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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2319-2332
Retroviral-Mediated Gene Transduction of c-kit Into Single
Hematopoietic Progenitor Cells From Cord Blood Enhances Erythroid
Colony Formation and Decreases Sensitivity to Inhibition by Tumor
Necrosis Factor- and Transforming Growth Factor- 1
By
Li Lu,
Michael C. Heinrich,
Li-Sheng Wang,
Mu-Shui Dai,
Amy J. Zigler,
Lin Chai, and
Hal E. Broxmeyer
From the Departments of Microbiology and Immunology, Medicine
(Hematology/Oncology), the Walther Oncology Center, Indiana University
School of Medicine, Indianapolis, IN; the Walther Cancer Institute,
Indianapolis, IN; and the Division of Hematology and Medical Oncology,
Department of Medicine, and Portland Veterans Affairs Medical Center,
Portland, OR.
 |
ABSTRACT |
The c-kit receptor and its ligand, steel factor (SLF), are
critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34+ cells were sorted as a
population or as 1 cell/well for cells expressing high levels of
CD34+++ and different levels of c-kit
(++, +, Lo/ ), transduced
and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3),
IL-6, erythropoietin (Epo) +/ SLF in the absence of serum. At a
single-cell level, transduction with c-kit, but not with
control (neo only), virus significantly increased colony formation,
especially by erythroid and multipotential progenitors. The enhancing
effect of c-kit transduction was inversely correlated with
expression of c-kit protein before transduction. The greatest
enhancing effects were noted in CD34+++
kitLo/ cells transduced with c-kit. The
stimulating effect was apparent even in the absence of exogenously
added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo.
Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse
transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA
expression in CD34+ cells, and use of neutralizing
antibodies to SLF and/or c-kit suggested the presence of
endogenous, although probably very low level, expression of SLF by
these progenitor cells. Transduction of c-kit significantly
decreased sensitivity of progenitor cells to the inhibitory effects of
transforming growth factor- 1 and tumor necrosis factor- .
c-kit-transduced cells had increased expression of
c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into
selected progenitor cells can enhance proliferation and decrease
apoptosis and that endogenous SLF may mediate this effect.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC CELL proliferation,
differentiation, migration, and apoptosis are mediated via the
interaction of various hematopoietic stimulatory and inhibitory
cytokines and their receptors. Among these, SLF and its receptor,
c-kit, are critical for maintenance of steady-state
hematopoiesis.1 c-kit, a 145-kD transmembrane glycoprotein, is the normal cellular counterpart of the viral oncogene
v-kit, and a member of the receptor tyrosine kinase subclass III family
that includes receptors to platelet-derived growth factor (PDGF),
macrophage colony-stimulating factor (M-CSF), and flt3
ligand.2-4 The c-kit gene product is expressed on a
variety of murine5-7 and human8-10
hematopoietic stem/progenitor cells, mast cells, and germ cells. The
ligand of c-kit exists in both transmembrane and soluble forms
and is known as mast cell growth factor (MGF), SLF, stem cell factor
(SCF), and kit ligand (KL).11-15 We and others have
shown that SLF is produced by human and murine hematopoietic stromal
cells including fibroblasts, endothelial cells, and bone marrow
(BM)-derived stromal cells.16,17
The dependence of normal hematopoiesis on an intact c-kit/SLF
axis is demonstrated in mice that have specific mutations of either the
c-kit (W) or SCF (SL) gene loci. These mice
exhibit BM failure, as well as white spotting, sterility, and a
profound decrease in tissue mast cells.1 Studies of various
W mutations in mice have suggested that the severity of the
observed phenotype correlates with residual functional activity of the
c-kit polypeptide and that there may be a threshold level of
c-kit kinase activity required for functional
hematopoiesis.18 Additional evidence supporting the
importance of c-kit in regulating hematopoiesis has been
provided by in vivo or in vitro experiments in which c-kit
protein functional activity is blocked with neutralizing antibodies.
Treatment of mice with an anti-c-kit monoclonal antibody that
blocks SLF binding results in aplastic anemia.5 Several groups of investigators have demonstrated that treatment of murine or
human long-term BM culture (LTBMC) with a c-kit neutralizing monoclonal antibody completely inhibits the production of
differentiated hematopoietic progenitors and mature myeloid blood
cells. After removal of the antibody, hematopoiesis recovers and the
number of transplantable stem cells in antibody-treated cultures is not different than that in treated cultures. It has been suggested that
functional c-kit is necessary for the generation of
differentiated hematopoietic progenitors and mature myeloid blood
cells, but may not be necessary for the survival of stem
cells.19,20 In addition, treatment of LTBMC with
c-kit antisense oligonucleotides causes suppression of the
generation of burst-forming unit-erythroid (BFU-E) which
continues for at least 3 weeks.21
Transforming growth factor-1 (TGF-1), a multifunctional cytokine,
affects the proliferation and differentiation of a variety of tissue
and cell types including cells of the hematopoietic system.22-25 TGF-1 is produced by both hematopoietic
cells and stromal cells of the hematopoietic
microenvironment,22,26 and inhibits hematopoietic
progenitor cells (HPC) in LTBMC.22,27 In LTBMC,
addition of TGF-1 to the medium blocks the recruitment of HPC into
cell cycle, and addition of neutralizing antibody to TGF-1 increases
the percentage of primitive progenitors in cell cycle.22,27
TGF-1 has also been reported to inhibit the proliferation of
progenitors in colony assays in vitro,23,28,29 and
treatment of mice with TGF-1 suppresses blood cell formation in
vivo.30 TGF-1 inhibits the expression of receptors on
progenitor cells for hematopoietic growth factors including receptors
for interleukin-1 (IL-1), IL-3, SLF, and granulocyte-macrophage
colony-stimulating factor (GM-CSF),31,32 increases
expression of cyclin-dependent kinase inhibitors (CDKI) such as
p27kip1, p21cip1, and
p15INK4B,33-36 and reduces
phosphorylation of the Retinoblastoma protein (Rb).37
Tumor necrosis factor- (TNF-) is another bifunctional cytokine
that is able to interact with many growth factors and their receptors.38-42 It has been reported to upregulate the
expression of IL-3, IL-5, and GM-CSF receptors40 and
downregulate receptors for GM-CSF and SLF.41,42 The
mechanism by which TNF- inhibits the proliferation of hematopoietic
stem/progenitor cells remains largely unknown, but may involve
induction of apoptosis and/or cell-cycle arrest. Some investigators
have found that TNF- decreases c-kit expression on both
normal and leukemic CD34+ cells,42 whereas
others have reported that TNF- enhances the expression of
c-kit in acute myelogenous leukemia (AML) cells through posttranscriptional stabilization of the c-kit
transcripts.43 TNF- decreases expression of
c-kit on murine HPC in a manner analogous to
TGF-1.44
We have previously described a subpopulation of CD34+ cells
expressing a high level of this antigen (CD34+++) from cord
blood (CB), which are enriched for primitive HPCs and can be isolated
and studied as single cells.45 These cells can be
transduced with retroviral vectors at both a population and a
single-cell level.10,46-49 In the present report, we
examined if enforced expression of c-kit by retroviral gene
transduction in CD34+++ CB cells could change the
proliferation capacity of these cells to growth cytokines and evaluated
their responsiveness to the inhibitory effect of TGF-1 and TNF-.
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MATERIALS AND METHODS |
Cells and cell separation.
Cells were obtained from normal human umbilical CB scheduled for
discard after delivery of the infant and after prior need for samples
for clinical study had been satisfied. CD34+++ cells were
obtained after sorting magnetic activated cell separation (MACS) beads (Miltenyi Biotec Inc, Auburn, CA) separated
CD34+ cells (90% to 95% pure) on a flow cytometer
(FacStarplus; Becton Dickinson, San Jose, CA) as previously
described.45 These cells are enriched for stem and immature
subsets of progenitor cells.45 The kit++,
kit+, and kitLo/ subsets of cells were
sorted from CD34+++ cells based on expression of
c-kit using a phycoerythrin-conjugated monoclonal antibody
against human c-kit (CD117/PE) (PharMingen, San Diego, CA) as
previously described.10 Sorting gates are shown in
Fig 1. Cells sorted by CD34 and
c-kit expression were either directly sorted by an autoclone
device as 1 cell/well into single wells containing 0.1 mL semisolid
culture medium (with a single cell per well verified as described
previously), or cells were sorted into tubes as populations of cells
and assayed for colony formation as previously
described.46-49
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| Fig 1.
Representative analysis of c-kit (CD117)/PE and
CD34/fluorescein isothiocyanate (FITC) immunofluorescence on gated high
densities of CD34+ (CD34+++) cells.
Sorting windows shown as ++, +, and Lo/ are for
CD34+++ kit++, kit+,
and kitLo/ cells, respectively.
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Colony assay.
Cultures contained Iscove's modified Dulbecco's medium (IMDM;
GIBCO-BRL, Gaithersburg, MD), 1% methylcellulose, 30% fetal calf
serum (FCS; Hyclone Laboratory, Logan, UT), and 0.1 mmol/L hemin
(Eastman Kodak Co, Rochester, NY). Serum-depleted medium contained
bovine serum albumin (1 mg), iron-saturated transferrin (300 µg),
cholesterol (7.8 µg), and CaCl2 (200 µg)/mL instead of
serum as described.29 Recombinant human SLF, IL-3, and
GM-CSF were kind gifts from Immunex Corporation (Seattle, WA) and were used per/mL, respectively, at 0 to 50 ng, 200 U, and 200 U. Recombinant human IL-6 was a gift from Genetics Institute (Cambridge, MA) and was
used at 10 ng/mL. Recombinant human erythropoietin (Epo) was purchased
from Amgen Corp (Thousand Oaks, CA) and was used at 1 U/mL. TGF-1
and TNF- were purchased from R&D Systems (Minneapolis, MN) and
Genentech, Inc (San Francisco, CA), respectively, and added at 5 ng and
20 ng/mL, respectively. After gene transduction at a population level,
TGF-1 and TNF- were added at the initiation of the colony assays.
After gene transduction at the single-cell level, TGF-1 and TNF-
were added 1 day after transduction to avoid the inhibitory effect on
cell growth, which could have resulted in a decrease in the gene
transduction efficiency. Cells were incubated under humidified
conditions at 37°C, 5% O2 and 5% CO2 for
14 days.
Retroviral vectors and packaging cell line.
To construct the retroviral vector encoding human (h) c-kit, a
polymerase chain reaction (PCR) was used with pCVN hc-kit
plasmid as a template. This plasmid encodes a full-length human
c-kit cDNA that contains the 12-bp insert encoding an
asparagine-rich insert in the extracellular domain (kit A+
isoform).50 The sense primer sequence was CTC GAG GCC
GCC ATG AGA GGC GCT CGC GGC GCC TGG. This primer contains a
synthetic XhoI site and Kozak consensus sequence (underlined).
The c-kit protein initiation codon is shown in bold. The
antisense primer used had a sequence of GGA TCC TCA
GAC ATC GTC GTG CAC AAG CAG and contains a synthetic BamHI site
(underlined). Bases complementary to the c-kit stop codon are
shown in bold. The product of the PCR reaction was cloned into the
pCRII vector (Invitrogen, Carlsbad, CA) and the insert sequence was
verified by automated DNA sequencing. The insert from this plasmid
clone was cloned into the BamHI site of the M5g Neo vector to
yield M5g hkit Neo. The M5g Neo vector was constructed by excision of
the murine c-kit sequence from the plasmid
M5gNeo-kitwt (a generous gift of Dr Hitoshi Kitayama, Osaka
University Medical School, Osaka, Japan).50 The M5g Neo
vector contains the long terminal repeat (LTR) of the
murine myeloproliferative sarcoma virus (MPSV) and encodes the sequence
for the bacterial neomycin phosphotransferase gene (Neo). This plasmid
has been previously described.51 Two transcripts are
derived from the M5g hkit Neo vector: a monocistronic message
containing sequences for human c-kit coding sequence as well as
Neo gene and an alternatively spliced message containing only the
Neo sequences (Fig
2).52 Vector lacking hkit cDNA (M5g Neo) was used as mock
virus control. The retroviral packaging cell lines,  -2 and PA317,
were maintained in Dulbecco's modified Eagle's medium, supplemented
with 10% FCS. High titer amphotropic PA317 packaging cells for M5g Neo
or M5g hkit Neo were produced by standard techniques.47 The
viral titers for both the PA317 M5g Neo and PA317 M5g hkit Neo
packaging cells were in the range of 1 to 3 × 106
G418 resistant (G418R) colony-forming unit (CFU)/mL on
NIH3T3 cells. Viral supernatant was harvested and filtered through
0.45-µm and 0.22-µm filters and stored at 80°C until
use.
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| Fig 2.
Diagram of retroviral vectors encoding human
c-kit cDNA used in the studies. (A) M5g Neo vector. (B) M5g
hkit Neo vector. The positions of the PCR primers and expected size of
amplified products are shown.
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Retroviral transduction protocol.
For population cell gene transduction, sorted CD34+++ cells
and their subsets of kit++, kit+, or
kitLo/ cells were prestimulated with IL-3 (200 U),
GM-CSF (200 U), Epo (1 U), and IL-6 (10 ng)/mL for 2 days and
transduced with viral supernatant as described.46-49 The
cells were then washed twice and plated for colony formation at 200 or
300 cells/mL for serum-containing or 800 cells/mL for serum-depleted
cultures in the presence of GM-CSF, IL-3, Epo, and IL-6, minus and plus
varying concentrations of SLF (2, 10, and 50 ng/mL). An alternative
protocol was used for single-cell gene transduction. Single cells were
directly sorted into single wells containing 0.1 mL methylcellulose in the presence of GM-CSF, IL-3, Epo, IL-6 at the same concentrations as
mentioned above, minus and plus varying concentrations of SLF (2, 10, and 50 ng/mL) in the serum-containing or serum-depleted cultures for 2 days. Viral supernatant was added once at 20 µL/well with polybrene
(8 µg/mL) as described previously.46-48 The cells remained in the same growth factor medium throughout the study. Two
days after addition of viral supernatant, fewer than 2% of the cells
had formed doublets and the predicted short half-life of the virus
preparation suggested that in more than 95% of the cases, single cells
had been transduced.
Statistics.
The probability of significant differences between cells plated at 200 or 800/mL (expressed as mean ± 1 standard error of mean
[SEM]) was determined by Student's t-test, and
that between groups in the single-cell experiments was determined by
 2 test.
PCR analysis.
Genomic DNA was isolated from individual colonies as previously
described.46-48 Briefly, individual colonies were removed
from methylcellulose culture medium and washed with 1 mL of
phosphate-buffered saline (PBS). Cell pellets were resuspended in the
small volume of remaining PBS to which 200 µL of a chelex 100 solution (200 to 400 mesh; BioRad, Richmond, CA) was added. Cells were
mixed well with chelex and lysed by boiling for 5 minutes, chilled on ice for 5 minutes, and pelleted for 30 seconds at 2,800g. A
total of 10 µL of supernatant from the lysate was used for PCR. The plasmid DNA of M5g hkit Neo was used as PCR positive controls. As a
negative control, DNA was obtained from cells incubated with viral
supernatant collected from packaging cell lines that had been
transfected with the retroviral vector lacking the hkit cDNA insert,
M5g Neo (mock control). A pair of primers was used at 1 µmol/L in the
PCR reaction (see Fig 2 for PCR primer positions). The following
primers were used: 5' TCT GCT TGT GCA CGA CGA TGT CT 3'
(residues: 2928-2950 sense strand from human c-kit cDNA sequences according to the numbering convention of Yarden
et al2) and 5' CAT GCG AAA CGA TCC TCA TCC 3'
(residues: 5464-5484 antisense strand from M5g Neo sequences). Each
sample was amplified with AmpliTaq (Perkin-Elmer/Cetus, Roche Molecular
Systems, Inc, Branchburg, NJ) using a DNA thermocycler
(Perkin-Elmer/Cetus) for 35 cycles (94°C for 30 seconds to denature
the DNA, 55°C for 30 seconds for primer annealing, and 72°C for
1 minute for primer extension) resulting in a 959-bp product. Ten
microliters of reaction mixture was electrophoresed on a 1% agarose
gel. To ensure that amplified products were the correct sequences,
Southern blot analysis was performed by standard methods. A 3.4-kb
fragment of human c-kit was isolated from pM5g hkit Neo plasmid
digested with BamHI and XhoI and used as probe.
Reverse transcriptase (RT)-PCR analysis.
RNA was extracted from CD34+ cells or individual colonies
as described previously.46-48 Briefly, colonies were
removed, cell pellets were resuspended in 20 µL double distilled
water treated with dimethyl pyrocarbonate (DEPC), 100 U RNasin, and 1 µg tRNA, cooled on ice for 30 minutes, and pelleted for 30 seconds. A
9-µL supernatant of the resulting RNA solution was used in the
reverse transcription reaction. To eliminate contaminating DNA, the RNA extracts were treated with 1 U DNase I for 15 minutes at room temperature. The DNase was inactivated by adding 1 µL of 20 mmol/L EDTA solution to the reaction mixture and heating at 65°C for 10 minutes before reverse transcription. A total of 20 µL of the RNA
lysate in RT buffer (RT buffer: 25 mmol/L Tri-HCL [pH 8.3], 37.5 mmol/L KCL, 1.5 mmol/L MgCl, 5 mmol/L dithiotreitol
[DTT], 10 mmol/L deoxyribonucleoside triphosphate
[dNTP] mixture), 0.5 µg oligo (dT)15
primer, 26 U RNasin, and 200 U murine Molony leukemia virus reverse
transcriptase (Promega Corp, Madison, WI) were used for reverse
transcription. After cDNA synthesis, 10 µL of the DNA solution was
used for the RT-PCR reaction. The primers and PCR conditions for the
RT-PCR reaction were the same as for PCR. To detect mRNA of SLF on
CD34+++ cells and their subsets, RNA was isolated from
cells and reverse transcription was performed. The following primers
were used: 5' TGG ATA AGC GAG ATG GTA GT 3' (residues:
388-407 sense strand from human SLF cDNA) and 5' TGG GTA GCA AGA
ACA GAT AAA (residues: 1124-1144 antisense strand from hyman SLF cDNA)
for PCR analysis. Each sample was amplified for 35 cycles (94°C for
1 minute to denature the DNA, 60°C for 1 minute for primer
annealing, and 72°C for 2 minutes for primer extension) resulting
in 757-bp and 673-bp products, respectively, representing soluble and
membrane-bound forms of SLF as described previously.16 A
1.0-kb fragment of human SLF was isolated from pBluescript SK-plasmid
digested with BamHI and HindIII and used as
probe.16 To detect mRNA c-kit expression on
CD34+ cells and their subsets, RNA was isolated and reverse
transcription was performed as above. The following primers were used:
5' CGT TGA CTA TCA GTT CAG CGA G 3' (residues: 843-864 sense strand from human c-kit cDNA) and 5' CTG GGA ATG
TGT AAG TGC CTC C (residues: 1180-1201 antisense strand from human
c-kit cDNA) for PCR analysis. Each sample was amplified for 35 cycles (94°C for 30 seconds to denature the DNA, 55°C for 30 seconds for primer annealing, and 72°C for 2 minutes for primer
extension) resulting in 360-bp products as
described.53 Human stromal cells
(NFF-6)16 were used as positive control for RT-PCR reaction
for SLF and c-kit, and PCR reaction reagents were used as
negative control for PCR analysis. -Actin was used as internal
control. The primers were as follows: 5'ATC TGG CAC CAC ACC TTC
TAC AAT GAG CTG CG 3' (sense) and 5' CGT CAT ACT CCT GCT
TGC TGA TCC ACA TCT GC 3' (antisense). The -actin primers were
used to amplify for 35 cycles (94°C for 45 seconds to denature the
DNA, 60°C for 45 seconds for primer annealing, and 72°C for 2 minutes for primer extension) resulting in an 838-bp fragment.
Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine
triphosphate (dUTP) nick end-labeling (TUNEL) assay.
Apoptosis was determined using the In Situ Cell Death Detection Kit
(Boehringer Mannheim, Indianapolis, IN) as described by the
manufacturer to detect the incorporation of labeled nucleotides to DNA
strand breaks by TUNEL assay. Briefly, cells were washed with PBS
containing 1% bovine serum albumin (BSA) and fixed by 2%
paraformaldehyde solution (Sigma, St Louis, MO) for 30 minutes at room
temperature and permeabilization was performed with 0.1% Triton-X-100
(Bio-Rad Laboratories, Richmond, CA) in 0.1% sodium citrate (Sigma) at
4°C for 2 minutes. The cells were then washed and labeled with
TUNEL reaction mixture at 37°C for 60 minutes in the dark. After
washing, cells were resuspended in cold PBS and analyzed by a FACScan
flow cytometer (Becton Dickinson).54
Enzyme-linked immunosorbent assay (ELISA).
To measure the soluble form of SLF endogenously produced and released
into the cultures, ELISA assay was applied using polyclonal antibody
against SLF with an ELISA kit (R&D Systems) as described.52 To measure soluble SLF, medium conditioned by CD34+, sorted
CD34+++kit+, and
CD34+++kitLo/ cells at 106
cells/mL or no cells were cultured in IMDM containing 10% FCS or
serum-depleted medium in the presence of IL-3, GM-CSF, IL-6, and Epo at
200 U, 200 U, 1 U, and 10 ng/mL for 72 hours at 37°C, 20%
CO2 and 5% O2. Medium was collected as
conditioned medium (CM) by centrifugation at 3,000 rpm
for 10 minutes. To measure cell-associated SLF, MACS-separated
CD34+ cells were washed extensively and whole cell lysates
were prepared using 1 mL of lysis buffer (20 mmol/L TRIS [pH 7.4], 1 mmol/L EDTA, 0.1% sodium dodecyl sulfate [SDS], 1% Triton X-100,
0.02% sodium azide, 1 mol/L NaCl). A postnuclear fraction of the whole cell lysate was obtained by pelleting the nuclei in a microcentrifuge and then collecting the supernatant. Soluble and cell-associated SLF
was measured using a commercially available ELISA kit (R&D Systems)
according to the manufacturer and read by a microplate reader (Titetek
Multiskan Plus) at optical density (OD) 450 nm. Standards
for soluble SLF were prepared using culture medium diluent, whereas
standards for cell-associated SLF were prepared using lysis buffer as a
diluent. The minimum detectable level of SLF is about 9 pg/mL.
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RESULTS |
Influence of c-kit gene transduction on colony formation by
CD34+++ cells.
CD34+++ cells were separated into 3 subsets denoted as
kit++, kit+, and kitLo/ cells
based on cell surface expression of c-kit protein (Fig 1).
Cells were transduced with c-kit or mock control virus using either bulk population or single-cell gene transduction
methods.46-49 Bulk population transduced cells were plated
in the presence of Epo (1 U/mL), IL-3 (200 U/mL), IL-6 (10 ng/mL),
GM-CSF (200 U/mL) without or with SLF at 2, 10, or 50 ng/mL in the
presence of FCS or in serum-depleted cultures.
Figure 3 shows the results from a
representative of 3 separate experiments using a serum-depleted culture
assay and cells plated at 800 cells/mL. Transduction of these cells
with c-kit, but not mock (neo only), retrovirus significantly increased colony formation by BFU-E and sometimes CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) in the
presence of GM-CSF, IL-3, IL-6, Epo with or without addition of SLF.
Transduction with c-kit cDNA had little or no effect on numbers
of colonies formed by CFU-GM. There was no difference between Neo only
virus and medium without virus (data not shown). The enhancing effect of transduction inversely correlated with c-kit protein
expression before transduction; the greatest effects were noted in
kitLo/ cells transduced with c-kit, either
without or with addition of SLF (2 to 50 ng/mL). For example,
transduction of kitLo/ cells with c-kit cDNA
followed by culture in the presence of GM-CSF + IL-3 + IL-6 + Epo
increased BFU-E and CFU-GEMM colonies up to 256% and 300%,
respectively, compared with the mock virus-transduced cells. In
contrast, transduction of c-kit cDNA and culturing in the same
cytokine combination resulted in an increase of 48% for BFU-E in
kit+ and 17% in kit++ cells and an increase of
54% for CFU-GEMM in kit+ and 38% in kit++
cells, respectively. Less enhancing effects were obtained when SLF was
added at 50 ng/mL. Similar results were observed in the presence of
serum (data not shown). It has been reported that the action of SLF is
particularly critical when levels of other growth factors are
limited.55 We thus tested to see if the effects would be
greater when one fourth concentrations of GM-CSF (50 U), IL-3 (50 U),
IL-6 (2.5 ng), and Epo (0.25 U)/mL were used. Similar, but not greater,
increases were noted in the absence or presence of different
concentrations of SLF (data not shown).
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| Fig 3.
Influence of cytokines on colony formation by
CD34+++kit++, kit+, and
kitLo/ cells transduced with c-kit cDNA. Sorted
cells were prestimulated with full dosages of IL-3 (200 U), GM-CSF (200 U), Epo (1 U), and IL-6 (10 ng/mL) and then transduced with
c-kit cDNA (M5g hkit Neo) or mock (M5g Neo) viruses as
described in Materials and Methods. After washing, cells were assayed
at 800 cells/mL for colony formation in semisolid culture in the
presence of IL-3, GM-CSF, and Epo with SLF at 0 (I), 2 (II), 10 (III),
and 50 (IV) ng per mL in the serum-depleted cultures as described in
Materials and Methods. Results are expressed as mean ± SEM from 1 representative of 3 separate experiments. Significant differences from
mock virus control are: aP < .01;
bP < .05.
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We previously reported that transduction and culture of single
CD34+++ cells with retroviruses encoding EpoR and/or IL-9R
increased Epo-dependent erythroid colony formation compared with mock
transduced cells.46-49 We therefore tested if transduction
of single cells with c-kit cDNA resulted in similar effects as
seen after transduction and culture of the bulk populations of cells.
The 3 subsets of c-kit expressing cells were directly deposited at a
frequency of 1 cell/well into wells containing full dosages of GM-CSF,
IL-3, IL-6, and Epo without or with SLF (2 to 50 ng/mL) in the presence or absence of serum for prestimulation. Single-cell gene transduction was performed as described previously.46-48 The results
from a total of up to 192 wells per point for 3 separate experiments using serum-depleted cultures are shown in
Fig 4. The increase in colony numbers of
progenitor cells transduced with c-kit cDNA was confirmed at
the single-cell level in the absence of serum. These effects were most
notable for BFU-E. This increase was more apparent in
kitLo/ cells, especially in the absence of
exogenously added SLF or the low concentration of SLF (2 ng/mL).
Similar results were obtained using serum-containing cultures (data not
shown). Our results suggest that c-kit transduction has a
direct growth enhancing effect on progenitors.
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| Fig 4.
Influence of cytokines on colony formation by single
sorted CD34+++ kit++,
kit+, and kitLo/ cells
transduced at the single-cell level with c-kit cDNA or mock
viruses. Single cells were directly sorted into wells as 1 cell/well
containing 0.1 mL of semisolid culture in the presence of full dosages
of IL-3 (200 U), GM-CSF (200 U), Epo (1 U), IL-6 (10 ng) with SLF at 0 (I), 2 (II), 10 (III), and 50 (IV) ng per mL in the serum-depleted
culture as described in Materials and Methods. Results are expressed as
percent wells with growth for a total of 192 wells per point from 3 separate experiments. Significant differences from mock virus control
are: aP < .05.
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c-kit transduction not only increased clonogenic efficiency of
progenitors, but also increased the size of individual BFU-E and
CFU-GEMM colonies (Fig 5). The number of
cells per BFU-E (106 × 103) and CFU-GEMM (109 × 103) colony from c-kit transduced cells was greater
than that of the mock-transduced cells (29 × 103 and
46 × 103). Interestingly, the number of cells per
CFU-GM colony was significantly increased as well in c-kit
transduced cells (5.2 × 103) compared with that of
mock-transduced cells (2.9 × 103).
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| Fig 5.
Influence of c-kit cDNA transduction on the size
of colonies derived from CD34+++ cells in the
presence of full dosages of IL-3, GM-CSF, Epo, and SLF (10 ng) per mL.
The 30 largest CFU-GM, BFU-E, and CFU-GEMM colonies from
c-kit-cDNA or mock-transduced cells were removed from a total
of 2 experiments and the cell numbers per colony were counted.
Significant differences from mock virus control are:
aP < .0001; bP < .05.
|
|
Influence of c-kit transduction on responsiveness of
kit++, kit+, and
kitLo/ cells to the inhibitory effects of
TGF-1 and TNF-.
One of the known effects of TGF-1 and TNF- on progenitor cells is
to suppress c-kit protein.52,56 Therefore, we
assessed if enforced expression of c-kit in progenitors could
influence the sensitivity of c-kit transduced cells to these
inhibitory cytokines. We tested the inhibitory effects of TGF-1 and
TNF- on colony formation using dosages ranging from 10 pg to 5 ng/mL and 62.5 U to 1,000 U/mL, respectively, added to cultures containing full dosages of SLF, IL-3, GM-CSF, and Epo using 3 subsets of CD34+++ cells (kit++, kit+, and
kitLo/), transduced in bulk cell populations with
c-kit cDNA or mock virus. We found a dose-dependent inhibitory
effect of TGF-1 and TNF- on colony formation in the
kit++, kit+, and kitLo/
populations of cells transduced with the mock control (M5gNeo) virus
and grown in the presence of serum (data not shown). Significant inhibition was detected at concentrations as low as 50 to 100 pg/mL of
TGF-1 and 125 U/mL of TNF- in kit++,
kit+, and kitLo/ cells. However, the
inhibitory effect was significantly reduced by 50% to 75% in cells
transduced with c-kit compared with the mock virus transduced
cells at the same dosages of TGF-1 and TNF- (data not shown). The
effects of c-kit transduction in reducing the sensitivity of
progenitor cells to TGF-1 was similar between the subsets of
kit++, kit+, and kitLo/ cells.
These results suggested that transduction with c-kit cDNA
reduced the sensitivity of progenitors to the growth inhibitory effects
of TGF-1 and TNF-. To further test this hypothesis, we transduced
single cells with c-kit cDNA and cultured them under serum-depleted conditions. The results summarized from 3 separate experiments are shown in Fig 6. Using
kitLo/ cells in the presence of full dosages of
GM-CSF, IL-3, IL-6, Epo, and SLF, transduction of c-kit reduced
the inhibitory effect of TGF-1 from 74% in mock virus-transduced
cells to 45% in c-kit-transduced cells (Fig 6I). Using
kit+ cells, similar, but slightly less, reduced inhibitory
effects were detected in c-kit transduced cells (48% decrease)
compared with mock-transduced cells (63% decrease) (Fig 6II). In
contrast, no significant reduction in inhibitory effects by TGF-1
was noticed in Kit++ cells transduced with either
c-kit (47% decrease) or mock (51% decrease) virus (Fig 6III).
Similar results were obtained when lower dosages of SLF (10 ng/mL) were
used (data not shown), and the same pattern was noticed in the presence
of serum (data not shown).
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| Fig 6.
Inhibitory effects of TGF-1 and TNF- on colony
formation from single sorted
CD34+++kitLo/ (I), kit+
(II), and kit++ (III) cells transduced with
c-kit cDNA or mock control and growth in the presence of full
dosages of IL-3, GM-CSF, Epo, and SLF with or without TGF-1 (5 ng)
or TNF- (20 ng) per mL in the serum-depleted cultures. Results are
expressed as percent wells with growth from a total of 96 wells per
point from 3 separate experiments. Significant differences for cells
transduced with c-kit cDNA from that with mock control;
aP < .05; significant differences for cells
incubated with TGF-1 or TNF- from medium control;
bP < .05.
|
|
Using kitLo/ cells in the presence of full dosages
of GM-CSF, IL-3, IL-6, Epo, and SLF and in the absence of serum,
transduction of c-kit reduced the inhibitory effect by TNF-
from 63% in mock-transduced cells to 48% in c-kit-transduced
cells (Fig 6I). Using kit+ cells, similar, but slightly
less, reduced inhibitory effects were detected in
c-kit-transduced cells (41% decrease) compared with mock
virus transduced cells (54% decrease) (Fig 6II). However, no
significant reduction in inhibitory effects by TNF- was obtained in
Kit++ cells transduced with c-kit (42% decrease)
compared with that of mock virus-transduced cells (42% decrease) (Fig
6III). Similar results were obtained when SLF was used at 10 ng/mL
(data not shown), and the same pattern was noticed in the presence of
serum (data not shown).
Proviral integration and expression of c-kit cDNA by PCR and RT-PCR
analysis.
Proviral integration and gene expression of c-kit cDNA were
determined from individual colonies by PCR and RT-PCR analysis using
primers that contained both c-kit cDNA and viral vector sequences as shown in Fig 2. Figure 7 shows
a sample of DNA integration and mRNA expression of the transduced
c-kit gene in cells from individual colonies derived from
single sorted kit++ and kit+ cells transduced
with c-kit cDNA and growth in the presence of full dosages of
GM-CSF, IL-3, IL-6, Epo, and SLF. DNA and RNA were extracted from
individual colonies derived from single sorted kit++ and
kit+ cells. Integration and expression of retroviral
c-kit was found in 47.6% and 50.5%, respectively, from a
total of 105 and 97 colonies analyzed. There was no difference in
integration or expression for kit++ and kit+
cells.
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| Fig 7.
Examples of integration and expression of transduced
c-kit cDNA by PCR (A) and RT-PCR (B) analysis. Sorted single
CD34+++kit++ and kit+
cells were prestimulated, transduced with c-kit cDNA or mock control
viruses, and growth in the presence of cytokine combination as
described in Materials and Methods. Individual BFU-E colonies were
removed and DNA and RNA were extracted for PCR and RT-PCR analysis. The
PCR generates a 959-bp fragment.
|
|
Detection of endogenous hSLF in cultures.
We noted that c-kit-transduced cells had enhanced colony
formation even in the absence of exogenously added SLF, suggesting that
endogenous SLF might be present in the cultures. Neutralizing antibodies against human SLF and c-kit (R&D Systems) were
assessed for their capacity to neutralize endogenous SLF activity
potentially present in our cultures. Sorted kit++,
kit+, and kitLo/ subsets of
CD34++ cells transduced with c-kit cDNA were
incubated with neutralizing antibodies against SLF (1 µg/mL) and/or
c-kit (100 ng/mL) in the presence and absence of serum with
full dosages of GM-CSF, IL-3, IL-6, and Epo/mL at room temperature for
1.5 hours. No exogenous SLF was added. The cells were then assayed at
300 cells/mL for serum-containing or 800 cells/mL for serum-depleted
cultures in the presence of the same cytokine combination and
antibodies. Results from 1 of 2 representative serum-depleted
experiments are shown in Fig 8. Either
anti-SLF or anti-c-kit partially blocked colony
formation stimulated by the cytokine combination in both c-kit
or mock transduced cells, suggesting the presence of endogenous SLF in
the cultures. Slightly greater, but not complete, neutralizing activity
was noted using anti-c-kit than anti-SLF. No further inhibition was seen using both anti-SLF and anti-c-kit
antibodies. Similar results were obtained in 2 experiments for
serum-containing cultures (data not shown). These results indicate that
endogenous SLF might contribute to the enhancing effects of
c-kit transduction in these cultures even in the absence of the
addition of SLF, but in the presence of GM-CSF + IL-3 + IL-6 + Epo.
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| Fig 8.
Effects of neutralizing antibodies against human SLF
and/or c-kit on colony formation by CD34+++
kitLo/ (I), kit+ (II), and
kit++ (III) cells transduced with c-kit cDNA in
the serum-depleted cultures. Transduced cells were treated with or
without neutralizing antibodies at room temperature for 1.5 hours and
assayed for colony formation at 800 cells/mL in the presence of full
dosages of IL-3, GM-CSF, IL-6, and Epo. Results are expressed as mean ± SEM from 1 of 2 representative experiments. Significant differences
for cells transduced with c-kit cDNA from that with mock virus control,
aP < .05. Significant differences for cells
treated with neutralizing antibodies from that of medium without added
antibodies as control, bP < .05.
|
|
To determine whether the cells were releasing soluble SLF protein,
conditioned medium (CM) was collected from 106
MACS-separated CD34+ cells or no cells in the presence or
absence of serum and presence of GM-CSF, IL-3, IL-6, and Epo and
assayed for SLF using an ELISA method. In 3 separate experiments, SLF
levels were below the level of assay sensitivity with or without serum
or cells. We then sought to determine if cell-associated SLF was
expressed by these cells. Whole cell lysates were prepared from
MACS-separated CD34+ cells and measured by ELISA. An
average of 60.4 ± 14.4 pg (n = 4) of SLF was detected in the
lysates from 3 × 105 cells per sample. The results
suggest that cell-associated SLF may contribute to the enhancing
effects of tranduction of c-kit. Because only small numbers of
cells were available, we were unable to compare the level of SLF in the
3 subsets of CD34+++ cells. Therefore, RT-PCR was used to
assess SLF expression in CD34+++ cells and their subsets.
Results from 4 experiments show that SLF expression was detected in all
3 subsets. Figure 9A shows a sample of SLF
expression by RT-PCR in the 3 subsets of cells. Interestingly, it seems
that the expression level of SLF was higher in
kitLo/ cells (lane 3) than in the other 2 subsets
(lanes 1 and 2). No obvious differences were noted in the level of SLF
in these 3 populations before and after c-kit gene transduction
(data not shown). We were unable to detect SLF expression on the cell
surface using a flow cytometric-based assay system.52
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| Fig 9.
Examples of expression of SLF (A) and c-kit (B)
by RT-PCR in c-kit subsets of CD34+++ cells in 1 representative of 4 experiments. RNA was extracted from cells and
reverse transcription was performed. PCR for SLF was performed using a
pair of primers as described in Materials and Methods, which generate
757-bp and 673-bp products, respectively, representing soluble and
membrane-bound SLF (A). PCR analysis for c-kit was performed
using a pair of primers as described in Materials and Methods, which
generates a 360-bp product (B). -Actin, used as internal control,
was performed using a pair of primers as described in Materials and
Methods, and generates an 838-bp product. RNA extracts
from human stromal cells (NFF) were used as positive control (+), and
PCR reaction reagents were used as negative control (). Lanes 1 through 3 are samples from CD34+++
kit++, kit+, and kitLo/
cells, respectively.
|
|
Influence of transduction of c-kit on expression of c-kit protein on
CD34+ cells and subsets by flow cytometer analysis.
The number of CD34+++ cells available was too small to
allow serial measurement of c-kit expression over time.
Therefore, MACS-separated CD34+ cells (90% to 95% purity
and with a 40% cloning efficiency for progenitors under optimal
cytokine combinations) were used for flow cytometry analysis with
monoclonal antibody against c-kit. Results from 5 separate
experiments showed that CD34+ cells expressed c-kit
on 64.2% ± 10% of the cells and this percent expression
declined to 40.2% ± 7% after 2 days of
prestimulation with full dosages of GM-CSF, IL-3, IL-6,
and Epo, but without the addition of SLF. After gene transduction, the
cells were incubated with the same cytokines for 24, 48, and 72 hours.
The percent of c-kit expressing cells transduced with
c-kit (80.2% ± 8%) was significantly greater than the
mock virus-transduced cells (50.2% ± 7%) at 48 hours
posttransduction. No significant decreases in percent of c-kit
expressing cells were seen by TGF-1 or TNF- in cells transduced
with either c-kit or control virus (data not shown). To compare
the influence of c-kit gene transduction on the c-kit
protein expression in subsets of CD34+++ cells,
kit++, kit+, and kitLo/
cells were prestimulated and transduced with c-kit cDNA.
c-kit expression on kitLo/ cells could also
be detected by RT-PCR analysis (Fig 9B). After transduction, the cells
were incubated with the same cytokine combination (without SLF) for 48 hours, and c-kit expression was analyzed by flow cytometry.
Results from 4 separate experiments are summarized in
Table 1. The percent of c-kit
expressing cells was increased in all 3 subsets of cells transduced
with c-kit cDNA compared with the mock virus-transduced cells.
The greater increase was seen in transduced kit+ (118%)
and kitLo/ (101%) cells as compared with the
kit++ cells (17%). Figure 10
shows a representative experiment of c-kit expressing cells in
the 3 c-kit subsets of CD34+++ cells transduced with
c-kit cDNA or mock virus as analyzed by flow cytometer. Not
only was the percent of c-kit expressing cells increased in the
3 subsets, but the fluorescence intensity was increased as well. The
mean peak fluorescence channel was increased by 24% ± 11%, 37% ± 22%, and 34% ± 10%, respectively, in
c-kit-transduced kit++, kit+, and
kitLo/ cells compared with mock virus-transduced
cells. The results indicate that transduction of c-kit
increased c-kit protein expression on the cell surface and
increased c-kit fluorescence intensity as well.
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|
Table 1.
Effect of Transduced c-kit on c-kit
Expression on Subsets of CD34+++ CB Cells Analyzed by
Flow Cytometry
|
|
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| Fig 10.
Representative analysis of c-kit protein
expression on CD34+++ kit++,
kit+, and kitLo/ cells transduced with
c-kit cDNA by flow cytometer. Sorted cells were prestimulated
with 10% FCS and full dosage of IL-3, GM-CSF, IL-6, and Epo and
transduced with c-kit cDNA (M5g hkit Neo) or mock (M5g Neo) control
viruses as described in Materials and Methods. The cells were washed
and incubated with the same cytokine combination for 48 hours and then
stained with monoclonal CD117/PE (solid line) or mouse isotope control
(dashed line) and analyzed by flow cytometer.
|
|
Influence of transduction of c-kit on apoptosis in MACS-separated
CD34+ cells as assessed by TUNEL assay.
One reported function of SLF is to inhibit
apoptosis.57 We tested whether enforced
expression of c-kit altered cellular susceptibility to
apoptosis. Summarized results from 4 experiments are shown in
Table 2. In mock virus-transduced cells,
TGF-1 and TNF- significantly increased the percent of apoptotic
cells from 28.6% ± 6% to 44.1% ± 4% and 36.2% ± 5%,
respectively, in the absence of SLF, but in the presence of full doses
of GM-CSF, IL-3, and Epo. Addition of SLF at 50 ng/mL resulted in
reduction of apoptotic cells to 14.8% ± 4% in mock
virus-transduced cells. Addition of TNF-, but not TGF-1, to
cultures of mock virus-transduced cells cultured with SLF slightly, but
significantly, increased apoptosis. Transduction of c-kit
induced a marked reduction of apoptotic cells (4.4% ± 3%) in the
absence of exogenously added SLF, and no further reduction was seen
with addition of SLF at 50 ng/mL to cultures of
c-kit-transduced cells. These results suggest that transduced
c-kit, even without exogenous addition of SLF, plays an
important role in preventing apoptosis, possibly due to the small
amount of endogenous cell-associated SLF expressed by these cells.
| |
DISCUSSION |
Studies of mice with partial or complete inactivating mutations of the
c-kit (W locus) or SLF (Sl locus) genes have highlighted the
essential role of an intact SLF/c-kit axis in maintaining steady state hematopoiesis. Mice with a complete deficiency of wild-type c-kit protein product are not viable due to severe anemia. Mice with partial function of c-kit protein are viable, but
have varying degrees of macrocytic anemia and tissue mast cell
deficiency.18 These studies of c-kit function in
murine hematopoiesis suggest that under certain conditions the amount
of functional c-kit protein is limiting to stem/progenitor cell proliferation.
We tested the hypothesis that increasing c-kit expression on
CD34+++ cells and their subsets with a human c-kit
encoding retrovirus would alter proliferation and/or differentiation.
Transduction of CD34+++kit++ cells with a
c-kit encoding retrovirus had only a small effect on the total
number of colonies formed compared with mock virus-transduced cells,
while transduction of CD34+++kit+ or
CD34+++kitLo/ cells with the
c-kit encoding retrovirus resulted in substantial increases in
total colony number when cells were plated in the presence of a
combination of cytokines in the presence and absence of serum. The
increase in colony numbers was due to a marked increase in BFU-E- and
a smaller increase in CFU-GEMM-derived colonies. Transduction of
c-kit did not significantly increase CFU-GM proliferation even
in the starting population of
CD34+++kitLo/ cells. In the absence of
exogenously added SLF, but in the presence of added GM-CSF, IL-3, and
Epo, the enhancing effects of transduced c-kit were more
significant in all 3 subsets of the cells than in the presence of added
GM-CSF, IL-3, Epo, and SLF. Moreover, the enhancing effect of
transduced c-kit on colony formation was confirmed at the
single-cell level and in the absence of serum. The largest effects of
the transduced c-kit were seen in
CD34+++kitLo/ cells grown in the
presence of GM-CSF, IL-3, and Epo, but in the absence of exogenously
added SLF under serum-depleted culture conditions.
The exact mechanism for the increase of erythroid and multipotential
progenitor cell proliferation in c-kit-transduced cells is not
clear. However, it is highly likely that this is directly due to the
increased c-kit protein expression by c-kit-transduced cells. The percent of c-kit expressing cells was significantly increased in the 3 subsets of CD34+++ cells transduced with
c-kit cDNA. Among the 3 subsets, the increase of c-kit
expressing cells was greater in the lower density of c-kit
(kit+ and kitLo/) expressing cells
before c-kit transduction. Not only was the percent of c-kit
expressing cells increased, but the fluorescence intensity was
increased as well. Likewise, the enhancing effect of c-kit
transduction on colony formation was greater in
c-kit-transduced kit+ and
kitLo/ cells than in kit++ cells.
We hypothesize that optimal hematopoiesis might require a certain
minimal level of c-kit function and that by increasing
c-kit expression in kit+ and
kitLo/ cells, a threshold level of expression is
reached thereby recruiting additional progenitors into a proliferative
state. However, there may be an upper limit to c-kit expression
above which no further biologic response is elicited due to maximal
stimulation of downstream signal transduction pathways. Alternatively,
heterogeneity of c-kit expression in CD34+++ cells
may also reflect differences in differentiation status. We have
reported previously that CD34+++kit++ cells are
enriched for CFU-GEMM and BFU-E, CD34+++kit+
cells are enriched for more primitive high proliferative
potential-colony-forming cell (HPP-CFC), while
CD34+++kitLo/ cells are enriched for
CFU-GM.10 Thus, transduction of c-kit into
different classes of HPC may result in disparate effects on differentiation.
Transduction of progenitors with c-kit may increase colony
formation at least in part by inhibition of apoptosis.57
Our data clearly show that apoptosis in the c-kit-transduced
cell population (4.4% ± 2%) was lower than in the mock
virus-transduced cell population (28.6% ± 6%), even in the
absence of exogenously added SLF. Our data suggest that SLF has
different dose response effects on apoptosis versus proliferation. In
c-kit-transduced cells, endogenous SLF is sufficient to
maximally inhibit apoptosis even in the presence of the inhibitory
cytokines TNF- and TGF-1. However, maximal colony growth is
obtained only with the addition of exogenous SLF.
Our colony formation experiments and apoptosis assays indicated a
biologic effect of c-kit transduction even in the absence of
exogenous SLF. The enhancing effects of c-kit transduction on
colony formation were blocked by neutralizing anti-SLF and/or anti-c-kit antibodies indicating that the observed
effect was likely due to interaction of SLF with its receptor. Similar
results were obtained in serum-depleted cultures indicating that the
progenitor cells rather than the serum was the source of endogenous SLF
in our system. Indeed, any SLF that might be present in our
serum-containing culture medium was below the level of detectability,
nor could we detect soluble SLF in conditioned medium from progenitor
cells. However, endogenous cell-associated SLF was detected in
MACS-separated CD34+ cells by ELISA assay. The results
suggest that cell-associated SLF is the major source of SLF in our
culture system and likely contributes to the enhancing effects of
transduced c-kit gene seen in the absence of exogenous SLF.
Because of the small number of subsets of CD34+ cells, we
were unable to compare the level of cell-associated SLF between the 3 subsets of cells using ELISA assay. However, using RT-PCR analysis,
transcripts for both the soluble and membrane-bound isoforms of SLF
were found in CD34+ cells and all 3 subsets of
CD34+++ (kit++, kit+, and
kitLo/) cells.
Taken together, our antibody neutralization studies, ELISA, and RT-PCR
experiments strongly suggest that endogenous SLF expression by
CD34+ cells may play a role in regulating progenitor
proliferation and/or apoptosis. Our results are in agreement with those
of Ratajczak et al58 who previously reported
that CD34+ cells transcribe SLF mRNA and that treatment of
such cells with antisense oligonucleotides complementary to SLF mRNA
reduced BFU-E and CFU-Mix, but not CFU-GM, colony formation.
TGF-1 appears to be a critical negative regulator of hematopoiesis.
One reported effect of TGF-1 on HPC is to repress expression of
c-kit protein by accelerating the degradation of c-kit
transcripts.9,52,56 Because the effects of c-kit on
CDK1 and Rb phosphorylation are potentially antagonistic to the effects
of TGF-1 on cell-cycle progression, we reasoned that the effect of
TGF-1 on c-kit expression might be critical to the overall
inhibitory effect of TGF-1 on HPC
proliferation/differentiation.35-37,59,60 Our data provide evidence that enforced expression of c-kit antagonizes the
antiproliferative effects of TGF-1.
TNF- is another complex regulator of hematopoiesis. The actions of
TNF- on HPC are biphasic and can be either stimulatory or inhibitory
depending on the target cell type. Additional complexity arises because
TNF- is a potent inducer of hematopoietic growth factor release by
accessory cells.61 A physiological role in regulating
primitive HPC proliferation is suggested by the report of significantly
increased numbers of Lin Sca1+
kit+ HPC in the bone marrow of mice with deficiency of
TNF-receptor p55 protein.62 The actions of TNF- on HPC
are poorly understood, but might be mediated via cell-cycle arrest or
increased apoptosis, as reported by several groups.63
Another reported action of TNF- is to accelerate the degradation of
c-kit transcripts.43,44 Signaling through the
c-kit receptor is associated with increased cell-cycle
progression and is antiapoptotic, especially for erythroid progenitors.
Our data provide evidence that enforced expression of c-kit
antagonizes the antiproliferative and/or antiapoptotic effects of
TNF-.
In summary, our experiments have demonstrated that transduction of
CD34+++ cells and their subsets with a c-kit
encoding retrovirus increases growth factor-dependent erythroid and
multipotential progenitor cell proliferation. These data are consistent
with other data concerning the in vitro and in vivo biological effects
of c-kit and further support the notion that optimal growth of
HPC may require c-kit receptor. Transduction of progenitors
with c-kit cDNA was associated with increased expression of
c-kit protein, decreased spontaneous or cytokine-induced
apoptosis, and expression of endogenous cell-associated SLF. In
addition, c-kit transduction of CD34+++ cells and
their subsets decreased the sensitivity of these cells to the
inhibitory effects of TGF-1 and TNF-.
| |
ACKNOWLEDGMENT |
We thank Rebecca Miller and Linda Cheung for secretarial assistance and
Drs Jie He, Yung-xing Li, and Hong-Jun Liu for technical assistance.
| |
FOOTNOTES |
Submitted November 10, 1998; accepted June 4, 1999.
L.L. and M.C.H. contributed equally to this work.
Supported by Public Health Service Grants No. RO1 HL 56416, RO1 HL
54037, RO1 DK 53674 and a project in RO1 HL-53586 from the National
Institutes of Health (to H.E.B.), grants from the Phi Beta Psi Sorority
and Genetics Institute (to L.L.), and a VA Merit Review Grant from the
Department of Veterans Affairs (to M.C.H.).
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 reprint requests to Li Lu, MD, the Walther Oncology Center,
Indiana University School of Medicine, 1044 W Walnut St, Room 302, Indianapolis, IN 46202-5254.
| |
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ABSTRACT
INTRODUCTION