results (Fig 4A) indicated that three
IGF-1-independent ELM clones (like two stroma-independent clones, I/1
and I/2, isolated previously16) contained readily
detectable levels of activated ERK2 (and to a lesser extent ERK1) when
assayed in the absence of serum, in contrast to ELM cells freshly
removed from stroma.
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| Fig 4.
Levels of activated MAP kinase, IGF-1 receptors, and
IGF-1 receptor mRNA in ELM cells grown long-term on stroma or in SCF or
IGF-1. (A) Phosphorylation status of MAP kinase (ERK1) in three IGF-1-selected clones (20.1b, 20.2b, and 20.2g) or two
stroma-independent variants isolated previously.16 Cells
were starved in 0.5% serum medium for 6 hours before analysis. As a
positive control, serum-starved ELM cells were stimulated with 20%
serum for 10 minutes. Activated ERK1 and ERK2 were determined by
Western blotting with antibodies against the activated form of MAP
kinase (top panel) or with antibody against total MAP kinase as a
loading control (bottom panel). (B) Levels of IGF-1 receptors in ELM
clones isolated by long-term growth in IGF-1, determined by Western
blotting. (C) Numbers and affinities of IGF receptors in ELM clones
selected by long-term growth in SCF or IGF-1, compared with ELM cells
growing on stroma, as determined by IGF-1-binding studies. Top panel,
typical Scatchard plots for ELM cells freshly removed from stroma, and
two ELM clones maintained long-term in either IGF-1 (20.2B) or SCF
(20.1D). Bottom panel, average numbers of receptors/cell and affinities
(Kd) calculated from the Scatchard plots of two separate
experiments. (D) Levels of IGF mRNA by RT-PCR.
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One obvious mechanism whereby growth of ELM cells in IGF-1 could result
in outgrowth of IGF-1-independent cells might be overexpression of
IGF-1 receptors or autocrine production of IGF-1. However, IGF-1-selected ELM cells have the same levels of receptors, whether assayed by Western blotting (Fig 4B) or IGF-1-binding studies (Fig
4C). Scatchard analysis of the binding studies also shows that the
affinities of binding of IGF-1 to its receptor is approximately the
same in ELM clones isolated on stroma, in SCF, or in IGF-1 (Fig 4C).
This suggests that IGF-1-independent clones do not survive because the
IGF-1 receptor has become more sensitive to traces of IGF-1 in the
serum. Another explanation for the generation of IGF-1-independent
cells might be that the ELM cells grown long-term in IGF-1 are induced
to produce IGF-1 constitutively. However, this does not appear to be
the case, as judged by measurement of IGF-1 mRNA levels by RT-PCR (Fig
4D) or Northern analysis (data not shown). Other experiments have shown
that IGF-1- or stroma-independent clones do not produce SCF (data not
shown). At present, we have been unable to identify the molecular
mechanism(s) whereby such cells have a constitutively active MAP kinase
pathway, except in one variant, which has an activating mutation in the
SCF receptor (N. Leslie, unpublished data).
Differentiation capacity of ELM cells maintained long-term in SCF or
IGF-1.
To test whether long-term growth in SCF or IGF-1 affected the ability
of ELM cells to undergo erythroid differentiation, growth factors were
removed from several clones of ELM cells grown long-term in SCF and the
levels of globin mRNA were measured by Northern analysis. ELM cells
freshly removed from stroma were used for comparison. The response of
ELM cells to EPO and IL-3 was also determined in the presence of stroma
or SCF. The results show several interesting features
(Fig 5). First, ELM cells removed from
stroma or SCF differentiate spontaneously. As described earlier, ELM
cells grown long-term in IGF-1 become independent of IGF-1 for growth
and do not differentiate if IGF-1 is removed. Second, EPO/IL-3 can
induce differentiation of ELM cells in the presence of SCF or IGF-1,
whereas growth on stroma prevents EPO/IL-3-induced differentiation.
This suggests that one mechanism whereby stroma maintains ELM cells in
vivo or in vitro is by preventing their differentiation. This is not
due to integrin signaling because ELM cells still differentiate in
response to EPO and IL-3, even if grown in SCF on ECM-coated plates
(data not shown). In contrast, the differentiation arrest by SCF can be
overridden by EPO/IL-3.
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| Fig 5.
Inducibility of globin mRNA by EPO and IL-3 in ELM cells
grown with or without stroma or ELM clones grown continuously in SCF
(20.5D, 13.1C) or IGF-1 (20.2B, 20.5B). ELM cells were grown for 4 days
in the growth factors shown, RNA extracted and 10 µg analyzed by
Northern transfer for globin mRNA expression by probing with an
-globin cDNA probe or 7S ribosomal RNA as a loading control. The
amounts of growth factors used were: SCF, 10 ng/mL; IGF-1, 100 ng/mL;
EPO, 5 U/mL; IL-3, 10 U/mL.
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Tumorigenicity of ELM cells grown under different conditions.
To test whether growing ELM cells long-term in SCF or IGF-1, as
opposed to normal stroma, affected their tumorigenicity, several ELM
clones selected in SCF or IGF-1 cells were injected via the tail vein
into syngeneic C3H mice and the growth of the cells intrasplenically
measured by the increase in spleen weight. Our previous studies have
shown that the increase in spleen size is due to growth of the injected
ELM cells.16 However, maintaining ELM cells long-term in
SCF or IGF-1 did not affect their tumorigenicity compared with
injection of cells maintained on stroma (data not shown).
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DISCUSSION |
Bone marrow-, spleen- or fetal liver-derived stromal cells have been
shown to support the growth of various normal hematopoietic stem cells
and progenitors,22,23 including erythroid
progenitors,24-26 which are often found in association with
macrophages in bone marrow27,28 or in long-term Dexter
cultures set up to favor erythropoiesis.29 There is
increasing evidence that stromal derangements can contribute to the
evolution of leukemias (reviewed by Duhrsen and
Hossfeld30). Some primary leukemic cells still maintain
this stroma-dependence,23,31-33 but there is evidence that
different types of stroma can favor the growth of normal versus
leukemic progenitors.34-38 However, most in vitro cultures of leukemic cells, including retrovirus-induced murine
erythroleukemias,8-10 have lost their dependence on stroma
and grow readily in suspension culture with or without added growth
factors. The ELM erythroleukemia model is one of the few examples where
bone marrow-derived stromal cells are required for long-term
maintenance of the cell in culture,11-13 whereas clonal
extinction occurs after a limited period of growth after
stroma withdrawal, even in the presence of conditioned medium from
stromal cells.14
Therefore, it is of considerable interest to define the cell
interactions and signaling events responsible for this long-term maintenance of ELM cells by stroma. We have previously shown that one
component of this is the interaction between SCF receptor on ELM cells
and membrane-presented SCF by the stroma cells (Friel et al, manuscript
submitted). Our present results extend this analysis
further and define other components of the ELM-stroma interaction. Our
antibody blocking experiments show that growth of ELM cells on stroma
is mediated by a combination of SCF, IGF-1, and integrin signaling. The
involvement of the SCF receptor is also supported by the fact that
Sl/Sl stroma, which does not express any form of SCF, is less
efficient than normal stroma at supporting ELM cell growth or cloning
long-term. SCF and integrin signaling in cooperation with CD44 have
been shown to be involved in the interactions of stromal cells with
normal erythroid progenitors.39-42 IGF-1 is also required
for growth and differentiation of normal erythroid progenitors in the
absence of accessory cells.43 Thus, the ELM erythroleukemia
model is unusual in retaining many of the signaling pathways of normal
erythroid progenitors.
It is obviously of practical importance for basic research and clinical
applications to find ways of maintaining erythroid progenitors
long-term under defined conditions and to determine whether such
conditions affect the progression of erythroleukemic cells to a more
aggressive phenotype. We therefore investigated whether combinations of
growth factors could support the proliferation of ELM cells, either in
normal serum medium or serum-free media without generating abnormal
phenotypes. The outcome of all of these experiments showed that SCF
alone is capable of maintaining ELM cells for many months, without
generating SCF- or stroma-independent variants and without increasing
their tumorigenicity in vivo compared with ELM cells maintained on
stroma. The main phenotypic difference we have found between ELM cells
grown long-term in SCF and stroma is that growth on stroma blocks their
differentiation in response to EPO/IL-3, whereas growth in SCF does
not. This is somewhat different to the situation with normal human
erythroid cells and K562 cells where SCF has been reported to block
erythroid differentiation.44,45 ELM cells maintained in SCF
also differ from cells maintained on stroma in that they lose their
responsiveness to IGF-1.
Normal stroma produce two forms of SCF, a membrane inserted form and a
soluble, secreted form.46 Because we found that ELM cells
can be grown indefinitely in soluble SCF alone, we were initially
surprised that neither conditioned medium from normal stroma, nor
Sl/Sld stroma, which expresses soluble SCF, was able to
support long-term maintenance or cloning of ELM cells. We believe the
most likely reason for this is that the amounts of SCF produced by
stroma may be suboptimal, or SCF may diffuse away from the ELM
cell/stroma monolayer into the medium too quickly. Another factor may
be that the signal produced by the interaction of SCF receptor with the membrane form of SCF is reported to be longer-lived than the signal from the interaction with soluble SCF.47 This may be
important, as differentiation or proliferation of, for example,
pheochromocytoma PC12 cells in response to growth factors is believed
to be governed by the duration of the period of activation of the MAP
kinase pathway they induce.48
In contrast to SCF, IGF-1 does not maintain ELM cells long-term, and
after forced adaptation to growth in IGF-1, they, in fact, become
IGF-1-independent for growth. It is presently unclear how these
IGF-1-independent ELM cells arise. Two different explanations could be
envisaged. One explanation might be that growth in IGF-1 induces
permanent changes in growth factor or growth factor receptor expression, which allows the cells to grow autonomously. Beug et
al49,50 have provided evidence that relatively late chicken erythroid progenitors can be converted into more immature progenitors with high proliferative capacity by growth in SCF, transforming growth
factor (TGF), estradiol, and an unknown serum factor and this is
associated with upregulation of the TGF receptor. Kamai et
al51 have also reported that long-term growth of
preadipocyte cells in IGF-1 and growth hormone permanently altered the
splicing pattern of IGF-1 transcripts to give an IGF-1 mRNA encoding
secreted IGF-1, which then supported cell growth in a
paracrine/autocrine manner. However, in the ELM system, we have been
unable to find changes in the expression of IGF-1 or SCF or their
receptors in IGF-1-independent variants, although some change
resulting in constitutive activation of MAP kinases does occur. The
alternative explanation we favor for how the IGF-1-independent ELM
variants arise is that they are mutants generated spontaneously during culture. This is consistent with the fact that the cloning efficiency of ELM cells in IGF-1 is very low and with our previous estimate that
stroma-independent variants arise at a frequency of about 10-5.14,16 In contrast, we did not isolate a
single independent variant in all of the SCF cloning experiments
performed in the course of this work, and the reason for this is
probably that the growth rate/cloning efficiency of ELM cells in SCF is
sufficiently high to prevent outgrowth of any independent cells in the
population; whereas in IGF-1, ELM cells die out, leaving any
independent cells to overgrow.
| |
FOOTNOTES |
Submitted July 31, 1997;
accepted October 17, 1997.
Supported by the Cancer Research Campaign, UK, and the Deutsche
Forschungsgemeinschaft, Germany.
Address reprint requests to Paul R. Harrison, PhD, The
Beatson Institute for Cancer Research, CRC Beatson Laboratories,
Garscube Estate, Switchback Rd, Bearsden, Glasgow G61 1BD, Scotland.
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.
| |
ACKNOWLEDGMENT |
We thank Prof J. Wyke and Dr I. Pragnell for their critical reading of
the manuscript.
| |
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Abstract
Introduction
Abstract