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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 527-536
D3: A Gene Induced During Myeloid Cell Differentiation of
Linlo c-Kit+ Sca-1+ Progenitor
Cells
By
Sarah R. Weiler,
John M. Gooya,
Mariaestela Ortiz,
Schickwann Tsai,
Steven J. Collins, and
Jonathan R. Keller
From the Laboratory of Molecular Immunoregulation, Division of Basic
Sciences, and the Intramural Research and Support Program, Science
Applications International Corp-Frederick, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick,
MD; and the Fred Hutchinson Cancer Research Center, Seattle, WA.
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ABSTRACT |
In an effort to characterize molecular events contributing to
lineage commitment and terminal differentiation of stem/progenitor cells, we have used differential display reverse
transcription polymerase chain reaction (DDRT-PCR) and
cell lines blocked at two distinct stages of differentiation. The cell
lines used were EML, which is representative of normal multipotential
primitive progenitors (Sca-1+, CD34+,
c-Kit+, Thy-1+) able to differentiate into
erythroid, myeloid, and B-lymphoid cells in vitro, and MPRO, which is a
more committed progenitor cell line, with characteristics of
promyelocytes able to differentiate into granulocytes. One clone
isolated by this approach was expressed in MPRO but not in EML cells
and contained sequence identical to the 3 untranslated region of
D3, a gene cloned from activated peritoneal macrophages of unknown
function. We have observed a novel pattern of D3 gene expression and
found that D3 is induced in EML cells under conditions that promote
myeloid cell differentiation (interleukin-3 [IL-3], stem cell factor
[SCF], and all-trans-retinoic acid [atRA]) starting at 2 days,
corresponding to the appearance of promyelocytes. D3 RNA
expression reached a maximum after 5 days, corresponding to the
appearance of neutrophilic granulocytes and macrophages, and decreased
by day 6 with increased numbers of differentiated neutrophils and
macrophages in vitro. Induction of D3 RNA in EML was dependent on IL-3
and was not induced in response to SCF or atRA alone or SCF in
combination with 15 other hematopoietic growth factors (HGF) tested.
Similarly, D3 was not expressed in the normal bone marrow cell (BMC)
counterpart of EML cells, Linlo c-Kit+
Sca-1+ progenitor cells. D3 RNA expression was induced in
these cells when cultured for 7 days in IL-3 plus SCF. A comparison of
the expression of D3 RNA in cell lines and normal BMC populations demonstrated that D3 is induced during macrophage and granulocyte differentiation and suggests a potential physiological role for D3 in
normal myeloid differentiation.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
HEMATOPOIETIC development results in the
formation of eight morphologically and functionally distinct blood cell
lineages from pluripotential hematopoietic stem cells (PHSC). Mature
cells have a limited life span and are continuously replenished from a
pool of PHSC throughout an individual's lifetime. Significant progress
has been made in characterizing the PHSC and in understanding the
growth factors/cytokines required for their survival, growth, and
differentiation. Although less is known about the molecular events
involved in PHSC differentiation, progress has been made in identifying
lineage- and stage-specific genes and transcription factors
contributing to the development and differentiation of normal
PHSC.1-3 Genes involved in hematopoiesis have been
identified using a variety of techniques. For example, studies of
naturally occurring mutations in the mouse and oncogene-induced
malignancy have elucidated critical roles for c-myb, c-myc, c-kit, and
c-fos in hematopoiesis.3 Other genes have been identified
using molecular approaches such as polymerase chain reaction (PCR) or
low stringency hybridization to isolate genes with sequence similarity
to known genes (eg, MZF-1) or by using subtractive hybridization
techniques. Other genes, for example, AML1 and PLZF, have been
identified by mapping sites of chromosome translocations or by their
aberrant expression in leukemias.3
Several properties of stem cells have made the isolation and
characterization of genes involved in stem cell development difficult. First, PHSC are present at low frequency in the bone marrow (0.01% to
0.005% of nucleated bone marrow cells).4 Second, even
while these cells can be greatly enriched, they may still be
heterogeneous. Therefore, to circumvent these difficulties, we used a
murine lymphohematopoietic progenitor cell line, EML,5 to
identify developmentally regulated genes. This cell line was generated by insertion of a retrovirus carrying a dominant negative retinoic acid
receptor gene (RAR 403) into bone marrow cells. EML cells are
phenotypically similar to primitive murine hematopoietic stem cells,
defined by Li and Johnson6 as Linlo
c-Kit+ Sca-1+ cells that are highly enriched
for PHSC. EML cells are stem cell factor (SCF)-dependent and can
differentiate into erythroid, myeloid, megakaryoctye, mast, and
B-lymphoid cells in vitro. Myeloid differentiation is blocked in EML
due to the expression of RAR403, but the block can be overcome by
treatment with supraphysiological concentrations of all-trans-retinoic
acid (atRA). A similarly derived cell line, MPRO,7 is
granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent
and is blocked at a more committed, promyelocyte stage of
differentiation. MPRO cells possess morphology (enlarged cytoplasm) and
cell surface marker expression (Mac-1 and 8C5 positive) characteristic
of promyelocytes. Thus, the EML and MPRO cell lines are temporally
related (separated by approximately 48 hours of differentiation) and
provide a unique model to examine differences in gene expression
between two distinct stages of myeloid differentiation.
In this study, we determined (1) whether the EML and MPRO cell lines
could be used as a model system to isolate novel and potentially
important genes involved in stem/progenitor cell growth and
differentiation and (2) the expression pattern of such isolated genes
in various lineages and whether these genes were regulated during
myeloid cell differentiation. To accomplish this, we used differential
display reverse transcription-polymerase chain reaction (DDRT-PCR).
This method was designed to analyze changes in gene expression by
amplifying short cDNA sequences representative of mRNAs present in cell
populations. The amplified cDNAs are displayed on a sequencing gel,
allowing both comparative analysis of gene expression during
differentiation and the reamplification and cloning of differentially
expressed cDNAs.8 Two clones isolated by this procedure
have novel 3 sequence: one is expressed in EML but not MPRO, and
one is expressed at higher levels in EML than MPRO. A third clone,
expressed in MPRO but not in EML, was identified as the previously
described gene, D3.9 The function of D3 is unknown, and its
potential role in myeloid differentiation has not been characterized;
therefore, we chose to focus on the regulation of this gene during EML
cell and normal hematopoietic cell differentiation.
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MATERIALS AND METHODS |
Cell lines and cytokines.
EML cells were maintained in Iscove's modified Dulbecco's medium
(IMDM), 20% horse serum (GIBCO-BRL, Gaithersburg, MD), 2 mmol/L
L-glutamine, and 1% penicillin-streptomycin supplemented with 8% to
15% BHK/MLK conditioned media (CM), as a source of SCF, or 100 ng/mL
recombinant SCF (Peprotech, Rocky Hills, NJ) (EML complete medium). To
induce the differentiation of EML cells, complete media was
supplemented with 30 ng/mL interleukin-3 (IL-3; Peprotech) and
10 5 mol/L atRA (Sigma, St Louis, MO). MPRO and
EPRO5 cells were maintained in DMEM, 10% fetal calf serum
(FCS; Gemini Bio-Products, Calabasas, CA), 2 mmol/L L-glutamine, and
1% penicillin-streptomycin, supplemented with 20 ng/mL recombinant
murine GM-CSF (Peprotech), and were differentiated with addition of
atRA (105 mol/L). The following cell lines were
maintained in RPMI 1640, 10% FCS, 2 mmol/L L-glutamine, and 1%
penicillin-streptomycin, and supplemented as indicated: FDC-P1 (IL-3
dependent),10 32D-Cl3,11 32D-Cl23,11 DA-3,12 and M-NFS-60
(ATCC, Manassas, VA), with 10% Wehi-3b conditioned media;
FDC-P1 (IL-4 dependent) with 100 ng/mL IL-4 (Peprotech); and
CTLL-213 with 20 ng/mL IL-2 (Cetus, San Francisco, CA).
HCD-5714 was maintained in IMDM, 30% FCS, 5 × 105 mol/L 2-mercaptoethanol, 2 mmol/L L-glutamine,
1% penicillin-streptomycin, and 5 U/mL recombinant human
erythropoietin (EPO; R.W. Johnson Pharmaceutical Research
Institute, Raritan, NJ). NIH 3T3 (ATCC) cells were maintained in DMEM,
10% calf serum (GIBCO-BRL), 2 mmol/L L-glutamine, and 1%
penicillin-streptomycin.
Isolation and purification of normal bone marrow cells (BMC).
Animal care was provided in accordance with the procedures outlined in
the "Guide for the Care and Use of Laboratory Animals," NIH
Publication No. 86-23, 1985. Normal murine BMC were obtained by
aspirating the femurs of 12-week-old female Balb/c mice. Light-density BMC (LDBMC) were separated by centrifugation on lymphocyte separation medium (Organon Teknika Corp, Durham, NC). Cells were washed twice in
IMDM and resuspended in IMDM supplemented with 10% FCS (complete IMDM). LDBMC were purified to remove committed hematopoietic cells according to a previously described protocol.4 Briefly,
LDBMC were resuspended in complete IMDM and incubated at 4°C for 30 minutes with a cocktail of antibodies, Ly-6G (Gr-1), CD4 (L3T4), CD45R
(B220), CD11b (Mac-1), and CD8 (all from Pharmingen, San Diego, CA).
Cells were washed twice and resuspended in complete IMDM and magnetic
beads (Dynal, Great Neck, NY) were added at a ratio of 40:1
(beads/cell). The mixture was incubated for 30 minutes at 4°C and
the cells were separated using a magnetic particle concentrator
resulting in a lineage-low (Linlo) cell population.
To isolate Linlo c-Kit+ progenitors,
Linlo cells were directly labeled with phycoerythrin
(PE)-conjugated monoclonal antibodies (MoAbs) that recognize c-Kit or
isotype-matched control antibodies (Pharmingen). Cells were incubated
for 30 minutes at 4°C with the recommended concentration of
antibodies, washed twice in complete IMDM, resuspended in the same
medium to approximately 2 to 5 × 107 cells/mL, and
separated by fluorescence-activated cell sorting (FACS; Becton
Dickinson Co, San Jose, CA). To isolate Linlo
c-Kit+ Sca-1+ progenitor cells,
Linlo cells were further purified by two-color FACS using
fluorescein isothiocyanate (FITC)-conjugated c-Kit
antibodies and PE-conjugated Ly6A/E (Sca-1) MoAbs (Pharmingen).
Normal B220+ B cells were purified by flow cytometry from a
fresh suspension of mouse spleen cells using FITC-conjugated anti-B220 MoAbs or isotype-matched control antibodies (Pharmingen).
GR-1+ granulocytes were isolated by flow cytometry from
freshly aspirated cell suspensions of unfractionated mouse BMC using
FITC-conjugated anti-8C5 MoAbs (Pharmingen). To isolate
Ter119+ erythroid cells, unfractionated mouse BMC were
labeled with biotinylated MoAbs that recognize Ter119 or
isotype-matched control, stained with avidin-conjugated PE, and
separated by FACS.
Soft agar colony formation.
To measure colony formation of EML cells in vitro, cells were suspended
in 1 mL of EML complete media and 0.3% sea plaque agarose (FMC,
Rockland, ME) with cytokines and plated in 35-mm Lux petri dishes
(Miles Scientific, Naperville, IL), incubated at 37°C in 5%
CO2 for 7 to 10 days, and scored for colony growth (>50
cells).
Proliferation assays.
To measure the proliferative response of EML cells to cytokines, cells
were washed three times in complete media and plated at 1 × 104 cells/mL in microtiter plates in triplicate with single
cytokines: 100 ng/mL SCF, 30 ng/mL IL-3, 50 ng/mL GM-CSF, 100 ng/mL
IL-7 (Peprotech), 100 ng/mL IL-6 (Peprotech), 5 U/mL EPO, 50 ng/mL granulocyte colony-stimulating factor (G-CSF; Amgen, Thousand Oaks,
CA), 100 ng/mL macrophage colony-stimulating factor (M-CSF; Peprotech),
20 ng/mL IL-1 (Hazleton, Vienna, VA), 500 U/mL interferon- (IFN-; Biosource Int, Camarillo, CA), and 20 ng/mL transforming growth factor  (TGF; Oncogene Corp, Seattle, WA).
Cells were incubated for 48 hours and pulsed with 1 µCi
3H-thymidine (6.7 Ci/mmol/L; NEN, Boston, MA) for the last
6 to 8 hours of incubation and then harvested (Tomtech Harvester 96; Tomtech, Inc, Orange, CT) onto glass-fiber filter paper (Filtermat A;
Wallac Oy, Turku, Finland). Filter strips were dried and counted by
liquid scintillation.
RNA isolation, electrophoresis, and Northern blotting.
Total RNA was purified using RNeasy (Qiagen, Chatsworth, CA) as
outlined by the manufacturer. Separation of RNA samples (10 to 15 µg)
by electrophoresis was performed on 1% agarose, 5.2% formaldehyde
(37% solution), 1× MOPS gels. The running buffer was 1×
MOPS, 5% formaldehyde. RNA was transferred to nylon membrane (MSI,
Westboro, MA) and hybridized at 60°C in Church-Gilbert
hybridization solution15 (0.5 mol/L NaPO4, pH 7.0, 1 mmol/L
EDTA, 1% bovine serum albumin [BSA], 7% sodium dodecyl sulfate
[SDS]). cDNA probes were random prime labeled using Prime-It II
(Stratagene, La Jolla, CA) following the manufacturer's instructions.
To quantitate the levels of expression, Northern blots were stripped
and reprobed with either hu--actin or G3PDH (Clontech, Palo Alto,
CA). Signals were quantitated, relative to controls, by scanning
densitometry.
DDRT-PCR and cDNA sequencing.
Total RNA from EML and MPRO was isolated as described above and treated
with DNase I to remove DNA using MessageClean (GenHunter Corp,
Brookline, MA). DDRT-PCR was performed using RNAimage
(GenHunter) as described by the manufacturer. First, in three separate
reactions using one of three primers (H-T11G,
H-T11A, and H-T11C), RNA samples were reverse
transcribed. Each of the first-strand cDNA pools was then amplified in
the presence of -35S-dATP in 4 separate reactions with
either H-AP1 (5-AAGCTTGATTGCC-3), H-AP2
(5-AAGCTTCGACTGT-3), H-AP3
(5-AAGCTTTGGTCAG-3), or H-AP4 (5-AAGCTTCTCAACG-3) in combination with the primer used
to reverse transcribe the sample. PCR products were separated on 6%
denaturing polyacrylamide gels that were dried onto 3M
paper (3MM Whatman, Maidstone, UK) and exposed to film.
Bands that consistently appeared to be differentially expressed in two
separate PCR reactions were isolated, reamplified, and cloned into the
vector, pCR II (Invitrogen, Carlsbad, CA). The inserts were sequenced
by automated sequencing (ABI Model 373, Foster City, CA)
using M13 primers.
RT-PCR.
Total RNA was isolated as described, and 100 ng was reverse transcribed
in 5 mmol/L MgCl2, 1× PCR buffer, 1 mmol/L dCTP, 1 mmol/L dGTP, 1 mmol/L dATP, 1 mmol/L dTTP (Perkin Elmer Core Reagents; Roche, Branchburg, NJ), 1 U/µL RNase Inhibitor (Promega, Madison, WI), and 2.5 U/µL Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL). cDNA from 4 to 5 ng of total RNA input was then amplified by PCR using Perkin Elmer Core reagents as specified by the
manufacturer. For amplification of actin, the primer sequences used
were as follows: 5 GAC ATG GAG AAG ATC TGG CAC 3 and
5 GAT CTT CAT GGT GCT AGG AG 3. Standard conditions for
PCR were denaturation at 94°C for 2 minutes; 25 cycles of 94°C
for 1 minute, 60°C for 1 minute, and 72°C for 1 minute; and
extension at 72°C for 7 minutes. For amplification of D3/204, the
primers used were as follows: 5 TCG GCT AAG AAC CAA AAA TCA CA
3 and 5 CAC TCC CCA CAA CTT CTA TCC TTC 3. The
full-length cDNA clones for D3 and 204 were generous gifts from Dr
Thomas Hamilton (Research Institute, Cleveland Clinic Foundation,
Cleveland, OH) and Dr Peter Lengyel (Department of Biophysics and
Biochemistry, Yale University, New Haven, CT), respectively. Standard PCR conditions were as indicated
above, except that cycle number was increased to 30.
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RESULTS |
Isolation of cDNA clones by DDRT-PCR.
To isolate and characterize genes that are regulated during myeloid
differentiation, DDRT-PCR was employed using EML and MPRO cell lines.
Using 12 different primer pairs for comparison, 15 differentially
expressed cDNA clones were obtained, 3 of which were confirmed to be
differentially expressed in the EML and MPRO cell lines by Northern
blot analysis (Fig 1A). One cDNA clone, designated EG4C1, hybridized to an 8.7-kb transcript expressed in EML
cells that was absent in MPRO cells. The second cDNA clone, EG4C2,
hybridized to transcripts of 2.7 and 1.5 kb that are downregulated in
MPRO cells compared with EML cells. The third cDNA clone, MC2C5, hybridized to a 1.6-kb transcript expressed in the MPRO cell line that
was absent in EML cells.
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| Fig 1.
(A) Northern blot analysis of RNA extracted from the EML
and MPRO cell lines. The blots were probed with partial cDNA clones
isolated by DDRT-PCR and reprobed with human -actin. (B) Northern
blot analysis of RNA extracted from MPRO, EPRO, and EML cell lines and
EML cells treated with IFN for 16 hours or IL-3/SCF/atRA for 6 days.
The blot was probed with the 3 UTR of the D3 gene and reprobed
with G3PDH. (C) Northern blot analysis of RNA extracted from EML cells
treated for 1 or 2 days with (+) or without ( ) CM, SCF, IL-3, and
atRA. The blot was probed with the 3 UTR of D3 gene and reprobed
with G3PDH. (D) Northern blot analysis of RNA extracted from EML cells
cultured for 0 to 144 hours in IL-3/BHK CM/atRA. The blot was probed
with D3 (top), MPO (center), and G3PDH (bottom).
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The nucleotide sequences of the three differentially expressed cDNAs
were determined and compared with sequences in gene databases. No
significant homology was found for the EG4C1 and EG4C2 clones, whereas
the nucleotide sequence of the MC2C5 clone was identical to the
3 untranslated region (UTR) of the previously described gene,
D3, of unknown function. The D3 gene was initially shown to be induced
during macrophage activation in response to IFN-, IFN-, and
lipopolysaccharide (LPS)16 and is a member of a family of
genes called the 200 family.
D3 gene expression is induced in response to IL-3/SCF/atRA in EML.
To determine whether D3 is induced during myeloid cell differentiation,
EML cells were induced to differentiate by culturing with SCF, IL-3,
and atRA. D3 gene expression was analyzed by Northern blots
with RNA from MPRO, EPRO, EML, and EML cells cultured for 6 days in
IL-3, BHK conditioned media (CM) as a source of SCF, and atRA that were
probed with the 3 UTR specific to D3 (Fig 1B). The EPRO cell
line is similar to MPRO, except that it was derived directly from the
EML cell line by inducing EML differentiation with atRA for 2 days,
generating cells blocked at the promyelocyte stage.5 As
predicted, EPRO cells express approximately the same level of D3 RNA as
MPRO cells, whereas EML cells show no detectable expression. However,
D3 RNA is induced in EML cells cultured in IL-3, SCF (BHK CM), and atRA
for 6 days. Furthermore, because D3 was originally shown to be rapidly
induced by IFN-, EML cells were also cultured in SCF and IFN- for
16 hours. Lane 1 of Fig 1B demonstrates that, in EML cells, D3 is not
induced in response to IFN-.
To determine what combinations of SCF, IL-3, and atRA were required to
induce D3 expression, EML cells were cultured in recombinant SCF (rSCF)
in the presence and absence of IL-3 and atRA. First, Northern blot
analysis confirmed that D3 gene expression was induced with rSCF plus
IL-3 and atRA to the same extent and with the same kinetics (27%
between days 1 and 2) as with CM plus IL-3 and atRA (28% between days
1 and 2; Fig 1C). Second, neither CM nor rSCF alone or in combination
with atRA was able to induce D3 expression. Third, IL-3 alone was not
able to induce D3 expression within 48 hours of IL-3 stimulation (data
not shown). Finally, low levels of D3 gene expression were observed
when EML cells were cultured in a combination of IL-3 and rSCF for 2 days. Thus, IL-3 in combination with SCF is required to induce D3 RNA
expression in EML cells. This effect is greatly enhanced in the
presence of atRA.
To investigate whether other hematopoietic growth factors (HGF), with
positive and negative effects on proliferation of EML cells and normal
progenitor cells, could induce D3 expression, EML cells were cultured
in rSCF in the presence or absence of atRA in combination with other
HGF (GM-CSF, EPO, G-CSF, M-CSF, IL-7, IL-6, IL-1, IFN-, or TGF)
for 2 days. To determine whether EML cells have biologically active
receptors for these HGF, proliferation assays were performed with
single HGF. Table 1 shows that EML cells
respond to SCF, IL-3, GM-CSF, IL-6, IL-7, and TGF, indicating the
presence of functional receptors for these HGF. Only the combination of
IL-3 and SCF was sufficient to activate D3 gene expression, with much higher levels observed in the presence of atRA.
Kinetics of D3 gene expression in EML during myeloid differentiation.
To determine the kinetics of D3 gene expression, RNA was obtained from
EML cells treated with SCF, IL-3, and atRA for 0 to 6 days. Northern
blot analysis demonstrated that D3 expression was induced beginning at
day 2 (48 hours), with maximal expression observed at day 5 (70%
increase in expression between days 2 and 5) that decreased after 6 days (Fig 1D). When D3 gene expression was compared with myloperoxidase
(MPO), a granule protein known to be expressed in maturing neutrophils,
MPO gene expression showed two peaks of expression. First, there was a
rapid increase in MPO RNA levels 2 to 4 hours after treatment that
returned to baseline levels of expression after 8 hours of treatment in
culture. Then, a second peak of MPO RNA expression occurred at day 4, which decreased at day 5, and further decreased to levels below
background by day 6 in culture. Thus, D3 is not induced rapidly (within
8 hours) in response to growth and differentiation signals, but rather corresponds to the second peak of MPO expression that correlates with
the appearance of maturing granulocytes in culture5 (see below).
D3 expression in hematopoietic cell lines.
To determine whether D3 expression is lineage restricted, RNA was
obtained from a panel of hematopoietic cell lines of various lineages
and Northern blots were hybridized with D3 specific probes. First,
seven myeloid progenitor cell lines were tested, each of which is
blocked at a different stage of differentiation, although all are
thought to be blocked at stages more primitive than promyelocytes. Of
the seven, only NSF-58 contained D3 RNA expression levels comparable to
MPRO cells (Table 2). Low but detectable
expression was observed in FDC-P1 cultured in IL-3. Second, two T-cell
lines (CTLL-2 and EL4) showed no D3 RNA expression, and no expression
was detected in an erythroid cell line (HCD-57). Finally, one (Wehi-3b)
of two myelomonocytic leukemia cells tested expressed D3 RNA. As a
positive control for D3 expression, the macrophage cell line GG2EE was
stimulated with IFN- to induce D3 mRNA expression. Thus, the
expression pattern in cell lines indicates that D3 is restricted to
cells of the myeloid lineage and that expression is absent in cells
that represent more primitive stages of myeloid differentiation.
D3 expression in normal tissues and normal hematopoietic cells.
This analysis was extended to determine the expression of D3 in normal
murine tissues. On multiple tissue Northern blots probed with a D3
specific probe, the highest mRNA levels were observed in the lung, with
lower levels of expression detected in heart, spleen, and skeletal
muscle (Fig 2A). D3 message was not
detected in the brain, liver, kidney, or testis.
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| Fig 2.
(A) Northern blot analysis of poly A+ RNA extracted
from normal mouse tissues. The blots were probed with a D3 specific
probe (top) and reprobed with human -actin (bottom). (B) Northern
blot analysis of RNA extracted from BMC, lineage-depleted BMC
(Lin), and Lin cells purified by flow
cytometry for expression of c-Kit (Lin
c-Kit+). The blots were probed with the D3 specific probe
(top) and equal loading of samples was verified by ethidium bromide
staining of rRNA (bottom). (C) RT-PCR analysis of D3 expression (left)
in Lin c-Kit+ Sca-1+ BMC
cultured 0, 3, and 7 days in IL-3/SCF. Amplification of actin from each
cDNA sample is shown (right).
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To determine the expression of D3 in normal hematopoietic cells, we
isolated RNA from normal unseparated BMC, Linlo progenitor
enriched BMC, and Linlo BMC further purified by flow
cytometry for cells that express c-Kit. Little or no D3 RNA was
detected in unseparated BMC; however, higher RNA levels were obtained
in the progenitor purified Linlo and in Linlo
c-Kit+ cells (Fig 2B). On normal BMC Northern blots probed
with the D3 specific 3 UTR probe, two bands (~1.6 and 1.8 kb)
were detected as opposed to the single, 1.6-kb transcript detected on
the cell line Northern blots.
We further purified the Linlo, c-Kit+
population using antibodies that recognize Sca-1 and flow cytometry to
enrich for the most primitive progenitor cells, which phenotypically
represent the normal counterparts of EML cells. Because low numbers of
highly purified cells were obtained by this purification process, a PCR approach was developed to detect D3 message. The primer pairs used for
the analysis amplify both D3 and the highly homologous family member,
204. However, the amplification products can be distinguished by
restriction enzyme digestion with Cla I, which digests D3 once
but does not cut within the 204 cDNA (demonstrated using plasmids
containing cDNAs for either gene; see Fig 4A). Linlo
c-Kit+ Sca-1+ cells were cultured for 0, 3, or
7 days in IL-3 plus SCF, after which RNA was isolated and reverse
transcribed with oligo (dT) and cDNA was amplified with the D3/204
primers (Fig 2C). To verify that equal quantities of cDNA were used in
each PCR reaction, cDNAs were titrated and amplified with
actin-specific primers and demonstrated to be equal within a linear
range of amplification (data not shown). Low levels of amplified
product were observed at days 0 and 3 in Linlo
c-Kit+ Sca-1+ cells, with a significant
increase detected after 7 days in culture using the D3/204
amplification primers. Similarly, low levels of D3 expression were
observed in EML cells at day 0, even though no D3 RNA can be detected
by Northern blot analysis. Next, to determine whether the amplification
product was D3 or 204, the amplification product from day 7 RNA was
reamplified in a second round of PCR to obtain adequate amounts of
material for the digests. The purified PCR fragments were then digested
with Cla I. Figure 3B shows that
the message amplified from Linlo c-Kit+
Sca-1+ day 7 was completely digested with Cla I,
indicating that the amplification product was D3 and not 204. In
summary, little or no D3 expression is detected in primitive
Linlo c-Kit+ Sca-1+ cells, but
treatment of cells with the combination of SCF plus IL-3 induces D3 RNA
expression, supporting the view that the EML cell line provides a model
for this progenitor cell stage.
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| Fig 3.
RT-PCR analysis of D3 expression in cell lines and in
normal hematopoietic cells. (A) Plasmids containing the full-length
cDNAs for D3 and 204 were purified and amplified using D3/204 primers,
which amplifies a 505-bp band visualized by ethidium bromide staining.
The amplification products were then purified and treated with (+) or
without () Cla I. RNA extracted from EML, EML cultured in
IL-3/SCF/atRA for 6 days, MPRO, and EPRO cell lines were reverse
transcribed with oligo (dT), amplified with D3/204 primers, and treated
with (+) or without () Cla I. (B) RNA from normal
hematopoietic cells was purified and analyzed for D3 expression by
RT-PCR as indicated above.
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Using the PCR-based detection method for D3, we examined RNA obtained
from normal T cells (thymocytes), B220+ B cells,
8C5+ granulocytes, and Ter-119+ erythroid
cells. Figure 3 shows that the PCR product amplified in MPRO and EPRO
cell lines is exclusively D3 (completely digested by Cla I),
whereas normal B220+ B cells exclusively express the 204 RNA species (completely undigested by Cla I). In contrast,
8C5+ granulocytes, T cells, and Ter-119E+
erythroid cells express both D3 and 204 RNAs.
Finally, to determine whether D3 is expressed and/or induced
during macrophage differentiation, we isolated total RNA from unseparated normal BMC and BMC cultured in M-CSF for 0 to 5 days (conditions that promote terminal unilineage macrophage differentiation in vitro). As shown in Fig 2, Northern blots probed with D3 detected little or no expression in unseparated normal BMC
(Fig 4, lane 1). In addition, high levels
of D3 RNA expression were observed after 1 day in cultures supplemented
with M-CSF that were sustained until day 5, when D3 RNA expression
levels decreased to the level observed in unfractionated BMC (Fig 4).
This correlated with the appearance of mature differentiated
macrophages (data not shown). Thus, D3 is upregulated during macrophage
differentiation and is downregulated upon maturation. As previously
reported, D3 expression can be induced again in mature macrophages when
they are activated with interferons.9
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| Fig 4.
Induction of D3 expression in differentiating bone marrow
macrophages. RNA was extracted from normal BMC treated 0 to 5 days in
M-CSF in vitro as described in Materials and Methods. RNA was analyzed
on Northern blots, which were probed with D3 (top), and equal loading
of RNA samples was verified by ethidium bromide staining of rRNA
(bottom).
|
|
Kinetics of morphological changes and colony formation in EML cells
during differentiation.
To determine whether there was a correlation between biological events
associated with EML cell differentiation and D3 gene expression, we
performed a kinetic analysis of morphological changes and the
production of more differentiated HGF-responsive colony-forming cells
in differentiating EML cell cultures. Morphological changes in EML
cells undergoing myeloid differentiation were assessed by culturing
cells for 0 to 6 days in SCF and atRA with or without IL-3. On days 0, 2, 4, and 6, cells were cytospun and stained with Wright-Giemsa stain.
Before treatment, approximately 95% of the EML cells were
undifferentiated blasts (myeloblast) and promyelocytes
(Table 3). By 2 days in IL-3, SCF, and
atRA, the percentage of undifferentiated blasts/promyelocytes decreased to 78%, with an increase in granulocytic neutrophils with segmented or
banded nuclei and macrophages with enlarged vacuolated cytoplasm (to
13% and 9%, respectively). After 4 days, 50% of the EML cells appear
as undifferentiated blasts/promyelocytes, whereas 35% of cells are
granulocytic neutrophils and 15% of EML are macrophages. On day 6, 34% of EML cells were undifferentiated blasts/promyelocytes, 44% were
granulocytic neutrophils, and 22% were macrophages. These morphological changes are dependent on the addition of IL-3. GM-CSF in
combination with SCF is unable to promote morphological changes associated with atRA-induced myeloid differentiation (data not shown).
Over the same time course, production of more differentiated
HGF-responsive colony-forming units-culture (CFU-c) was assessed by
taking aliquots of cells cultured in SCF and atRA with or without IL-3
and replating them in soft agar colony assays containing either GM-CSF,
EPO, or SCF (Fig 5A through C). The number
of GM-CSF-responsive CFU-c generated from 1 × 105
EML cells cultured in SCF, IL-3, and atRA went from 12 on day 0 to a
maximum of 2,000 on day 3. In contrast, the maximum number of
GM-CSF-responsive colonies produced in cultures containing only the
combination of SCF and atRA was 26 on day 3. The number of
EPO-responsive colonies produced in cultures of EML in IL-3, SCF, and
atRA reached a maximum of 850 on day 4 versus none generated by culture
with SCF and atRA alone. Lastly, the number of SCF-responsive colonies
remained fairly constant over time in cultures containing SCF/IL-3 and
atRA, whereas there was an increase in SCF-responsive colonies from 2.5 × 104 to 2 × 105 generated with SCF
plus atRA (the same increase was seen in cultures with SCF alone).
Taken together, using two different assays to measure differentiation
(morphology and generation of CFU-c), D3 RNA expression in EML cells
correlates with the appearance of morphologically differentiated
neutrophils and the generation of more mature cells responsive to
GM-CSF and EPO.
View larger version (14K):
[in this window]
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| Fig 5.
Induction of HGF-responsive CFU-c progenitors from EML
cells. EML cells were induced to undergo myeloid differentiation
according to the procedures described in Materials and Methods. (A) The
total number of GM-CSF-responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3.
(B) The total number of EPO-responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3.
(C) The total number of SCF-responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3.
( ) SCF + RA + IL-3; ( ) SCF + RA.
|
|
| |
DISCUSSION |
It is becoming increasingly clear that hematopoiesis is regulated, in
part, by proteins that regulate growth, commitment, and differentiation
and that aberrant expression of these regulatory proteins can
contribute to leukemogenesis. In this study, our goal was to address
whether the EML cell line could be used as a model to isolate and
characterize novel genes involved in normal hematopoiesis. To this end,
we performed DDRT-PCR using RNA from the EML and MPRO cell lines and
demonstrated that these cell lines are particularly suited to
differential screening techniques, because they represent clonal
populations of cells arrested at distinct, temporally related stages of
myeloid differentiation. The utility of these cell lines in allowing us
to isolate molecules involved in normal myeloid differentiation can be
quickly confirmed by comparing expression in normal stem/progenitor
cell populations. Also, because EML can be induced to differentiate
into other lineages depending on the culture conditions used, we
predict that the system described here may be similarly useful in
isolating and characterizing genes involved in erythroid,
megakaryocyte, mast, and lymphoid cell differentiation.
In this report, we demonstrated the isolation of two novel cDNAs and a
cDNA identical to a previously described gene, D3, of unknown function.
The D3 gene was originally isolated from peritoneal macrophages treated
with macrophage activating agents, including interferons and
LPS.16 D3 is a member of a multigene cluster of
interferon-inducible genes (200 family). There are at least four and
possibly six members in the mouse (p202, p204, p203, and D3) and three
human homologues (MNDA, IFI16, and AIM2) that map to a conserved
linkage group in human band region 1q21-32 and distal mouse chromosome
1.17-22 However, there is currently no known biological
function for any of the 200 family members of interferon-inducible
genes.
We have demonstrated here that D3 is induced during myeloid
differentiation by using both cell line models and normal cells allowing us to determine (1) the growth factors/cytokines involved in
the induction of D3 expression, (2) the kinetics of this expression during myeloid differentiation, and (3) the lineage restriction of D3
gene expression. D3 RNA is not expressed in the multipotential progenitor cell line, EML; however, it is expressed in the
promyelocytic cell line MPRO and can be induced in EML cells in culture
conditions that promote myeloid cell differentiation (IL-3 plus rSCF
plus atRA). We have demonstrated that, in EML cells, the induction appears to be independent of interferons and that D3 is not induced in
response to atRA alone, distinguishing it from retinoic acid-inducible genes. In light of the lack of effect observed on hematopoiesis in
IL-3-deficient mice,23 it is interesting to note that IL-3 appears to be required for EML cells to differentiate and for the
induction of D3, which cannot be substituted with GM-CSF or other HGF
tested to date. This suggests that either there are uncharacterized
factors in vivo with activities that overlap with IL-3 or that
dependence on IL-3 for differentiation may be a unique property of the
EML cell line.
The relevance of D3 expression to normal hematopoietic cell
differentiation was demonstrated using Linlo
c-Kit+ Sca-1+ BMC (EML cell equivalent) and
Linlo cells (MPRO cell equivalent). Like the EML cells,
Linlo c-Kit+ Sca-1+ cells do not
express D3; however, expression can be induced by culturing these cells
in medium containing IL-3 plus SCF. We are currently testing whether D3
RNA can be induced in normal Linlo c-Kit+
Sca-1+ primitive BMC in response to HGF other than the
combination of IL-3 and SCF. Like the MPRO and EPRO cell lines,
Linlo cells express high levels of D3 RNA. Our results
demonstrate that the EML and MPRO cell lines can be successfully used
to isolate novel and potentially important genes involved in normal
myeloid differentiation and that these cell lines are at least
partially representative of their normal cellular counterparts in terms of gene expression patterns.
EML cells undergoing myeloid differentiation begin to express D3 RNA
after 24 to 48 hours; expression peaks after 5 days and decreases by
day 6 with the appearance of increasing numbers of terminally
differentiated neutrophils and macrophages. These relatively slow
kinetics of IL-3/SCF-induced D3 RNA expression in EML cells (24 to 48 hours by Northern blot analysis) suggest that the regulation of D3 gene
expression is a complex process requiring multiple downstream events
from receptor engagement. For example, IL-3 in combination with SCF may
promote EML cells to synthesize transcription factors, receptors,
and/or cytokines that, in turn, induce D3 gene expression.
Whereas D3 RNA expression was clearly observed on Northern blots after
48 hours (Fig 1), with peak expression after 4 to 5 days in EML cells,
the onset of D3 expression in normal BMC treated with M-CSF occurred 24 hours earlier than in EML cells (Fig 4). Maximum D3 expression in
M-CSF-treated BMC cultures was observed between days 1 and 2, with low
levels of expression detected by day 5 when the culture consisted of
greater than 90% terminally differentiated macrophages. We believe
that differences in the kinetics of induction of D3 could be explained, in part, by the state of differentiation of EML progenitor cells (primitive) and M-CSF-induced BMC cultures (committed). Thus, D3 RNA
expression is temporally regulated during granulocyte and macrophage
differentiation.
In an effort to understand the tissue and lineage restriction of D3
expression, we have examined panels of tissue blots, cell lines, and
purified normal cells. Analysis of RNA obtained from normal mouse
tissues showed no D3 expression in brain, liver, kidney, or testis.
Modest levels of D3 were detected in the lung, with lower levels
observed in spleen, skeletal muscle, and heart. Because lung tissue
contains activated macrophages, we predicted this tissue to be positive
for D3. Also, because spleen contains a small percentage of myeloid
cells, this could account for the low expression seen in that site. The
expression of D3 in heart and skeletal muscle suggests a potential role
for this gene in these tissues.
Our survey of RNA expression in a wide range of hematopoietic cell
lines showed that D3 RNA expression is highly restricted to the myeloid
lineage. D3 gene expression was not detected in two T-cell lines tested
(EL4 and CTLL-2), and no expression was detected in an erythroid cell
line (HCD-57). Furthermore, D3 was not expressed in six of seven
myeloid progenitor cell lines. Although all of these cell lines are
blocked in differentiation at slightly distinct stages, they represent
myeloid cells developmentally more primitive than promyelocytes. This
suggests that not only is D3 expression restricted to cell lines of the
myeloid lineage, but also to myeloid cells at or beyond the
promyelocyte stage.
To determine whether D3 showed lineage restriction in normal
hematopoietic cells, RNA was obtained from purified cell populations of
various lineages and analyzed for D3 gene expression by RT-PCR. Using
this highly sensitive, nonquantitative analysis, we were able to
determine that splenic B220+ B cells do not express D3 RNA;
however, expression was detected in Gr-1+ granulocytes, Ter
119+ erythroid cells, and thymic T cells. Although D3 gene
expression could be detected by RT-PCR in thymocytes, we believe that
the level of expression is extremely low, because D3 was not detected by Northern blot analysis of thymocyte RNA (data not shown). Finally, D3 RNA expression was induced in nomal BMC cultured in M-CSF, which
promotes monocyte/macrophage lineage differentiation. Taken together,
these results indicate that D3 is expressed in differentiating granulocytes and macrophages and possibly in erythroid cells.
There is currently no known biological function for D3 or any of the
200 family members of interferon-inducible genes. All of the family
members are structurally related and contain one or two copies of a
highly homologous 200 amino acid domain. Like MNDA, D3 contains one of
the 200-amino acid homology domains, whereas the other family members
contain two of these domains.9 Although the subcellular
localization of D3 is unknown, other members of this family, including
p202, p204, MNDA, and IFI 16, localize to the
nucleus,24,25 and the open reading frame of D3 encodes a
highly basic protein of 425 amino acids containing putative nuclear
localization sequences.9 Interestingly, p202 has been
demonstrated to bind a variety of proteins involved in both cell cycle
regulation and transcription, including retinoblastoma protein
(pRB),26 p53-binding protein 1 (53BP-1),27
NF- B, and AP-1.28 The p202 protein has also been
demonstrated to inhibit E2F-mediated transcription.29,30
These results suggest that the 200 family proteins may mediate
pleotropic effects on growth and differentiation by binding DNA
directly or, more likely, through protein-protein interactions. We are
currently testing whether D3 has DNA and/or protein binding
activity.
The interferon family of cytokines has been shown to mediate a large
number of biological effects, including antiviral, antibacterial, immunomodulatory, and antiproliferative activity.31 These
effects are the result of receptor-mediated signaling events that lead to the induction of gene expression of a variety proteins, including the 200 family. For example, D3 can be induced in terminally
differentiated macrophages by exposure to interferons.9 In
addition to the immunomodulatory effects, interferons have been shown
to modulate cellular differentiation.31 In this regard, we
have shown here for the first time that D3 expression is induced during
normal myeloid cell growth and differentiation by HGF other than
interferons, including SCF and IL-3. It is currently not known what
role D3 and the other 200 family members play in regulating these
diverse effects, which include the immunomodulatory actions of the
interferons and the growth and differentiation effects induced by
SCF/IL-3. We are currently investigating whether the relationship
between D3 expression and myeloid differentiation is causal.
| |
ACKNOWLEDGMENT |
The authors are grateful to Drs Sally Spence, Joost Oppenheim, Peter
Donovan, Peter Johnson, and Simon Williams for their review of this
manuscript.
| |
FOOTNOTES |
Submitted June 17, 1998;
accepted September 22, 1998.
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the US Government.
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 Jonathan R. Keller, PhD, Intramural
Research and Support Group, Science Applications International
Corp-Frederick, National Cancer Institute-Frederick Cancer Research and
Development Center, Frederick, MD 21702-1201.
| |
REFERENCES |
1.
Kehrl JH:
Hematopoietic lineage commitment: Role of transcription factors.
Stem Cells
13:223, 1995[Abstract]
2.
Shivdasani RA, Orkin SH:
The transcriptional control of hematopoiesis.
Blood
87:4025, 1996[Free Full Text]
3.
Tenen DG, Hromas R, Licht JD, Zhang D-E:
Transcription factors, normal myeloid development, and leukemia.
Blood
90:489, 1997[Free Full Text]
4.
Spangrude GJ, Heimfeld S, Weissman IL:
Purification and characterization of mouse hematopoietic stem cells.
Science
241:58, 1988[Abstract/Free Full Text]
5.
Tsai S, Bartemez S, Sitnicka E, Collins S:
Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development.
Genes Dev
8:2831, 1994[Abstract/Free Full Text]
6.
Li CL, Johnson GR:
Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization.
Blood
85:1472, 1995[Abstract/Free Full Text]
7.
Tsai S, Collins SJ:
A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage.
Proc Natl Acad Sci USA
90:7153, 1993[Abstract/Free Full Text]
8.
Liang P, Pardee AB:
Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.
Science
257:967, 1992[Abstract/Free Full Text]
9.
Tannenbaum CS, Major J, Ohmori Y, Hamilton TA:
A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes.
J Leukoc Biol
53:563, 1993[Abstract]
10.
Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D:
Growth of factor-dependent hemopoietic precursor cell lines.
J Exp Med
152:1036, 1980[Abstract/Free Full Text]
11.
Greenberger JS, Eckner RJ, Sakakeeny M, Marks P, Reid D, Nabel G, Hapel A, Ihle JN, Humphries KC:
Interleukin-3-dependent hematopoietic progenitor cell lines.
Fed Proc
42:2762, 1983[Medline]
[Order article via Infotrieve]
12.
Ihle JN, Morse HC 3rd, Keller J, Holmes KL:
Interleukin-3 dependent retrovirus induced lymphomas: Loss of the ability to terminally differentiate in response to differentiation factors.
Curr Top Microbiol Immunol
113:86, 1984[Medline]
[Order article via Infotrieve]
13.
Gillis S, Ferm MM, Ou W, Smith KA:
T cell growth factor: Parameters of production and a quantitative microassay for activity.
J Immunol
120:2027, 1978[Abstract/Free Full Text]
14.
Ruscetti SK, Janesch NJ, Chakraborti A, Sawyer ST, Hankins WD:
Friend spleen focus-forming virus induces factor independence in an erythropoietin-dependent erythroleukemia cell line.
J Virol
64:1057, 1990[Abstract/Free Full Text]
15.
Church GM, Gilbert W:
Genomic sequencing.
Proc Natl Acad Sci USA
81:1991, 1984[Abstract/Free Full Text]
16.
Hamilton TA, Bredon N, Ohmori Y, Tannenbaum CS:
IFNgamma and IFN-beta independently stimulate the expression of lipopolysaccharide-inducible genes in murine peritoneal macrophages.
J Immunol
142:2325, 1989[Abstract]
17.
Seldin MF, Morse HC 3rd, Reeves JP, Scribner CL, LeBoeuf RC, Steinberg AD:
Genetic analysis of autoimmune gld mice I. Identification of a restriction fragment length polymorphism closely linked to the gld mutation within a conserved linkage group.
J Exp Med
167:688, 1988[Abstract/Free Full Text]
18.
Seldin MF, Morse HC, LeBoeuf RC, Steinberg AD:
Establishment of a molecular genetic map of distal mouse chromosome 1: Further definition of a conserved linkage group syntenic with human chromosome 1q.
Genomics
2:48, 1988[Medline]
[Order article via Infotrieve]
19.
Kingsmore SF, Watson ML, Howard TA, Seldin MF:
A 6000 kilobase segment of chromosome 1 is conserved in human and mouse.
EMBO J
8:4073, 1989[Medline]
[Order article via Infotrieve]
20.
Peters J
(ed):
Man and mouse homology map, in Mouse News Letter 84. Bristol, UK, Arrowsmith, 1989, p 52.
21.
Briggs RC, Briggs JA, Ozer J, Sealy L, Dworkin LL, Kingsmore SF, Seldin MF, Kaur GP, Athwal RS, Dessypris EN:
The human myeloid cell nuclear differentiation antigen gene is one of at least two related interferon-inducible genes located on chromosome 1q that are expressed specifically in hematopoietic cells.
Blood
83:2153, 1994[Abstract/Free Full Text]
22.
DeYoung KL, Ray ME, Su YA, Anzick SL, Johnstone RW, Trapani JA, Meltzer PS, Trent JM:
Cloning a novel member of the human interferon-inducible gene family associated with control of tumorigenicity in a model of human melanoma.
Oncogene
15:453, 1997[Medline]
[Order article via Infotrieve]
23.
Nishinakamura R, Miyajima A, Mee PJ, Tybulewicz VL, Murray R:
Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.
Blood
88:2458, 1996[Abstract/Free Full Text]
24.
Choubey D, Lengyel P:
Interferon action: Nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster.
J Cell Biol
116:1333, 1992[Abstract/Free Full Text]
25.
Choubey D, Lengyel P:
Interferon action: Cytoplasmic and nuclear localization of the interferon-inducible 52-kD protein that is encoded by the Ifi 202 gene from the gene 200 cluster.
J Interferon Res
13:43, 1993[Medline]
[Order article via Infotrieve]
26.
Choubey D, Lengyel P:
Binding of an interferon-inducible protein (p202) to the retinoblastoma protein.
J Biol Chem
270:6134, 1995[Abstract/Free Full Text]
27.
Datta B, Li B, Choubey D, Nallur G, Lengyel P:
p202, an interferon-inducible modulator of transcription, inhibits transcriptional activation by the p53 tumor suppressor protein, and a segment from the p53-binding protein 1 that binds to p202 overcomes this inhibition.
J Biol Chem
271:27544, 1996[Abstract/Free Full Text]
28.
Min W, Ghosh S, Lengyel P:
The interferon-inducible p202 protein as a modulator of transcription: Inhibition of NF-kB, c-Fos, and c-Jun activities.
Mol Cell Biol
16:359, 1996[Abstract]
29.
Choubey D, Li S-J, Datta B, Gutterman JU, Lengyel P:
Inhibition of E2F-mediated transcription by p202.
EMBO J
15:5668, 1996[Medline]
[Order article via Infotrieve]
30.
Choubey D, Gutterman JU:
Inhibition of E2F-4/DP-1-stimulated transcription by p202.
Oncogene
15:291, 1997[Medline]
[Order article via Infotrieve]
31.
Pestka S, Langer JA, Zoon KC, Samuel CE:
Interferons and their actions.
Annu Rev Biochem
56:727, 1987[Medline]
[Order article via Infotrieve]
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Top
Abstract
Introduction