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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 890-897
Stimulating Cell Proliferation Through the Pharmacologic Activation
of c-kit
By
Liqing Jin,
Haruhiko Asano, and
C. Anthony Blau
From the Department of Medicine, Division of Hematology, University
of Washington, Seattle.
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ABSTRACT |
Previous studies have shown that expression of a membrane targeted
chimeric protein containing the erythropoietin receptor (EpoR)
cytoplasmic domain fused to the FK506-binding peptide FKBP12 allowed
Ba/F3 cells to be rescued from interleukin-3 (IL-3) deprivation using a
dimeric form of FK506, called FK1012. In this report, a similar
approach is applied to the c-kit receptor. Expression of a
membrane targeted fusion protein containing the c-kit receptor linked to one or more copies of FKBP12 allowed Ba/F3 cells to be
switched from IL-3 dependence to FK1012-dependence. Similar results
were obtained using an alternative dimerizer of FKBP12 domains called
AP1510. Pharmacologic dimerization of chimeric proteins containing only
a single FKBP12 domain confirmed that receptor dimerization is
sufficient for proliferative signaling. Interestingly, while the
proliferative effects of both FK1012 and AP1510 were reversible,
FK1012-driven proliferation persisted for several days after drug
withdrawal. Furthermore, much higher concentrations of FK506 were
required to inhibit FK1012-mediated proliferation than were required to
inhibit AP1510-mediated proliferation. The persistence of FK1012's
effect appeared to be specific to clones expressing
c-kit-containing fusion proteins. These results suggest that
pharmacologically-responsive fusion proteins containing c-kit
may be useful for specifically and reversibly expanding genetically
modified hematopoietic cell populations.
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INTRODUCTION |
THE INEFFICIENCY of gene transfer into
human hematopoietic stem cells poses a major obstacle for gene therapy
of inherited and acquired blood cell disorders.1,2 The
development of therapeutic applications for stem cell gene transfer
depends on markedly increasing the proportion of genetically corrected
stem cells. One strategy for increasing the frequency of modified stem cells is to induce their preferential expansion through
selection.3 Providing the basis for selection are
bicistronic vectors in which the nonselectable therapeutic gene is
linked to a second gene encoding a selectable product. The frequency of
genetically corrected cells can be increased by applying selection
either ex vivo,4,5 or if a clinically tolerable regimen
were devised, selection could be applied in vivo. Current methods for
in vivo selection involve the transfer of a gene conferring drug
resistance such as multiple drug resistance-1 (MDR1) or dihydrofolate
reductase (DHFR), followed by the subsequent administration of the
corresponding cytotoxic drug in vivo.6
An alternative approach for selection would be to confer a direct
proliferative advantage on the genetically modified cell population.
The practical application of this strategy would require that the
proliferative stimulus (1) be restricted to the genetically modified
population and (2) be completely reversible. We have recently reported
the use of a new method that appears to meet these
requirements.7 This approach is based on the principle that
receptors for a variety of cytokines consist of single chains that are
activated upon ligand-induced homodimerization.8 Recently developed technology allows intracellular protein dimerization to be
reversibly activated in response to a lipid soluble dimeric form of the
drug FK506, called FK1012.7,9-15 FK1012 is used as a
pharmacologic mediator of dimerization to bring together two FK506
binding domains, taken from the endogenous protein FKBP12. Thus, fusion
proteins consisting of a cytokine receptor signaling domain linked to
an FKBP12 domain may be dimerized and thereby activated using
FK1012.9 This approach has been used to activate apoptosis
through the Fas signaling pathway15 and a related approach
has been used to inducibly activate synthesis of a reporter gene in
vivo.16
In recent studies,7 we have shown that FK1012-induced
aggregation of a fusion protein containing the intracellular portion of
the erythropoietin receptor (EpoR) allowed Ba/F3 cells, which are
normally dependent on interleukin-3 (IL-3), to proliferate in its
absence. This approach meets two criteria that are essential for gene
therapy applications: the proliferative stimulus is restricted to the
genetically modified cell population, and it is reversible. In the
context of the proper signaling molecule, a similar approach may be
envisioned as a means for expanding genetically modified hematopoietic
stem cells. Advantages of this system for in vivo applications are that
FK1012 lacks the immunosuppressive properties of FK5069 and
that it can be administered at biologically effective doses.15
Toward the goal of using this approach for the expansion of genetically
modified stem cells, we have tested the same strategy using a molecule,
which upon activation has the reported capacity for causing stem cells
to divide: c-kit.17-19 In this report, we show that
Ba/F3 cells expressing a membrane-targeted chimeric protein containing
the intracellular domain of c-kit linked to one or more copies
of FKBP12 are capable of FK1012-dependent proliferation in the absence
of IL-3. These results suggest that pharmacologic dimerization of
c-kit-containing fusion proteins may be useful for expanding
genetically modified hematopoietic stem cells.
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MATERIALS AND METHODS |
Plasmid construction.
F3, also designated pMF(PK)3E,12 is a modified form of
pMF3E.9 FKBP12 has been modified to contain the mutations
G89P and I90K. These mutations abrogate the ability of the FK506
complexes of this mutant FKBP to interact with
calcineurin20 and have a reduced propensity to interact
with cellular proteins.12 A 1296 or 1164 bp cytoplasmic
domain of the murine c-kit was amplified by polymerase chain
reaction (PCR) using Pfu polymerase, a plasmid containing the
c-kit cDNA (a gift from Alan Bernstein, University of Toronto,
Toronto, Canada) as a template and the following primers: 5 CCC
CTC GAG TAC AAA TAT TTG CAG AAA CC; 3 (432 amino acids): CCC CTC
GAG GGC ATC TTC GTG CAC; 3 (388 amino acids): CCC CTC GAG CTT
GGT GCT GTC CGA GAT. The PCR amplified fragments were digested using
Xho I, gel purified, and inserted into an Xho
I-digested and phophatased pBluescript vector. After sequence
confirmation using the PRISM system (Applied Biosystems, Foster City,
CA), the fragment was released from pBluescript by
Xho I digestion and ligated into the phophatased Sal I
digested plasmids F3 or F1 to generate the plasmids F3c-kit432,
F3c-kit388 and F1c-kit432 (Fig 1). Plasmids were
purified over two sequential cesium chloride gradients before
transfection.
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| Fig 1.
Plasmid constructs and Western blot. (A) Schematic
representation of constructs. F3, previously designated pMFPK3E
12 is a modified version of pMF3E,9 where the
FKBP12 domain has been modified to contain the mutations G89P and I90K.
Murine c-kit receptor sequences encode either the full-length
432-amino acid cytoplasmic domain, or the membrane proximal 388 amino
acid cytoplasmic domain. ( ), Myristylation peptide; ( ), FKBP12;
(), intracellular domain of c-kit receptor; ( ), HA
epitope tag. (B) Western blot of protein extracts from Ba/F3 cell
clones expressing the F3 and F3c-kit432 constructs display
protein bands of predicted sizes.
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Electroporation.
BaF3 cells were maintained in RPMI supplemented with glutamine,
pyruvate, penicillin, streptomycin, 10% fetal calf serum, and 10%
WEHI conditioned medium. Cells were split 1:2 on the day before
transfection. Electroporations were performed as described previously.7
Western blotting.
Cells were washed twice with phosphate-buffered saline (PBS), and
approximately 1 × 106 cells were lysed in 100 µL 50 mmol/L Tris pH8.0, 150 mmol/L NaCl, 1% NP40, 0.5% deoxycholate, 0.1%
sodium dodecyl sulfate (SDS), and placed on ice for 30 minutes. Western
assays were performed using the HA.11 antibody (Berkeley Antibody Co,
Berkeley CA), as previously described.7
Cell proliferation assay.
Cell proliferation assays were evaluated by using MTT
colorimetric assay (Sigma) as described previously.7 FK1012
was a gift of David Spencer (Baylor University, Houston, TX), and
AP1510 was a gift of Ariad Pharmaceuticals (Boston, MA).
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RESULTS |
Ba/F3 cells expressing F3c-kit432 proliferate in response to FK1012.
Studies were performed to determine whether Ba/F3 cells expressing a
c-kit/FKBP12 fusion protein can be induced to proliferate using
FK1012. Initial studies were performed using the construct F3c-kit432 (Fig 1A). This construct
was produced by inserting the cytoplasmic domain of the murine
c-kit receptor into the Sal I site of the plasmid F3
(Fig 1A). F3c-kit432 encodes a chimeric protein containing a
14-amino acid myristylation-targeting domain from
c-Src21 to direct localization to the inner
surface of the cell membrane, three copies of a 107-amino acid FKBP12
module22 to bind the drug FK506, the entire 432 amino acid
intracellular domain of the murine c-kit receptor, and a
9-amino acid influenza HA epitope tag23 to allow detection
of the recombinant protein by Western analysis.
Previous studies have shown that Ba/F3 cells expressing the full-length
c-kit receptor are capable of proliferation with stem cell
factor (SCF) in the absence of IL-3.24
Experiments were performed to determine whether Ba/F3 cells expressing
the F3c-kit432 fusion protein could, after withdrawal of IL-3,
be rescued by FK1012. Ba/F3 cells were cotransfected with the
F3c-kit 432 construct and a plasmid encoding neomycin
phosphotransferase. G418-resistant clones were expanded and evaluated
for expression of the chimeric protein by Western analysis (Fig 1B).
Three Ba/F3 cell clones expressing high levels of the
F3c-kit432 fusion protein were tested in cell proliferation
assays. Proliferation failed to occur in the absence of
IL-3-containing WEHI conditioned medium, and WEHI conditioned medium
stimulated cell proliferation in a concentration-dependent manner (data
not shown).
Proliferation assays were performed using the same clones in the
absence of IL-3, but with the addition of either FK1012 or a new
synthetic dimerizer of FKBP12 domains called AP1510. Concentrations of
drug ranged from 10-2 to 103 nmol/L. As shown
in Fig 2, both FK1012 and AP1510 exerted
concentration-dependent proliferative effects in all three clones. To
allow comparisons between the proliferative effects of FK1012, AP1510,
and WEHI conditioned medium, results shown in Fig 2 indicate absorbance as a fraction of that obtained in parallel cell proliferation assays
using 5% WEHI conditioned medium. For FK1012, cell proliferation was
observed at as low as 1 nmol/L, peaking at concentrations of 100 nmol/L. Proliferation also occurred in response to AP1510, although
approximately 10-fold higher concentrations of AP1510 were required to
achieve a similar proliferative response. To show that the
c-kit domain is required for mitogenic signaling, two control
BaF3 clones expressing high levels of the construct F3, which lacks the
c-kit domain (Fig 1), failed to proliferate in response to
either FK1012 or AP1510 (data not shown).
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| Fig 2.
Pharmacologic activation of c-kit stimulates cell
proliferation. MTT assays for three Ba/F3 clones expressing the
construct F3c-kit432. Cells were tested in the absence of IL-3
and in the presence of either FK1012 (upper panel) or an alternative
dimerizer of FKBP12 domains called AP1510 (lower panel) at
concentrations ranging from 10 2 nmol/L to
103 nmol/L. All Ba/F3 clones expressing F3c-kit432
showed a concentration-dependent proliferative response to both FK1012
and AP1510. Results are plotted as a fraction of OD570-630 nm values
obtained using the same clone cultured in 5% WEHI conditioned medium.
Data points indicate mean values of three separate experiments. Error
bars indicate standard deviations. Note that in comparison to FK1012, approximately 10-fold higher concentrations of AP1510 are required to
achieve equivalent proliferative effects.
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Dimerization of the FKBP12/c-kit fusion protein is sufficient for
proliferative signaling.
The presence of three FKBP12 domains in F3c-kit432 could allow
FK1012 or AP1510 to stimulate proliferation either through dimerization
or through oligomerization of the chimeric molecule. To distinguish
between these two possibilities for c-kit activation, a
construct was produced in which the 432-amino acid c-kit
receptor was linked to only a single copy of the FKBP12 domain
(F1c-kit432) (Fig 1 A). Proliferation in response to FK1012 or
AP1510 in cells expressing this fusion protein can be caused only by
dimerization. As shown in Fig 3, five Ba/F3
cell clones expressing F1c-kit432 exhibited proliferative
responses to both FK1012 and AP1510. In parallel assays, the level of
cell proliferation observed in these clones appeared to be slightly
reduced compared with clones expressing F3c-kit432. These
results confirm that dimerization of c-kit is sufficient for
mitogenic signaling.
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| Fig 3.
Dimerization of c-kit is sufficient for
proliferative signaling. MTT assays were performed on five Ba/F3 clones
expressing the construct F1c-kit432 in the absence of IL-3 and
in the presence of increasing concentrations of either FK1012 (upper
panel) or AP1510 (lower panel). Results are plotted as a fraction of
the OD570-630 measured from the same cells cultured in 5% WEHI
conditioned medium. Data points indicate mean values of three separate
experiments. Error bars indicate standard deviations. Note that the
level of cell proliferation observed is slightly reduced compared with Ba/F3 cell clones expressing the F3c-kit432 construct.
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FK506 efficiently competes FK506-mediated cell proliferation, but
inefficiently competes FK1012-mediated cell proliferation.
To test the hypothesis that FK1012 and AP1510 activate cell
proliferation by bringing together FKBP12 domains of adjacent FKBP12/c-kit fusion proteins, competition assays were performed using the monomer FK506. Cells were grown in the presence of either FK1012 (100 nmol/L), AP1510 (100 nmol/L), or WEHI conditioned medium
(1%), and a range of FK506 concentrations. Ba/F3 clones expressing
either F3c-kit432 or F1c-kit432 were tested, and
compared with a Ba/F3 clone expressing the construct
F3EpoR236,7 which contains the intracellular domain of the
murine erythropoietin receptor inserted into the corresponding
Sal I site of F3 (Fig 1A). In Ba/F3 cells expressing F3EpoR236,
FK506 exerted a concentration-dependent inhibition of FK1012-mediated
cell proliferation (Fig 4), identical to
results reported previously.7 Similar results were obtained in the presence of AP1510, although lower doses of FK506 were required
for complete inhibition, consistent with AP1510's approximately 20-fold lower binding affinity for FKBP12 relative to
FK506.24a As expected, FK506 exerted no effect on
IL-3-mediated proliferative signaling.
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| Fig 4.
FK506 efficiently competes AP1510-mediated cell
proliferation, but inefficiently competes FK1012-mediated cell
proliferation. MTT assays were performed using clonal populations of
Ba/F3 cells expressing F3EpoR236 (left column), F3c-kit432
(middle column), or F1c-kit432 (right column). Cells were grown
in the presence of 100 nmol/L FK1012 (upper panels), 100 nmol/L AP1510
(middle panels), or 1% IL-3-containing WEHI conditioned medium (lower panels), plus FK506 at the indicated concentrations. Note that in the
presence of WEHI conditioned medium, FK506 failed in all cases to
inhibit cell proliferation (lower panels). In contrast, for each clone
FK506 exerted a concentration-dependent inhibition of AP1510-mediated
cell proliferation (middle panels). Similarly, in a Ba/F3 cell clone
expressing F3EpoR236, FK506 produced a concentration-dependent inhibition of FK1012-dependent cell proliferation. In contrast, both
Ba/F3 cell clones expressing fusion proteins containing c-kit (F3c-kit432 and F1c-kit432) were resistant to
FK506-mediated inhibition of FK1012-dependent cell proliferation.
Repeat experiments using the same clones and different clones
expressing the same constructs yielded very similar results.
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Surprisingly, evaluation of Ba/F3 cells expressing FKBP12/c-kit
fusion proteins showed significantly different findings (Fig 4). In
Ba/F3 cells expressing F3c-kit432, equimolar concentrations of
FK506 failed to reduce FK1012-dependent cell proliferation, and a
significant degree of proliferation persisted despite the presence of a
10-fold molar excess of competing monomer. At a 100-fold excess of
FK506, complete inhibition of FK1012-dependent proliferation was
finally observed. The same results were obtained irrespective of
whether FK506 or FK1012 was added to the cells first. In contrast for
AP1510, equimolar concentrations of FK506 resulted in a nearly complete
inhibition of AP1510-mediated cell proliferation. These results suggest
that while FK506 efficiently competes with AP1510 for access to FKBP12
sites, the monomer appears to be surprisingly less efficient in
competing with its dimeric counterpart, FK1012. Similar results were
obtained using F1c-kit432: FK506 efficiently inhibited
AP1510-dependent cell proliferation, but inhibition of FK1012-mediated
proliferation was highly inefficient (Fig 4). As expected, the doses of
FK506 used had no significant effect on IL-3-mediated cell
proliferation.
Persistence of FK1012-mediated cell proliferation after drug
withdrawal.
To confirm that the proliferative effects of FK1012 and AP1510 are
reversible, cells initially cultured in the presence of either FK1012
or AP1510 were observed for cessation of cell growth after drug
withdrawal. Ba/F3 cells expressing the F3c-kit432 fusion protein were plated in media without IL-3 supplemented with either FK1012 or AP1510 (both 100 nmol/L). As shown in
Fig 5, cell proliferation was observed in
response to either compound, while in the absence of either drug or
IL-3, cells died over a period of 2 to 3 days. Similar to results
obtained in the MTT assays, FK1012 appeared to be a slightly more
potent stimulator of cell proliferation than was AP1510 at the
concentrations examined. After 3 days of culture, cells grown initially
in FK1012 or in AP1510 were washed extensively and then cultured either
in the presence or absence of added drug. Cells grown initially in
AP1510 died within 24 to 48 hours after drug withdrawal. In contrast,
cells grown initially in FK1012 continued to slowly proliferate for up
to 7 days after FK1012 was withdrawn. Thereafter, continued deprivation
of FK1012 eventually resulted in cell death (Fig 5). Similar results
were obtained with Ba/F3 cell clones expressing the F1c-kit432
fusion protein (data not shown). Taken together with results from the competition assays, these findings suggest that FK1012 binding to the
FKBP12/c-kit fusion protein may persist for several days after
drug withdrawal.
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| Fig 5.
Persistence of FK1012-mediated cell proliferation after
drug withdrawal. (A) A clonal population of Ba/F3 cells expressing F3c-kit432 proliferates in IL-3-deficient medium supplemented with either FK1012 ( ) or AP1510 ( ) (both 100 nmol/L). In the absence of either drug or IL-3 ( ), cells died over a period of 2 to
3 days. (B) The same cells grown initially in FK1012 or in AP1510 were
washed extensively and then cultured either in the presence or absence
of added drug. In the continued presence of FK1012 () or AP1510
(), continued cell proliferation was observed. After withdrawal of
AP1510 ( ), cell death occurred over 1 to 2 days. In contrast, cells
grown initially in FK1012 continued to proliferate for up to 1 week
after FK1012 was withdrawn (). Very similar findings were observed
in a repeat experiment.
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Deletion of the carboxy terminal end of the c-kit receptor reduces
responsiveness to FK1012 and AP1510.
Deletion of the carboxy-terminus of a related receptor c-fms,
results in an increased receptor sensitivity to ligand-dependent activation.25,26 To determine whether negative growth
regulatory domains reside in the carboxy terminal tail of
c-kit, the construct F3c-kit388 was produced, in which
the carboxy terminal 44 amino acids of c-kit were deleted (Fig
1A). Five BaF3 clones expressing the F3c-kit388 were generated.
As shown in Fig 6, both FK1012 and AP1510
exerted concentration-dependent proliferative effects in the absence of
IL-3. However, in parallel comparisons with BaF3 clones expressing
F3c-kit432, truncation of the c-kit receptor appeared
to significantly decrease responsiveness to FK1012 and AP1510. These
findings are not attributable to differences in the level of receptor
expression (data not shown).
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| Fig 6.
Deletion of the carboxy-terminal 44 amino acids of c-kit
reduces proliferation in response to FK1012 and AP1510. MTT assays were
performed on three Ba/F3 clones expressing F3c-kit388, in which
the carboxy terminal 44 amino acids of the c-kit receptor were
deleted. Each clone responded to both FK1012 (upper panel) and AP1510
(lower panel), however, the level of proliferation was less than in
clones expressing F3c-kit432 (Fig 2). Results are plotted as a
fraction of the OD570-630 value measured from the same cells cultured
in 5% WEHI conditioned medium. A second experiment yielded very
similar results.
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DISCUSSION |
Fundamental to the success of stem cell gene therapy is the development
of methods for increasing the frequency of genetically corrected stem
cells. This task may in theory be accomplished through selection.
Selection can be applied either ex vivo, or if a clinically tolerable
approach were devised, repeated cycles of selection could potentially
be applied in vivo. Current approaches for in vivo selection involve
the transfer of a drug resistance gene into a small population of
hematopoietic cells. Selective pressure is applied through in vivo
administration of the appropriate cytotoxic drug. Success requires that
the cytotoxic drug exert a proportionally greater toxic effect on the
population of unmodified marrow cells relative to their transduced
counterparts. Recent studies have underscored a major problem in using
drug resistance genes for in vivo selection: early hematopoietic cells
tolerate very high dosages of the chemotherapy drugs to which these
genes confer resistance.27,28 Thus, chemotherapy provides
little or no selective advantage to clonogenic progenitors bearing the drug resistance gene, as these early hematopoietic cells are normally highly drug-resistant.27-30
An alternative method for accomplishing selection would be to confer a
direct proliferative advantage on the genetically modified cell
population. The clinical applicability of this approach would mandate
that the proliferative advantage be reversible. The use of
pharmacologic inducers of dimerization7-13 may provide a
method for achieving this goal. Using fusion proteins containing the erythropoietin receptor, we have recently reported the development of a
"proliferation switch."7 In Ba/F3 cells expressing
the FKBP12/EpoR chimeric protein, the addition of FK1012 switches cell
proliferation to the "on" position, and withdrawal of FK1012
switches proliferation to the "off" position. Toward the goal of
testing whether a similar approach might be used for the expansion of
genetically modified hematopoietic stem cells, we tested this strategy
using c-kit.
Mutations at the white spotting (W) and steel
(Sl) loci provide the most compelling evidence for a biologic
role of c-kit and its ligand, respectively, in stem cell
regulation.31,32 Normal marrow cells introduced into
W/Wv mice can repopulate the entire hematopoietic
system without conditioning, indicating a severe defect in the
repopulating ability of stem cells in these animals.33
Further evidence comes from the severe pancytopenia that results from
administration of an antibody directed against c-kit in
mice.34 C-kit ligand (SCF) administration in vivo
stimulates the redistribution and expansion of
progenitors.35-37 Furthermore, SCF administration produces
a threefold expansion in the total number of transplantable stem cells
in mice when given alone17 and a 10-fold expansion when
given in combination with granulocyte colony-stimulating factor (G-CSF)
in splenectomized mice.19
In this report, we show that Ba/F3 cells expressing
FKBP12/c-kit fusion proteins can proliferate in response to
either FK1012 or a new synthetic FKBP12 dimerizer, called AP1510. After
IL-3 withdrawal, Ba/F3 cells expressing the F3c-kit432 fusion
protein could be rescued and maintained in either FK1012 or AP1510.
Approximately 10-fold higher concentrations of AP1510 were required to
achieve a level of proliferation similar to that observed with FK1012, possibly due to AP1510's 20-fold lower binding affinity for FKBP12 domains.24a
Several findings in this report differ from our previous observations
using the erythropoietin receptor. First, in the context of the
erythropoietin receptor, a fusion protein containing three FKBP12
domains was significantly more efficient in stimulating FK1012-mediated
cell proliferation than was a fusion protein containing only a single
FKBP12 domain.7 In the case of c-kit, the
difference in the level of proliferation observed in clones expressing
fusion proteins with a single FKBP12 domain appeared to be only
slightly reduced compared with clones with fusion proteins containing
three FKBP12 domains. These results suggest that dimers may be nearly as efficient as oligomers in the stimulation of c-kit-mediated proliferative signaling. Retaining only a single FKBP12 domain has
potential advantages for the development of retroviral vectors with
regard both to the reduction in insert size and to the avoidance of
repeated sequences that may promote recombination.
Unexpectedly, fusion proteins containing c-kit require
extremely high concentrations of FK506 to inhibit FK1012's
proliferative effect. Equimolar concentrations of FK506 had no
discernible effect on FK1012-dependent cell proliferation, and even a
10-fold molar excess of competing monomer failed to completely inhibit
FK1012's effect. In contrast, FK506 efficiently inhibited
AP1510-driven cell proliferation. Furthermore, FK506 efficiently
competed with FK1012 in parallel experiments using a clone expressing
F3EpoR236. These observations suggest that c-kit may in some
way stabilize the interaction between the FKBP12 moiety of the fusion
protein and FK1012. The hypothesis that FKBP12/c-kit fusion
proteins bind FK1012 in a highly stable manner is supported by our
observation that cell proliferation persists for up to 1 week after
FK1012 withdrawal (Fig 5). In this regard, it is noteworthy that after prolonged maintenance in IL-3-containing cultures (12 to 16 weeks), some Ba/F3 cell clones expressing FKBP12/c-kit acquire the
capacity for factor independent cell growth, but remain FK1012
responsive (data not shown). The significance of these findings awaits
testing in transduced primary murine bone marrow cells.
In view of the short in vivo half lives of both FK1012 and AP1510
(Spencer et al15 and Amara et al24a), drug
concentrations attainable in vivo are likely to be low. Thus, signaling
molecules that can be activated at very low drug concentrations may be
advantageous. Several mutations in the cytoplasmic domain of
c-kit have the capacity to trigger constitutive receptor
activation and neoplastic transformation.38,39 Sequence
differences between the feline v-kit oncogene and feline
c-kit include the replacement of the carboxy-terminal 44 amino
acids in c-kit by five unrelated amino acids in v-kit.
Other reports indicate that negative regulatory domains may reside
within the carboxy terminal end of another class III receptor tyrosine
kinase, c-fms.25,26 In our studies, deletion of 44 amino acids from the carboxy terminal end of c-kit failed to
augment responsiveness to either FK1012 or AP1510 and appear, in fact,
to have resulted in an impairment of receptor function. The lack of an
increase in receptor sensitivity is in agreement with a previous report
and suggest that remaining sequence differences between v-kit
and c-kit require evaluation.40
An eventual goal of our studies is to identify signaling domains that
stimulate stem cells to divide, but not to differentiate. Pharmacologic
activation of wild-type c-kit, which is also a major regulator
of mast cell differentiation,41-44 is unlikely to fulfill this goal. Urgently needed are approaches for the identification and,
if possible, the elimination of domains necessary for maturational signaling.
Although our studies show that FK1012 or AP1510 can rescue a
factor-dependent cell line, demonstrating a proliferative advantage in
genetically modified primary cells may be more difficult, particularly for in vivo applications. In Ba/F3 cells, withdrawal of IL-3 provides a
strong selective pressure that is not reproducible in vivo. Whether
pharmacologic inducers of dimerization can provide a proliferative stimulus in transduced primary cells beyond that provided by a physiologic milieu of cytokines remains to be determined.
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FOOTNOTES |
Submitted May 7, 1997;
accepted October 2, 1997.
Supported by Grants No. 1R01 DK52997-01, 5P01 HL53750, and 5P30 DK47754
from the National Institutes of Health, Bethesda, MD.
Address reprint requests to C. Anthony Blau, MD, Mail Stop
357710, Health Sciences Building, Division of Hematology, Department of
Medicine, University of Washington, Seattle, WA 98195.
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 |
The authors thank Alan Bernstein for his gift of the murine
c-kit cDNA, David Spencer for FK1012, and Mike Gilman and Jane Amara for AP1510 and for access to their results prior to publication.
| |
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Abstract
Introduction
Abstract