U.S. patent application number 10/295732 was filed with the patent office on 2003-06-05 for proteins suppressing proliferation of lympho-hematopoietic cells.
Invention is credited to Kincade, Paul W., Matsuzawa, Yuji, Oritani, Kenji, Tomiyama, Yoshiaki.
Application Number | 20030104569 10/295732 |
Document ID | / |
Family ID | 14454196 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104569 |
Kind Code |
A1 |
Oritani, Kenji ; et
al. |
June 5, 2003 |
Proteins suppressing proliferation of lympho-hematopoietic
cells
Abstract
A novel protein having the activity to suppress proliferation of
lympho-hematopoietic cells derived from BNS2.4 cells, its gene, a
method for preparing them and their uses are provided. The novel
protein has been identified from a stromal cell line BMS2.4 by
expression cloning targeting mouse myelomonocytic leukemia cell
line WEHI3. This protein and its gene are useful for treating
lympho-hematopoietic disorders.
Inventors: |
Oritani, Kenji; (Osaka,
JP) ; Tomiyama, Yoshiaki; (Hyogo, JP) ;
Matsuzawa, Yuji; (Hyogo, JP) ; Kincade, Paul W.;
(Oklahoma City, OK) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
14454196 |
Appl. No.: |
10/295732 |
Filed: |
November 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10295732 |
Nov 14, 2002 |
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09523686 |
Mar 13, 2000 |
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6518043 |
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Current U.S.
Class: |
435/69.1 ;
435/183; 435/320.1; 435/325; 435/7.23; 514/19.6; 514/7.5; 514/7.9;
536/23.2 |
Current CPC
Class: |
C07K 14/4703 20130101;
A61P 35/02 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61K
38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/7.23; 435/320.1; 435/325; 435/183; 514/12; 536/23.2 |
International
Class: |
G01N 033/574; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06; A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 1999 |
JP |
11/107246 |
Claims
What is claimed is:
1. A protein that suppresses the proliferation of
lympho-hematopoietic cells selected from the group consisting of:
(a) a protein comprising the amino acid sequence as set forth in
SEQ ID NO: 3; (b) a protein comprising a derivative of the amino
acid sequence set forth in SEQ ID NO: 3, in which one or more amino
acids are substituted, deleted, inserted, and/or added; and (c) a
protein encoded by a DNA hybridizing with the DNA comprising the
nucleotide sequence as set forth in SEQ ID NO: 1.
2. The protein according to claim 1, wherein the
lympho-hematopoietic cells are selected from the group consisting
of B lineage cell line, 1 A9, BC7.12, BC7.7, F10, 2E8, 18-81,
7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and
myelomonocytic leukemia cell line WEHI3.
3. A DNA encoding the protein according to claim 1.
4. The DNA of claim 3, comprising the coding region of the
nucleotide sequence as set forth in SEQ ID NO: 1.
5. A vector comprising the DNA of claim 3.
6. A host cell retaining the vector of claim 5.
7. A method for preparing the protein of claim 1, the method
comprising culturing the host cell of claim 6 and recovering
recombinant proteins expressed in the cell from the cultured cell
or culture supernatant thereof.
8. An antibody to the protein according to claim 1.
9. A peptide fragment of the protein according to claim 1.
10. A DNA specifically hybridizing with the DNA comprising the
nucleotide sequence as set forth in SEQ ID NO: 1 and comprises at
least 15 nucleotides.
11. A method for screening a compound binding to the protein
according to claim 1, the method comprising (a) contacting the
protein of claim 1 or its fragment with a test sample, (b)
detecting the binding activity of the test sample to the protein or
its fragment, and (c) selecting a compound that binds to the
protein or its fragment.
12. A compound that is isolable by the method according to claim 11
and binds to the protein of claim 1.
13. A method for screening a compound that interferes with the
activity of the protein according to claim 1 to suppress
proliferation of lympho-hematopoietic cells, the method comprising
(a) contacting the protein according to claim 1 with
lympho-hematopoietic cells in the presence of a test sample, (b)
detecting the proliferation of the cells, and (c) selecting a
compound that interferes with the activity of the protein according
to claim 1 to suppress proliferation of lympho-hematopoietic cells
as compared with a control where the detection is performed in the
absence of the test compound.
14. The method according to claim 13, wherein said
lympho-hematopoietic cells are selected from the group consisting
of B lineage cell lines, 1A9, BC7.12, BC7.7, F10, 2E8, 18-81,
7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and
myelomonocytic leukemia cell line WEHI3.
15. A compound isolable by the method according to claim 13, which
interferes with the activity of the protein according to claim 1 to
suppress proliferation of lympho-hematopoietic cells.
16. A pharmaceutical composition comprising the protein according
to claim 1 as an active ingredient.
17. The pharmaceutical composition according to claim 16, wherein
the composition is for treating lympho-hematopoietic disorders.
18. A pharmaceutical composition comprising the compound according
to claim 12 as an active ingredient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel polypeptide derived
from BMS2.4 cells, a gene thereof, a method for preparing the
polypeptide and the gene, and uses thereof.
BACKGROUND OF THE INVENTION
[0002] Production of blood cells is strictly regulated by various
stromal elements including adhesion molecules, extracellular
matrix, and cytokines. Complex interactions between stromal and
hematopoietic cells are essential for the movement of hematopoietic
stem/progenitor cells within or from bone marrow, for the control
of production of blood cells, and for the elimination of defective
and harmful cells.
[0003] Long-term bone marrow cultures are in vitro system that
mirror some in vivo relationships and provide an approach to define
molecular and cellular interactions that may regulate production of
blood cells. Pre-B cells could be displaced from the adherent layer
of long-term bone marrow cultures by addition of antibodies to very
late antigen-4 (VLA-4) or vascular cell adhesion molecule-1, and
these reagents completely blocked lymphopoiesis in Whitlock-Witte
(W/W) cultures. Antibodies to CD44 completely blocked production of
lymphoid and myeloid cells in long-term bone marrow cultures.
Addition of an antibody to CD9 cause strong adhesion between
myeloid and stromal cells, and blocked the production of myeloid
cells in Dexter cultures. These molecules might participate in
cell-cell interactions critical for adhesion and movement of
maturing hematopoietic cells in bone marrow.
[0004] Extracellular matrix delivers signals for survival and/or
expansion of lympho-hematopoietic cells as well as immobilizes
growth factors. Binding of fibronectin to integrins augments
responsiveness of hematopoietic stem/progenitors to
colony-stimulating factors. In contrast, interactions between
fibronectin and VLA-5 cause apoptosis in a myeloid cell line.
Hyaluronan, a ligand of CD44, forms viscous and hydrated gels and
facilitates cell-cell adhesion and cell migration. Thrombospondin
binds to hematopoietic progenitors, and hemonectin to myeloid
precursors. Matrix glycoprotein SC1/ECM2 binds to B lineage cells
and enhances their growth. Osteonectin not only acts as an
anti-adhesive molecule but also immobilizes platelet derived growth
factor.
[0005] Proliferation of primitive hematopoietic progenitors is
regulated by interacting groups of cytokines. Interleukin-3 (IL-3),
IL-4, and granulocyte/macrophage-colony-stimulating factor (GM-CSF)
support proliferation of progenitors that exit from the dormant
state. IL-6, IL-11, IL-12, granulocyte-CSF (G-CSF), and leukemia
inhibitory factor (LIF) work synergistically with IL-3, IL-4, and
GM-CSF to support proliferation of multipotential progenitors from
cell-cycle dormant progenitors. Stem cell factor (SCF) and
flt3-ligand not only support self-renewal of hematopoietic stem
cells but also function as co-factors with other cytokines in
promoting differentiation and expansion of hematopoietic
progenitors. On the other hand, transforming growth factor-.beta.
(TGF-.beta.), tumor necrosis factor-.alpha. (TNF-.alpha.),
interferon-.alpha./.beta. (IFN-.alpha./.beta.), and IFN-.gamma. are
downregulators of lympho-hematopoiesis. Growth arrest and/or
apoptosis of hematopoietic cells are induced by these factors.
These regulatory cytokines are typically made in extremely small
quantities in hematopoietic organs. Some of them are capable of
attachment to extracellular matrix, and certain other cytokines are
synthesized as transmembrane as well as soluble forms.
[0006] A number of genes that may be involved in
lympho-hematopoiesis have been identified by experiments using
cloned stromal cell lines that were originally selected for the
ability to support proliferation and/or differentiation of a
particular type of hematopoietic cells. A stromal cell line, BMS2,
that was established from adherent cells of long-term bone marrow
cultures has been known to have capacity to support growth of pre-B
cells (Pietrangeli, C. E. et al., Eur. J. Immunol., 18: 863-872,
1988). However, BMS2.4 cells, a subclone of BMS2, revealed unique
characteristics that they interfered with proliferation of
hematopoietic cells (Kincade, P. W. et al., Adv. Exp. Med. Biol.,
292: 227-234, 1991). However, its molecular mechanism is not known.
An understanding of these molecules may be informative about
negative regulator circuits that can potentially limit blood cell
formation under steady state. These molecules may be helpful for
understanding pathogenic mechanisms of lympho-hematopoietic
disorders or treating such diseases.
SUMMARY OF THE INVENTION
[0007] An objective of this invention is to provide a novel protein
derived from BMS2.4 cells that interferes with the proliferation of
lympho-hematopoietic cells, a gene thereof, a method for preparing
them, and uses thereof.
[0008] The present inventors transfected human renal carcinoma cell
strain 293T with a cDNA library derived from a mouse stromal cell
line, BMS2.4, and performed expression cloning based on growth
inhibitory effects of the culture supernatant of transformants on
myelomonocytic leukemia cell line, WEHI3. As a result, we succeeded
in isolating a gene encoding a novel protein, designated Blood Cell
Growth-Inhibiting Factor (BGIF) that interfered with proliferation
of WEHI3 cells in a similar manner as the culture supernatant of
BMS2.4 cells. A putative BGIF protein from the isolated gene is
homologous to IFN-.A-inverted. and IFN-, and expressed in stromal
cells in bone marrow and spleen.
[0009] The present inventors prepared a recombinant BGIF protein to
examine its effects on the growth of various lympho-hematopoietic
cells. The recombinant BGIF protein suppressed proliferation of
various (pre-)B lineage cell clones (1A9, BC7.12, BC7.7, F10, 2E8,
18-81, 7OZ/3, WEHI231, WEHI279, and SP2/0), T lineage lymphoma cell
line, BW1597, and multipotent cell line EML-C1, as well as WEHI3
cells. BGIF arrests the cell cycle of WEHI3 cells at the G0/G1
phase and prolongs the G1 phase. It induces apoptosis in BC7.12
cells. It also suppressed the establishment of functional adherent
layers in W/W culture.
[0010] Like type I IFNs, BGIF induced IFN regulatory factor-1
utlizing IFN-.A-inverted./ receptors, and activated JAK2 in
myelomonocytic leukocyte line.
[0011] BGIF protein and its gene of this invention are associated
with the lympho-hematopoietic system, and will be a useful tool for
elucidating pathogenic mechanisms of lympho-hematopoietic
disorders. BGIF protein of this invention would be applicable to
therapeutics for various disorders in which the protein is
involved.
[0012] Examples of disorders to be treated with the BGIF protein or
its gene of this invention include lymphocytoma/hematapostemia such
as acute lymphocytic or myelocytic leukemia, chronic lymphocytic or
myelocytic leukemia, and malignant lymphoma, collagen diseases such
as rheumatoid arthritis and systemic lupus erythematosus,
idiopathic thrombocytopenic purpura, etc. The protein or the gene
can also be used as immunoregulators. Disorders to be treated with
compounds that inhibit the activity of BGIF protein include those
accompanied by hematopenia such as aplastic anemia. These compounds
can also be used as immunoregulators.
[0013] This invention relates to a novel protein derived from
BMS2.4 cells that inhibits proliferation of lympho-hematopoietic
cells, a gene thereof, and a method for preparing the protein and
the gene, and uses thereof. More specifically, it relates to:
[0014] (1) a protein that suppresses the proliferation of
lympho-hematopoietic cells selected from the group consisting
of:
[0015] (a) a protein comprising the amino acid sequence as set
forth in SEQ ID NO: 3;
[0016] (b) a protein comprising a derivative of the amino acid
sequence set forth in SEQ ID NO: 3, in which one or more amino
acids are substituted, deleted, inserted, and/or added; and
[0017] (c) a protein encoded by a DNA hybridizing with the DNA
comprising the nucleotide sequence as set forth in SEQ ID NO:
1,
[0018] (2) the protein according to (1), wherein the
lympho-hematopoietic cells are selected from the group consisting
of B lineage cell line, 1 A9, BC7.12, BC7.7, F10, 2E8, 18-81,
7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and
myelomonocytic leukemia cell line WEHI3,
[0019] (3) a DNA encoding the protein according to (1),
[0020] (4) the DNA of (3) comprising the coding region of the
nucleotide sequence as set forth in SEQ ID NO: 1,
[0021] (5) a vector comprising the DNA of (3),
[0022] (6) a host cell retaining the vector of (5),
[0023] (7) a method for preparing the protein of (1), the method
comprising culturing the host cell of (6) and recovering
recombinant proteins expressed in the cell from the cultured cell
or culture supernatant thereof,
[0024] (8) an antibody to the protein according to (1),
[0025] (9) a peptide fragment of the protein according to (1),
[0026] (10) a DNA specifically hybridizing with the DNA comprising
the nucleotide sequence as set forth in SEQ ID NO: 1 and comprises
at least 15 nucleotides,
[0027] (11) a method for screening a compound binding to the
protein according to (1), the method comprising
[0028] (a) contacting the protein of (1) or its fragment with a
test sample,
[0029] (b) detecting the binding activity of the test sample to the
protein or its fragment, and
[0030] (c) selecting a compound that binds to the protein or its
fragment,
[0031] (12) a compound that is isolable by the method according to
(11) and binds to the protein of (1),
[0032] (13) a method for screening a compound that interferes with
the activity of the protein according to (1) to suppress
proliferation of lympho-hematopoietic cells, the method
comprising
[0033] (a) contacting the protein according to (1) with
lympho-hematopoietic cells in the presence of a test sample,
[0034] (b) detecting the proliferation of the cells, and
[0035] (c) selecting a compound that interferes with the activity
of the protein according to (1) to suppress proliferation of
lympho-hematopoietic cells as compared with a control where the
detection is performed in the absence of the test compound,
[0036] (14) the method according to (13), wherein said
lympho-hematopoietic cells are selected from the group consisting
of B lineage cell lines, 1A9, BC7.12, BC7.7, F10, 2E8, 18-81,
7OZ/3, WEHI231 and SP2/0, T lineage lymphoma cell line BW1597, and
myelomonocytic leukemia cell line WEHI3,
[0037] (15) a compound isolable by the method according to (13),
which interferes with the activity of the protein according to (1)
to suppress proliferation of lympho-hematopoietic cells,
[0038] (16) a pharmaceutical composition comprising the protein
according to (1) as an active ingredient,
[0039] (17) the pharmaceutical composition according to (16),
wherein the composition is for treating lympho-hematopoietic
disorders, and
[0040] (18) a pharmaceutical composition comprising the compound
according to (12) as an active ingredient.
[0041] Herein, .congruent.lympho-hematopoietic cells.congruent.
mean matured or precursor cells of erythrocytes, leukocytes or
platelets.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 illustrates that the clone 159-3-3 is carrying cDNA
that encodes growth inhibitor of WEHI3. The cloned 159-3-3 plasmid
or a control plasmid, pEF-BOS, were transfected into 293T cells
with a calcium phosphate precipitation method, and supernatants
from each transfectant were collected after 3 days cultures. BMS2
and BMS2.4 cells were cultured for 3 days after their confluent
condition, and their supernatants were collected. WEHI3 cells
(5.times.10.sup.3/well) were cultured in the presence of 10% of the
indicated supernatants for two days. Their proliferation was
evaluated by MTT assay. Statistically significant differences from
control values are indicated an asterisk (p<0.01).
[0043] FIG. 2 shows nucleotide and deduced amino acid sequence of
the 997 bp BGIF cDNA. The first and the second ATGs of BGIF are
indicated square boxes. The deduced amino acid sequence translated
from the first ATG is shown in an upper lane, and that translated
from the second ATG is shown in a lower lane. The polyadenylation
signal AATAAA is underlined.
[0044] FIG. 3 illustrates that a product translated from the second
ATG of BGIF is a major and functional protein. (A) shows relative
renilla luciferase activities of BMS2.4 and BMS2 cells that were
transfected with 1st-ATG/pRL-SV40 (open column) or the
2nd-ATG/pRL-SV40 (closed column), together with pGL-Control Vector
by lipofectamine transfection. After 2 days cultures, cells were
collected and subjected to luciferase assays. The relative renilla
luciferase activities were calculated by normalizing transfection
efficiency according to the firefly luciferase activities.
Statistically significant differences from control values are
indicated by an asterisk (p<0.01). (B) shows effects of culture
supernatants of 293T cells that were transfected with plasmids,
1st-ATG/pEFBOSX and 2nd-ATG/pEFBOSX, with a calcium phosphate
precipitation method. The transfectants were cultured for 3 days,
and then their supernatants were collected. WEHI3 cells
(5.times.10.sup.3/well) were cultured in the presence of 10% of the
indicated supernatants for 2 days. Their proliferation was
evaluated by MTT assay.
[0045] FIG. 4 shows amino acid sequence alignment of BGIF,
IFN-.alpha. and IFN-.beta.. Asterisks indicate amino acid
identities with BGIF. Dashed lines represent gaps introduced to
align sequences.
[0046] FIG. 5 shows expression of BGIF in lympho-hematopoietic
organs. (A) presents results of Northern blot analysis of
polyA.sup.+ RNAs (5 .mu.g/lane) isolated from WEHI3 and BMS2.4
cells. The lower panel is a control for equal loading where the
same blot was probed with .beta.-actin. (B) presents
electrophoretic patterns PCR products of total RNAs isolated from
various cells. Total RNAs (2.5 .mu.g) were isolated from the
indicated cells, and subjected to RT-PCR. The amplified products
were electrophoresed through a 1% agarose gel containing ethidium
bromide.
[0047] FIG. 6 illustrates that BGIF induces apoptosis in BC7.12
cells and G0/G1-arrest or G1-prolongation in WEHI3 cells. BC7.12 or
WEHI3 cells were cultured with 100 ng/ml of CD44-Ig or BGIF-Ig for
the indicated periods, and then subjected to cell viability (panel
A), nuclear DNA content (panel B), and DNA fragmentation analysis
(panel C). The results are representative of three similar
experiments.
[0048] FIG. 7 illustrates that BGIF selectively inhibits the
proliferation of normal lympho-hematopoietic progenitors. BGIF-Ig
or CD44-Ig (100 ng/ml) was added to CFU-IL-7 and CFU-B (panel A)
and CFU-mix, CFU-GM, and BFU-E (panel B) colony assays. The results
are shown as means .+-.SD of triplicate cultures. Statistically
significant differences from control values are indicated by one (p
<0.05) or two (p <0.01) asterisks. Similar results were
obtained in five independent experiments.
[0049] FIG. 8 shows effects of BGIF on LTBMC. Panel A presents that
BGIF inhibits the production of B lymphocytes in W/W cultures.
Replicate cultures of mouse bone marrow cells were prepared and
maintained in the continuous presence of 100 ng/ml BGIF -Ig,
CD44-Ig or medium alone. Numbers of non-adherent cells collected at
weekly intervals were expressed as means per flask. Similar results
were observed in three independent experiments. Panel B shows that
BGIF inhibits the establishment of adherent layers in W/W cultures.
Phase-contrast micrographs (photographed at 20.times.
magnification) are shown of six weeks of W/W and Dexter cultures.
Each figure shows one of three similar experiments.
[0050] FIG. 9 illustrates that BGIF utilizes the IFN-.A-inverted./
receptors. Panel A shows that BGIF influences B lineage lymphocytes
via the IFN-.A-inverted./ receptors. Bone marrow cells
(5.times.10.sup.4) were prepared from wild type (WT) or
IFN-.A-inverted./ receptor knock out mice
(IFN-.A-inverted./R.sup.0/0), and subjected to CFU-IL-7 colony
assays in the presence of 100 ng/ml of BGIF-Ig or CD44-Ig. The
results are shown as means .+-.SD of triplicate cultures.
Statistically significant differences from control values are
indicated by two (p<0.01) asterisks. Similar results were
obtained in two independent experiments. Panel B shows the results
of Northern blot analysis indicating IRF-1 induction by BGIF. WEHI3
cells (1.times.10.sup.7) were serum-starved for 1 h and then
stimulated with 100 ng/ml CD44-Ig or BGIF -Ig for the indicated
periods. Total cellular RNAs were isolated using TRIzol Reagent,
and 15 :g of each sample was electrophoresed on formaldehyde
agarose gels. The filters were hybridized with .sup.32P-labeled
probes for the indicated genes. Each figure shows one of three
similar experiments.
[0051] FIG. 10 shows signal transduction pathway utilized by BGIF.
WEHI3 (panel A) and BC7.12 (panel B) cells (1.times.10.sup.7) were
respectively serum-starved for 1 h and then stimulated with 100
ng/ml CD44-Ig or BGIF -Ig for 10 min. Total cell lysates were
immunoprecipitated with the indicated antibodies and blots were
probed with an anti-phosphotyrosine antibody. Filters were then
stripped and reprobed with the indicated antibodies. Similar
results were obtained in four independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0052] This invention relates to a novel protein designated as
BGIF, which is derived from BMS2.4 cells and inhibits proliferation
of lympho-hematopoietic cells. The nucleotide sequence of the
isolated BGIF cDNA, which is included in this invention, is shown
in SEQ ID NO: 1, and amino acid sequence of BGIF protein encoded by
said cDNA is represented in SEQ ID NO: 3.
[0053] The gene encoding BGIF protein was isolated by transfecting
human renal carcinoma cell line 293T with a cDNA library derived
from the mouse stromal cell line BMS2.4, and conducting the
expression cloning of cDNA based on growth inhibitory effects of
the culture supernatant of the transformed 293T cells on a
myelomonocytic leukemia cell line WEHI3.
[0054] The mouse BGIF gene of this invention contains an open
reading frame encoding a protein comprising 182 amino acid residues
(FIG. 2). The protein has a highly hydrophobic stretch of 21 amino
acid residues at the N-terminal end, but lacks an internal membrane
spanning domain, suggesting that BGIF is a secreted protein. The
amino-acid sequence of the protein contains one Asn-X-Ser/Thr
potential N-linked glycosylation sites at the position of amino
acid 68, consistent with the hypothesis that BGIF may be
glycosylated. As a result of computer search of BGIF sequence using
FASTA and BLAST programs, BGIF has a similarity with IFN-.alpha.
(31.9% identity in 166 amino acids overlap) and IFN-.beta. (25.9%
identity in 166 amino acids overlap) at the amino acid level (FIG.
4). However, six cystaine residues in the BGIF protein were not
identical to those of IFN-.alpha. or IFN-.beta..
[0055] Northern blot analysis revealed that the message size of
BGIF was approximately 1 kb and that BMS2.4, but not WEHI3,
expressed BGIF mRNA (FIG. 5A). In addition, RT-PCR was used to
survey expression in mouse bone marrow, spleen adherent cells as
well as freshly isolated bone marrow or spleen cells (FIG. 5B).
Therefore, BGIF mRNA is expressed in bone marrow and spleen, at
least by some of bone marrow and spleen stromal cells.
[0056] A BGIF fusion protein suppressed proliferation of B lineage
cell lines (1A9, BC7.12, BC7.7, F10, 2E8, 18-81, 7OZ/3, WEHI231,
SP2/0), a T lymphoma cell line (BW1597), and a myelomonocytic
leukemia cell line (WEHI3 and WEHI279) and a mouse multipotent cell
line (EML-C1). While most of the cell lines did not die, three of
long-term bone marrow culture derived pre-B cell clones (1A9,
BC7.12, 2E8) lost their viability by the treatment of the BGIF
fusion protein. In contrast, lymphoma cell lines (BCL1 and EL4) and
a myeloid cell line (M1) were not affected by the BGIF fusion
protein (Table 1). Therefore, BGIF suppresses growth of a variety
of lympho-hematopoietic cell lines.
[0057] Semisolid agar cloning assays were then used to evaluate the
direct influence of BGIF on colony formation of
lympho-hematopoietic progenitors. The cloning efficiency of IL-7
responding pre-B cells (CFU-IL-7) was decreased by addition of the
BGIF fusion protein (FIG. 7). BGIF induced G0/G1-arrest or
G1-prolongation in WEHI3 cells, and apoptosis in BC7.12 cells (FIG.
6). BGIF also suppressed the establishment of adherent layers in
W/W cultures (FIG. 8). Therefore, BGIF suppresses proliferation of
not only transformed but also normal lympho-hematopoietic
cells.
[0058] BGIF, like type I IFN, also induced the IFN regulatory
factor 1 using IFN-.A-inverted./ receptors, while it activated JAK2
in myelomonocytic leukemia cell lines (FIG. 9).
[0059] These facts indicate that the BGIF protein is a novel
humoral factor that suppresses proliferation of
lympho-hematopoietic cells. BGIF would thus be helpful for
understanding pathogenic mechanisms of lympho-hematopoietic
disorders and can be used as a drug for patients with
lympho-hematopoietic diseases such as leukemias and malignant
lymphomas, and for those with collagen diseases.
[0060] Collagen diseases are disorders which damage various organs
by producing an autoantibody that recognizes the body's own cells.
In rheumatoid arthritis and some other diseases, the activation of
polyclonal B lymphocytes is observed. Since BGIF suppresses B
lineage lymphocytopoiesis, it can be one of drugs for collagen
diseases by suppressing either the production of an autoantibody or
the activation of B lineage lymphocytopoiesis. In addition, the
deficiency in a negative regulatory factor for the
lymphocytopoiesis might be a cause of collagen diseases, and the
reduction of BGIF production may thus be a cause of collagen
diseases.
[0061] Proteins structurally similar to the mouse BGIF protein are
also included in this invention as long as they have an activity to
suppress proliferation of lympho-hematopoietic cells. Such
structurally analogous proteins also include variants of BGIF
protein and BGIF proteins derived from other organisms.
[0062] One skilled in the art would readily prepare these proteins
using, for example, standard mutagenesis methods. Known methods for
altering amino acids in proteins include site-specific mutagenesis,
for example, the method of preparing deletion-mutant (Kowalski, D.,
et al., 1976, J. Biochem., 15, 4457; McCutchan, T. F., et al.,
1984, Science, 225, 626-628), Kunkel's method (Kunkel, T. A., 1985,
Proc. Natl. Acad. Sci. USA, 82: 488-492; Kunkel, T. A. et al.,
1987, Methods Enzymol., 154: 367-382), Gapped-duplex method
(Kramer, W. and Fritz, H.-J. 1987, Methods Enzymol., 154: 350-367;
Zoller, M. J. and Smith, M., 1983, Methods Enzymol., 100: 468-500;
Hirose, S., 1991, Muramatsu, M. & Okayama, H., eds., Jikken
Igaku, extra issue, Genetic Engineering Handbook, Yodosha,
pp246-252), PCR method (Muramatsu, M., ed., Laboratory manual,
Genetic Engineering, 3rd edition, Maruzen, 1996, pp227-230),
cassette alteration method (Kishimoto, T., 1991, Muramatsu, M.
& Okayama, H., ed., Jikken Igaku, extra issue, Genetic
Engineering Handbook, Yodosha, pp253-260), etc. A Transformer.TM.
Site-Directed Mutagenesis Kit (CLONTECH #K1600-1), for example, may
be used.
[0063] In artificial alteration of amino acids in proteins, the
number of amino acid residues to be altered is usually 30 or less,
preferably 10 or less, and more preferably 5 or less. Alteration of
amino acids in proteins could occur spontaneously. Such proteins
having amino acid sequences different from that of the natural
mouse BGIF protein (SEQ ID NO: 3) due to artificial or spontaneous
substitution, deletion, addition and/or insertion of amino acid
residues, are also included in this invention as long as they have
an activity to suppress proliferation of lympho-hematopoietic
cells.
[0064] An amino acid has similar properties to that of the amino
acid to be substituted is preferably used for substitution. Since
Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are, for example, all
classified into the non-polar amino acid, they are considered to
have similar properties. Non-charged amino acids include Gly, Ser,
Thr, Cys, Tyr, Asn, and Gln. Acidic amino acids include Asp and
Glu, while basic amino acids include Lys, Arg and His.
[0065] The proteins formed by deleting some amino acid residues
from the mouse BGIF according to this invention include proteins in
which the signal sequence (SEQ ID NO: 4) is deleted. Proteins
formed by adding amino acid residues to the mouse BGIF protein
include a fusion protein of the mouse BGIF protein with other
peptides.
[0066] Proteins structurally similar to the mouse BGIF protein
having an activity to suppress proliferation of
lympho-hematopoietic cells can be prepared using the known
hybridization technique (Cell Engineering, extra issue, New Cell
Engineering Experimental Protocol, 1991, Shujunsha, pp. 188-193,
and Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor
Laboratory Press (1989), 8.46-8.52) and polymerase chain reaction
(PCR) technique (Cell Engineering, extra issue, 8, New Cell
Engineering Experimental Protocol, 1991, Shujunsha, pp. 171-186;
Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor
Laboratory Press (1989), 14.1-14.35). It is routine for one skilled
in the art to isolate DNA highly homologous to mouse BGIF cDNA from
various other organisms using the mouse BGIF cDNA (SEQ ID NO: 1) or
portions thereof as a probe and oligonucleotides specifically
hybridizing with the mouse BGIF cDNA as a primer to obtain proteins
structurally similar to the mouse BGIF protein from the isolated
DNA.
[0067] Proteins encoded by DNAs hybridizing with the mouse BGIF
cDNA are included in this invention as long as they suppress
proliferation of lympho-hematopoietic cells. Other organisms used
for isolating such proteins include, for example, humans, monkeys,
rats, rabbits, goats, cattle, pigs, etc., but are not limited
thereto. DNAs encoding such proteins can be isolated from such
sources as bone marrow, spleen cells, lympho-hematopoietic cells,
and cultured bone marrow/spleen stromal cells.
[0068] DNAs encoding the BGIF protein derived from animals other
than mice are usually highly identical with the nucleotide sequence
(SEQ ID NO: 1) of mouse BGIF cDNA. "Being highly identical" means
at least 60% or more, preferably 80% or more, and further
preferably 90% or more sequence identity at the amino acid level.
Sequence homology can be determined by the homology search program
of DNA Data Bank of Japan (National Institute of Genetics: Yata
1111, Mishima, Shizuoka 411, Japan).
[0069] One skilled in the art can readily determine conditions for
hybridization to isolate DNAs encoding proteins functionally
equivalent to the mouse BGIF protein. One example of hybridization
conditions is as follows. After the pre-hybridization at 42.degree.
C. overnight in a hybridization solution containing 25% formamide
(50% formamide under the stringent conditions), 4.times.SSC, 50 mM
Hepes pH 7.0, 10.times.Denhardt's solution and 20 :g/ml denatured
salmon sperm DNA, a labeled probe is added, and the hybridization
is performed by maintaining the reaction mixture at 42.degree. C.
overnight. Then, successive washings were performed at room
temperature with 2.times.SSC and 0.1% SDS (at 50.degree. C.,
0.5.times.SSC and 0.1% SDS under the stringent conditions). In this
case, although plural factors including the temperature,
concentration of formamide, salt concentration, etc. are thought to
influence the stringency of hybridization, one skilled in the art
would readily determine the stringent conditions similar to above
by suitably selecting these factors (Cell Technology, extra issue
8, New Cell Engineering Experimental Protocol Protocol.
[0070] The protein of this invention can be prepared as either
natural proteins or recombinant proteins utilizing gene
recombination techniques. Natural proteins can be prepared by
subjecting tissue extracts that are supposed to contain the BGIF
protein (for example, bone marrow and spleen cells) to affinity
chromatography using the antibody to the BGIF protein as described
below. On the other hand, recombinant proteins may be prepared by
culturing cells transformed with DNA encoding the BGIF protein,
allowing the transformants to express the protein, and recovering
the protein.
[0071] The proteins of this invention also include partial peptides
of the above-described proteins. Partial peptides of the proteins
of this invention include, for example, those corresponding to the
binding site of the protein to its receptor. Such partial peptides
can be administered to the body to serve as agonists and
antagonists of the receptors for the proteins of this invention, or
as competitive inhibitors for the proteins. These partial peptides
are useful as activators and inhibitors for the signal transduction
mediated by the proteins of this invention. Partial peptides of
this invention also include, for example, the N-terminal or
C-terminal region of the protein of this invention in which the
signal sequence is deleted, and said peptides can be used for the
preparation of antibodies. Partial peptides comprising the amino
acid sequence specific to the protein of this invention have at
least 7, preferably at least 8, more preferably at least 9 amino
acid residues. Partial peptides of this invention can be produced
by genetic engineering techniques, known peptide synthetic methods,
or by digestion of the protein of this invention with appropriate
peptidases.
[0072] This invention also relates to DNAs encoding the proteins of
the invention. DNAs encoding the protein of this invention are not
particularly limited as long as they can encode the proteins of
this invention, including cDNAs, genomic DNAs, and synthetic DNAs.
DNAs having any desired nucleotide sequences based on the
degeneracy of genetic codes are also included in this invention as
long as they can encode the proteins of this invention.
[0073] cDNAs encoding the proteins of this invention can be
screened, for example, by labeling cDNA of SEQ ID NO: 1 or segments
thereof, RNAs complementary to them, or synthetic oligonucleotides
comprising partial sequences of said cDNA with .sup.32P, etc., and
hybridizing them with a cDNA library derived from tissues (e.g.,
bone marrow, spleen, etc.) expressing the proteins of this
invention. Such cDNAs can be cloned by synthesizing
oligonucleotides corresponding to nucleotide sequences of these
cDNAs, and amplifying them by PCR with cDNA derived from suitable
tissues, (e.g. bone marrow, spleen, etc.) as a template. The
genomic DNA can be screened, for example, by labeling cDNA of SEQ
ID NO: 1 or segments thereof, RNAs complementary to them, or
synthetic oligonucleotides comprising partial sequences of said
cDNA with .sup.32P, etc., and hybridizing them with a genomic DNA
library. Alternatively, cDNAs encoding the proteins of this
invention can be cloned by synthesizing oligonucleotides
corresponding to nucleotide sequences of these cDNAs, and
amplifying them by PCR with a genomic DNA as a template. Synthetic
DNAs can be prepared, for example, by chemically synthesizing
oligonucleotides comprising partial sequences of cDNA of SEQ ID NO:
1, annealing them to form double strand, and ligating them with DNA
ligase.
[0074] These DNAs are useful for the production of recombinant
proteins. The proteins of this invention can be prepared as
recombinant proteins by inserting DNAs encoding the proteins of
this invention (e.g. DNA of SEQ ID NO: 1) into an appropriate
expression vector, transforming suitable host cells with the
vector, culturing the transformants, and purifying proteins
expressed. When the proteins of this invention is a secretory
protein, it can be prepared, for example, by expressing it in
mammalian cells to be secreted therefrom.
[0075] Expression vectors to be specifically used in E. coli
include, for example, pKK223-3, pKK233-2, pJLA502, etc. The
proteins of this invention can be expressed, for example, as fused
proteins with other proteins. Vectors for expressing such fusion
proteins include, for example, pRIT2T, pGEX-2T, pGEX-3X, etc. These
fused proteins can be easily collected using an affinity column.
Only a desired protein can easily excised from the fusion protein
when a vector is designed to provide the thrombin- or factor
Xa-cleaving site between the desired protein and a partner protein
in the fused protein. Vectors for secreting the proteins
extracellularly or into the periplasm include pKT280, pRIT5, etc.
(Okada, M. and Miyazaki, K. ed., Invincible Biotechnical Series,
Protein Experimental Note, 1 st volume, Extraction and
Separation/Purification, Yodosha, 1996, pp.139-149).
[0076] It is also possible to express the proteins of this
invention in insect and mammalian cells using baculoviruses.
Baculovirus vectors used in mammalian cells are, for example,
pAcCAGMCS1 (Muramatsu, M., ed., Labomanual Genetic Engineering, 3rd
ed., Maruzen, 1996, pp. 242-246).
[0077] Recombinant proteins expressed in host cells can be purified
by known methods. The protein of this invention expressed in the
form of a fused protein, for example, with a histidine residue tag
or glutathione-S-transferase (GST) attached at the N-terminus can
be purified by a nickel column or a glutathione sepharose column,
etc.
[0078] DNAs encoding the proteins of this invention can be applied
to gene therapy for disorders caused by the mutation thereof.
Vectors used for gene therapy include, for example, virus vectors
such as retrovirus vector, adenovirus vector, adeno-associated
virus vector, vaccinia virus vector, lentivirus vector, herpesvirus
vector, alphavirus vector, EB virus vector, papilloma virus vector,
foamy virus vector, etc., and non-viral vectors such as cationic
liposomes, ligand-DNA complexes, gene guns, etc. (Y. Niitsu, M.
Takahashi, Molecular Medicine, Vol. 35, No. 11, 1385-1395, 1998).
Gene transfer can be carried out in vivo and ex vivo. The DNA
encoding the protein of this invention can be co-transferred with
other cytokine genes.
[0079] The present invention also relates to DNAs specifically
hybridizing with the DNA comprising the nucleotide sequence of SEQ
ID NO: 1 and containing at least 15 nucleotides. "Specifically
hybridizing" means that DNA does not significantly cross-hybridize
with DNAs encoding other proteins under usual hybridization
conditions as described above, preferably under stringent
hybridization conditions. Such DNAs include probes, primers,
nucleotides or nucleotide derivatives (e.g. antisense
oligonucleotides and lipozymes, etc.), which specifically hybridize
with DNAs encoding the proteins of this invention, or DNAs
complementary to said DNAs.
[0080] cDNAs encoding the proteins of this invention or
oligonucleotides comprising partial sequences thereof can be used
for cloning genes and cDNAs encoding the proteins of this
invention, or amplifying them by PCR. The cDNAs and
oligonucleotides can also be utilized for detecting polymorphism or
abnormality (gene diagnosis, etc.) of the gene or cDNA by the
restriction fragment length polymorphism (RFLP) method, single
strand DNA conformation polymorphism (SSCP) method, etc.
[0081] This invention also relates to antibodies binding to the
proteins of the invention. Antibodies of this invention include
both polyclonal and monoclonal antibodies. Polyclonal antibodies
can be prepared, for example, according to the method described in
Institute of Medical Science, Section of Carcinostasis, University
of Tokyo, ed., Cell Engineering, extra issue 8, New Cell
Engineering Experimental Protocol, 1993, Shujunsha, pp. 202-217).
The purified proteins of this invention, partial peptides thereof,
or peptides synthesized based on amino acid sequences of the
proteins of this invention are injected to animals to be immunized
such as rabbits, guinea pigs, mice, chickens, etc.
introperitoneally, subcutaneously, intramuscularly, or into the ear
vein, groin, the back of appendicular nail, etc. Antigenic proteins
may be administered together with Freund's complete or incomplete
adjuvants. Antigen is usually administered every several weeks. The
titer can be elevated by the booster injection. Blood is
periodically collected to confirm the titer elevation by ELISA,
etc. After the final immunization, the blood is collected from
immunized animals to obtain antisera. Antisera are purified by
salting out, ion exchange chromatography, HPLC, etc. to obtain an
IgG fraction. The antibody can be further purified by affinity
chromatography using the immobilized antigen.
[0082] A monoclonal antibody can be prepared, for example,
according to the method described in Koike, T. & Taniguchi, M.
1991, Jikken Igaku, extra issue, Genetic Engineering Handbook,
Muramatsu, M. & Okayama, H., eds., Yodosha, pp 70-74). Animals
are immunized with the protein of this invention or partial
peptides thereof in a similar manner as described above, and, after
the final immunization, spleen or lymph node is excised from
immunized animals. Antibody-producing cells contained in the spleen
or lymph node are fused with myeloma cells using a fusing agent
such as polyethylene glycol, to prepare hybridomas. Desired
hybridomas are screened and cultured to prepare a monoclonal
antibody from the culture supernatant. The monoclonal antibody is
purified by salting out, ion exchange chromatography, HPLC, etc. to
obtain an IgG fraction. The resulting fraction can be further
purified by affinity chromatography using the immobilized
antigen.
[0083] Antibodies thus prepared are used for the affinity
purification of the proteins of this invention. They can also be
used for the test and diagnosis of disorders caused by abnormal
expression and structural abnormality of the proteins of this
invention and for detection of the expression level of the protein.
Abnormality in expression and structure of the proteins of this
invention can be examined and diagnosed by extracting proteins
from, for example, tissues, blood or cells, and detecting the
proteins of this invention using Western blotting,
immunoprecipitation, ELISA, etc. Antibodies of this invention can
be applied to the antibody treatment. For antibody treatment, they
are preferably humanized or human antibodies.
[0084] Humanized monoclonal antibodies can be prepared according
to, for example, a method described in H. Isogai, 1988, Jikken
Igaku, Vol. 6, No. 10, 55-60. A method using molecular biological
techniques as described in T. Tsunenari et al., 1996, Anticancer
Res., 16, 2537-2544 can also be used. Human antibodies can be
prepared by immunizing mice, in which the immune system is replaced
by the human system, with the protein of this invention.
[0085] The present invention also relates to a method for screening
a compound binding to the protein of this invention. The screening
method of this invention comprises: (a) contacting the protein of
this invention or partial peptides thereof with a test sample, (b)
detecting the binding activity of the test sample to the protein of
this invention or partial peptides thereof, and (c) selecting a
compound that binds to the protein of this invention or partial
peptides thereof.
[0086] Proteins binding to the protein of this invention can be
screened, for example, by applying culture supernatants or extracts
of cells, which are expectedly express proteins binding to the
protein of this invention, to an affinity column to which the
protein of this invention is attached (immobilized), and purifying
proteins specifically binding to the column.
[0087] Alternatively, proteins binding to the protein of this
invention can be screened by the West Western blotting method or
two hybrid system. In the former method, a cDNA library using a
phage vector is prepared from tissues or cells (for example,
lympho-hematopoietic cells, etc.) which expectedly express proteins
binding to the protein of this invention, and proteins are
expressed from cloned cDNA on agarose, transferred to filter,
fixed, and reacted with the labeled protein of this invention to
detect plaques expressing binding proteins. In the latter method,
the protein of this invention is expressed as a fusion protein with
a test protein such as GAL4 DNA-binding region and GAL4
transcription activating region, and the binding of the protein of
this invention to the test protein is detected by the expression of
a reporter gene linked to the downstream from a promoter having the
binding sequence of the GAL4 DNA-binding protein.
[0088] Other screening methods include the method comprising
contacting synthetic compounds, natural proteins or random phage
peptide display library with the immobilized protein of this
invention and detecting binding molecules and the method comprising
isolating compounds binding to the protein of this invention using
a high-through put based on combinatorial chemical technique.
[0089] Test samples used for screening include, for example, cell
extracts, expression products of a gene library, synthetic low
molecular weight compounds, synthetic peptides, natural compounds,
etc., but are not limited thereto. Those test samples used for
screening may be labeled prior to use as the occasion demands.
Labels include, for example, radioactive and fluorescent ones,
etc., but are not limited to them.
[0090] This invention also relates to a method for screening
receptors of the protein of the invention. The results obtained in
examples below showed that many lympho-hematopoietic cells have
responsiveness to the protein of this invention, suggesting that
these cells are assumed to express receptors of the protein of this
invention. Such receptors would thus be isolated using the proteins
of this invention.
[0091] For example, receptors of the protein of this invention can
be obtained by collecting proteins from cells which expectedly
express a receptor of the protein of this invention, and subjecting
the proteins to the above-described affinity chromatography. A DNA
encoding the receptor can be isolated by raising an antibody to the
receptor of the protein of this invention and screening a cDNA
expression library prepared from cells which expectedly express the
receptor of the protein of this invention using the antibody.
[0092] Alternatively, a DNA encoding the receptor of the protein of
this invention can be screened by the subtractive hybridization
method comprising preparing cDNAs from mRNA of cells responsive to
the protein of this invention, hybridizing the cDNAs with mRNA of
other cells unresponsive to the protein of this invention, and
subtracting cDNAs hybridizing with mRNAs from both responsive and
unresponsive cells to screen desired cDNAs. A DNA encoding the
receptor of the protein of this invention can also be isolated by
preparing cDNAs from cells which expectedly express the receptor of
the protein of this invention, transforming cells with the cDNAs,
and screening expressed proteins using the protein of this
invention as a ligand, or the antibody to the receptor of the
protein of this invention. It is also possible to prepare cDNAs
from cells which expectedly express the receptor of the protein of
this invention, to transform COS cells with the cDNAs to screen a
DNA by monitoring the transient expression. Another method
comprises preparing mRNA from cells which expectedly express the
receptor of the protein of this invention, injecting the mRNA into
oocytes of Xenopus laevis, and functionally screening the receptor.
The DNA can also be cloned by hybridization and PCR based on
homology to the known receptors of cytokines that are homologous to
the protein of this invention (T. Yokota & K. Arai, eds.,
Jikken Igaku, extra issue, Biomanual seeries 3, Gene Cloning
Method, 1993, pp. 99-156.
[0093] The invention also relates to a method for screening a
compound that interferes with the activity of the protein of this
invention to suppress proliferation of lympho-hematopoietic cells.
This method uses the activity of the protein of this invention to
suppress proliferation of lympho-hematopoietic cells as an
indicator, and comprises the steps of (a) contacting a protein of
this invention with lympho-hematopoietic cells in the presence of a
test sample, (b) detecting proliferation of said cells, and (c)
selecting a compound that interferes with the activity of the
protein of this invention to suppress proliferation of said cells
as compared with the activity detected in the absence of the test
sample (control).
[0094] Test samples used for this screening method include, for
example, cell extracts, expression products of a gene library,
synthetic low molecular weight compounds, proteins, natural or
synthetic peptides, natural products, sera, etc., but are not
limited to them. The test samples can be compounds isolated by the
above-described screening method monitoring the binding activity of
the compounds to the protein of this invention. Proteins of this
invention used for the screening method may be purified ones or
culture supernatants of transformants secreting the proteins of
this invention.
[0095] Any lympho-hematopoietic cells can be used for the screening
method, without limitation, as long as their proliferations are
suppressed by the proteins of this invention, Preferable cells
include, for example, pre-B lineage cell lines, 1A9, BC7.12, BC7.7,
F10, 2E8, 18-81, 7OZ/3, WEHI231and SP2/0, T lineage lymphoma cell
line BW 1597, and myelomonocytic leukemia cell line WEHI3.
[0096] Growth of these cells is inhibited in the absence of a test
compound since the proteins of this invention suppress
proliferation of lympho-hematopoietic cells. When proliferation of
the cells is suppressed in the presence of a test compound, the
compound is judged as the compound that interfere with the activity
of the proteins of this invention to suppress proliferation of
lympho-hematopoietic cells. Herein, "interfering with the activity
to suppress proliferation" includes "completely inhibiting the
suppression of proliferation."
[0097] Compounds to be isolated by this screening method include,
for example, 1) compounds binding to the protein of this invention
to inhibit its activity, 2) compounds binding to the proteins of
this invention or receptors thereof to inhibit the binding between
the proteins of this invention and receptors thereof, 3) compounds
binding to the receptors of proteins of this invention to inhibit
activation of the receptors, and 4) compounds inhibiting signal
transduction to express the phenotype of cell proliferation from
the receptors of the proteins of this invention. These compounds
can be used as drugs for preventing or treating disorders caused by
abnormality of the signal transduction system mediated by the
proteins of this invention (for example, diseases caused by
abnormality of lympho-hematopoietic system).
[0098] When the proteins of this invention or compounds isolated by
the screening methods of this invention are used as drugs, they may
be administered to patients as they are or as pharmaceutical
preparations produced by known methods. They can be formulated
together with, for example, pharmaceutically acceptable carriers or
media such as sterilized water, physiological saline, vegetable
oil, emulsifiers, suspending agents, surfactants, stabilizers, etc.
They may be administered to patients by methods well known in the
art, for example, by intra-arterial, intravenous, subcutaneous
injection. They can also be administered intranasally,
intrabronchially, intramuscularly, or orally. Doses may vary
depending on the body weight and age of patients as well as
administration method, and can be suitably selected by those
skilled in the art. When a DNA encoding the compound is known, gene
therapy may be performed by inserting the DNA into a vector for
gene therapy. Doses of DNA and method for its administration may
vary depending on the body weight, age and symptoms of patients,
but can be suitably selected by those skilled in the art.
[0099] BGIF proteins of this invention suppress proliferation of
lympho-hematopoietic cells, and can thus be applied to diagnosis
and treatment of lympho-hematopoietic disorders. BGIF proteins are
also useful as a tool for elucidating pathogenetic mechanisms of
lympho-hematopoietic disorders and for screening candidates of
drugs for the disorders. BGIF genes may also be applied to gene
therapy of the diseases. Thus, this invention enables novel
diagnosis and treatment of lympho-hematopoietic disorders.
[0100] The present invention is demonstrated with reference to the
following Examples, but is not to be construed as being limited
thereto.
EXAMPLE 1
Identification of a New Downregulator of Lympho-hematopoiesis
BGIF
[0101] 1.1 Cell Culturing
[0102] A human renal cell carcinoma cell line 293T, a mouse stromal
clone BMS2 (Pietrangeli, C. E. et al., 1988, Eur. J. Immunol. 18:
863-872) and its subclone, BMS2.4 (Kincade, K. W. et al., 1991,
Adv. Exp. Med. Biol. 292: 227-234), and mouse myeloid leukemia cell
lines WEHI3 (ATCC No. TIB-68) were maintained in Dulbecco's
modified Eagle's medium (Nakalai Tesque, Kyoto, Japan) supplemented
with 10% fetal calf serum (FCS; GIBCO, Grand Island, N.Y.).
[0103] 1.2 Screening of a BMS2.4 cDNA Library
[0104] A subclone of a bone marrow derived stromal cell line,
BMS2.4, produces soluble factors which inhibit proliferation of
several types of hematopoictic cell lines (Kincade, K. W. et al.,
1991, Adv. Exp. Med. Biol. 292: 227-234; Oritani, K. et al., 1999,
Blood 93: 1346-1354). Neutralizing antibodies to TNF-.alpha.,
TGF-.beta., or IFN-.beta. did not block the effects of BMS2.4
supernatant. Thus, the growth inhibitory effects were not readily
attributed to any of these factors. To identify the unique BMS2.4
products, we performed expression cloning on the basis of growth
inhibition.
[0105] Polyadenylated RNA was isolated from BMS2.4 cells using a
Fast Track mRNA isolation kit (Invitrogen, San Diego, Calif.).
Double-stranded cDNA was synthesized with a TimeSaver cDNA
systhesis kit (Pharmacia, Uppsala, Sweden), ligated with BstXI
adaptors (Invitorogen), and cloned into a mammalian expression
vector, pEF-BOS (S. Mizushima and S. Nagata, 1990, Nucl. Acids Res.
18: 5322). Plasmid cDNAs were purified from pools of a few hundreds
of clones, and were transfected into 293T cells by calcium
phosphate precipitation method. Supernatants from each transfectant
were recovered and examined for growth inhibitory effects.
[0106] A myelomonocytic leukemia cell line, WEHI3, was particularly
sensitive and was used as a target. Supernatants from the
transfectants of 293T cells were added into cultures of WEHI3
cells, and the proliferation of the cells was evaluated by
3-(4,5-dimethylthiazol)-2,5-d- iphenyl tetrazolium bromide rapid
colorimetric assay (MTT assay). The triplicate aliquots of cells
were cultured in 96-well, flat bottom microtiter plates. MTT (10
.mu.L of 5 mg/mL solution in PBS) was added for the final 4 hours
of cultures, and then 100 .mu.L of acid-isopropanol (0.04 N HCl in
isopropanol) was added and mixed. The optical density was measured
on the Microelisa plate reader (Corona Electric, Ibaraki, Japan)
with a test wavelength of 540 nm.
[0107] The library was screened based on inhibition of the growth
of WEHI3 cells. A positive pool was divided into progressively
smaller pools and rescreened until a single clone was isolated. We
screened approximately 1.times.10.sup.5 clones and isolated a
single clone (clone 159-3-3) whose insert DNA encoded a
growth-inhibitor of WEHI3 cells. As shown in FIG. 1, the
supernatant of 293T cells transfected with the cloned 159-3-3
plasmid as well as BMS2.4-supernatant suppressed proliferation of
WEHI3 cells.
EXAMPLE 2
Identification of BGIF Protein
[0108] 2.1 Primary structure
[0109] The insert of the isolated clone was subcloned into
pBluescript (Stratagene, La Jolla, Calif.), and nucleotide sequence
was determined using an automated DNA sequencer (Applied
Biosystems, Foster City, Calif.). The cloned plasmid 159-3-3
contains a 997 bp cDNA insert (FIG. 2, SEQ ID NO: 1). Nucleotide
data base searching was performed with BLAST and FASTA from the GGG
computer program (Genetics Computer Group, Madison, Wis.). No
previously reported cDNAs or genomic DNAs are identical to this
sequence, and we designated this cloned molecule as Blood Cell
Growth-Inhibiting Factor, BGIF. The predicted protein translated
from the first ATG is composed of 33 amino acids (SEQ ID NO: 2). In
contrast, the predicted protein translated from the second ATG is
composed of 182 amino acids (SEQ ID NO: 3), and has a highly
hydrophobic stretch of 21 amino acid residues (SEQ ID NO: 4) at the
N-terminal end that is appropriate for a signal peptide.
[0110] 2.2 Identification of Functional Protein
[0111] We analyzed which ATG of BGIF cDNA is functional. First, we
constructed the 1st-ATG/pRL-SV40 or the 2nd-ATG/pRL-SV40 plasmid to
produce renilla luciferase translated from the first or the second
ATG of BGIF under the control of the SV40 early enhancer/promoter,
respectively.
[0112] For the construct to produce renilla luciferase using the
first ATG of BGIF, BGIF cDNA was amplified by PCR with
5'-GGGCTGCAGTCAGCGAGCAAGAGCC- CGAAG-3' (SEQ ID NO: 5), and
5'-GGGGCTAGCCACAGGCAGCATGCTGAAGCTTGA-3' (SEQ ID NO: 6). For the
construct to produce renilla luciferase protein using the second
ATG of BGIF, BGIF cDNA was amplified by PCR with
5'-GGGCTGCAGTCAGCGAGCAAGAGCCCGAAG-3' (SEQ ID NO: 5) and
5'-GGGGCTAGCACAGGCAGCATGCTGAAGCTTGA-3' (SEQ ID NO: 7). The
amplified fragments were digested with PstI and NheI, and cloned
into the pRL-SV40 plasmid (Promega, Madison, Wis.) whose original
ATG site for the renilla luciferase protein had been destroyed by
direct site mutagenesis (1st-ATG/pRL-SV40 and 2nd-ATG/pRL-SV40).
The renilla luciferase protein was translated from the ATGs of BGIF
cDNA in the 1st-ATG/pRL-SV40 and the 2nd-ATG/pRL-SV40 under the
control of the SV40 early enhancer/promoter. All plasmid constructs
were confirmed by sequencing.
[0113] These plasmids were transfected into BMS2.4 or BMS2 cells to
perform luciferase assay. Luciferase assay was performed by using
Dual-Luciferase Reporter System (Promega, Madison, Wis.), in which
transfection efficiency was monitored by co-transfected pGL-Control
Vector (Promega), an expression vector of firefly luciferase. The
cultured cells were transfected with 10 .mu.g of 1st-ATG/pRL-SV40
or the 2nd-ATG/pRL-SV40 together with 5 .mu.g of pGL-Control Vector
by lipofectamine transfection method. The transfected cells were
lysed in lysis buffer supplied by the manufacturer, followed by
measurement of the firefly and the renilla luciferase activities on
luminometer LB96P (Berthold Japan, Tokyo, Japan). The relative
renilla luciferase activities were calculated by normalizing
transfection efficiency according to the fire fly luciferase
activities.
[0114] Renilla luciferase translated from the second ATG of BGIF
was mainly produced (FIG. 3A).
[0115] Next, we produced proteins that were translated from the
first or the second ATG.
[0116] To produce proteins that are translated from the first or
the second ATGs of BGIF, BGIF cDNA was amplified by PCR. The
oligonucleotide primers used for these reactions were as follows:
5'-GGGCTCGAGTCAGCGAGCAA- GAGCCCGAAG-3' (SEQ ID NO: 8) and
5'-GGGCTCGAGCTGGGCTGCAGCTCAGCA-3' (SEQ ID NO: 9) for the protein
that was translated from the first ATG;
5'-GGGCTCGAGAATCGTCAAGCTTCAGCA-3' (SEQ ID NO: 10) and
5'-GGGCTCGAGCTTCTCCTCATCTTGGGC-3' (SEQ ID NO: 11) for the protein
that was translated from the second ATG. The amplified fragments
were digested with XhoI, and cloned into a pEFBOSX plasmid that was
yielded by site-directed mutagenesis to remove the XhoI site at
3524 of pEF-BOS (1st ATG/pEFBOSX and 2nd-ATG/pEFBOSX). All plasmid
constructs were confirmed by sequencing.
[0117] These plasmides were transfected into 293T cells. Each
supernatant from the transfectants was recovered and added into
cultures of WEHI3 cells to examine their growth. Supernatant of
293T cells transfected with 2nd-ATG/pEFBOSX, but not that of 293T
cells trasnfected with 1st-ATG/pEFBOSX, inhibited proliferation of
WEHI3 cells (FIG. 3B). Therefore, a functional product of BGIF is
translated from the second ATG.
[0118] As shown in FIG. 2, the deduced BGIF protein has a highly
hydrophobic stretch of 21 amino acid residues at the N-terminal
end, but lacks an internal membrane spanning domain, suggesting
that BGIF is a secreted protein. The protein sequence contains one
Asn-X-Ser/Thr potential N-linked glycosylation sites at the
position of amino acid 68, consistent with the hypothesis that BGIF
may be glycosylated. As a result of computer search of BGIF
sequence using FASTA and BLAST programs, BGIF has a similarity with
IFN-.alpha. (31.9% identity in 166 amino acid overlap) and
IFN-.beta. (25.9% identity in 166 amino acid overlap) at the amino
acid level (FIG. 4). However, six cystaine residues in the BGIF
protein were not identical to those of IFN-.alpha. or
IFN-.beta..
EXAMPLE 3
Gene Expression of BGIF in Hematopoietic Organs
[0119] Gene expression of BGIF was examined by Northern blot
analysis. PolyA.sup.+ RNAs were isolated using a Fast Track mRNA
isolation kit (Invitrogen), electrophoresed through a formaldehyde
agarose gel, and transferred onto a nylon membrane (Amersham). The
cDNA fragments were labeled with .sup.32P-dCTP using a random
primed DNA labeling kit (Boehringer Mannheim, Indiapolis, Ind.) and
hybridized to the membrane. Blots were then washed and
autoradiographed.
[0120] Northern blot analysis revealed that the message size of
BGIF was approximately 1 kb and that BMS2.4, but not WEHI3,
expressed BGIF mRNA (FIG. 5A). In addition, RT-PCR was used to
survey expression in mouse bone marrow and spleen. Total RNAs (2.5
.mu.g) were reverse transcribed to cDNA in total reaction volume of
50 .mu.L comprised of M-MLV reverse transcriptase (GIBCO), oligo dT
(1 .mu.g), 0.1 M DTT, 10 mM each dNTP, and 1.times.RT buffer. To
perform PCR, 10 .mu.L of the above RT mixtures were added to PCR
buffer containing 1.5 mM MgCl.sub.2, 1 U Taq polymerase (Perkin
Elmer, Branchburg, N.J.), 2 mM each dNTP, and relevant sense and
antisense primers. The oligonucleotide primers used for these
reactions were as follows: 5'-TCCAGCGTCCAGCGCAGC-3' (SEQ ID NO: 12)
and 5'-AGCACTTGCAGCTCACGC-3' (SEQ ID NO: 13) for BGIF;
5'-CCTAAGGCCAACCGTGAAAAG-3' (SEQ ID NO: 14) and
5'-TCTTCATGGTGCTAGGAGCCA-- 3' (SEQ ID NO: 15) .beta.-actin. PCR
reaction mixtures were amplified under the following conditions: 28
cycles of 94.degree. C. for 1 min, 55.degree. C. for 2 min,
72.degree. C. for 3 min.
[0121] As shown in FIG. 5B, 509 bp of PCR-amplified band was
observed from the RT sample of BMS2.4 mRNA. Same size of products
were observed, when RNAs of cultured bone marrow or spleen adherent
cells as well as those of freshly isolated bone marrow spleen cells
were subjected to RT-PCR. This amplification was specific to BGIF,
because the PCR products were confirmed by sequencing.
[0122] Therefore, BGIF mRNA is expressed in bone marrow and spleen,
at least by some of bone marrow and spleen stromal cells.
EXAMPLE 4
Effects of BGIF on Lympho-hematopoietic Cell Lines
[0123] To analyze functions of BGIF, an Ig/pEFBOSX vector (Oritani,
K. and Kincade P. W., 1996, J. Cell. Biol. 134: 771-782) was used
to produce Ig fusion proteins that were composed of CH2+CH3
cassette of human IgGI. The entire coding region of BGIF cDNA was
amplified by PCR with 5'-GGGGCGGCCGCCGCAATCGTCAAGCTTCA-3' (SEQ ID
NO: 16) and 5'-GGGCTCGAGCTTGGGCCTCTTCTCGCAGA-3' (SEQ ID NO: 17) and
the PCR sample was digested with NotI and XhoI, and ligated into an
Ig/pEFBOS vector (BGIF-Ig/BOS). Plasmid constructs were confirmed
by sequencing.
[0124] The fusion protein prepared form BGIF and Ig (BGIF-Ig) was
purified with protein A column (Pierce, Rockford, Ill.) from the
supernatant of 293T cells transfected with the BGIF-If/BOS plasmid.
CD44-Ig (Oritani, K. and Kincade P. W., 1996, J. Cell. Biol. 134:
771-782) that was composed of CD44 and the constant region of human
IgG was prepared in the same way, and used as a negative
control.
[0125] These purified proteins were added to cultures of WEHI3
cells to examine their effects on the growth of WEHI3 cells.
BGIF-Ig, but not CD44-Ig, exhibited growth-inhibiting effects on
WEHI3 cells in a dose dependent manner and its maximal activity was
observed at the concentration of 10 ng/mL. CD44-Ig fusion protein
had no growth-inhibiting effects.
[0126] A large panel of lympho-hematopoietic cell lines were
cultured in the presence of 100 ng/mL of BGIF-Ig or CD44-Ig for 48
hours, and their proliferation and viability were evaluated (Table
1).
[0127] Mouse myeloid leukemia cell line, M1, was maintained in
Dulbecco's modified Eagle's medium (Nakalai Tesque, Kyoto, Japan)
supplemented with 10% fetal calf serum (FCS; GIBCO, Grand Island,
N.Y.). Mouse pre-B cell clones, 1A9, BC7.12, BC7.7, 2E8, and F10
were maintained in McCoy's 5A medium (GIBCO) supplemented with 5%
FCS and 5.times.10.sup.5 M 2-mercaptoethanol in the presence of 1
ng/mL IL-7 (R&D Systems, Minneapolis, Minn.). Mouse lymphoma
cell lines (7OZ/3, WEHI231, BW5147, BCL1, SP2/0, and EL4) and a
virus transformed pre-B cell line, 18.81, were maintained in RPMI
1640 medium (Nakalai Tesque) supplemented with 10% FCS and
5.times.10.sup.5 M 2-mercaptethanol. A mouse multipotent cell line,
EML-C1, was maintained in Iscove's modified Dulbecco's medium
(GIBCO) supplemented with 20% horse serum (ICN Biomedical Inc.
Costa Mesa, Calif.) and 10 ng/mL SCF.
[0128] Cell growth was analyzed by .sup.3H-thymidine incorporation
assay. The triplicate aliquots of cells were cultured in 96-well,
flat bottom microtiter plates. Each well was pulsed for 4 hours
with 0.5 .mu.Ci.sup.3H-thymidine (sp. act.: 5 Ci/mM; Amersham
International, Amersham, Bucks, UK). The cells were then harvested
with a semiauotmatic cell harvester (model 1295; Pharmacia LKB
Biotechnology, Piscataway, N.J.), and the .sup.3H-thymidine
incorporation was measured with a liquid scintillation counter.
[0129] The effect of BGIF proteins is expressed as the stimulation
index (S.I.) (thymidine uptake (cpm) in the presence of
BGIF-Ig/thymidine uptake (cpm) in the presence of CD44-Ig).
Viability of cells was assessed by the tripan blue exclusion assay.
All values represent mean of two independent experiments.
1TABLE 1 Cell line Histology Proliferation (S.I.) Loss of viability
(%) BC7.12 Pre-B (LTBMC-derived) 0.01** 98.2** 1A9 Pre-B
(LTBMC-derived) 0.10** 61.4** 2E8 Pre-B (LTBMC-derived) 0.12**
72.5** F10 Pre-B (LTBMC-derived) 0.32** 0.2 BC7.7 Pre-B
(LTBMC-derived) 0.59** 1.2 18.81 Pre-B (virus transformed) 0.61**
-0.5 70Z/3 B lymphoma 0.74** -2.3 WEHI231 B lymphoma 0.61** -1.2
BCL1 B lymphoma 0.98 -0.9 SP2/0 Myeloma 0.69** 1.6 EL4 T lumphoma
0.95 0.1 BW5147 T lumphoma 0.63** 1.3 EML-C1 Multipotent 0.86* 1.1
WEHI3 Myelomonocytic leukemia 0.19** 0.2 WEHI279 Myelomonocytic
leukemia 0.85* 2.8 M1 Myelomonocytic leukemia 1.01 0.0 Statiscally
significant differences from control are indicated by one (p <
0.05) or two (p < 0.01) asterisks.
[0130] As shown in Table 1, BGIF-Ig suppressed the proliferation of
B lineage cell lines (1A9, BC7.12, BC7.7, F10, 2E8, 18-81, 7OZ/3,
WEHI231, SP2/0), a T lymphoma cell line (BW1597), myelomonocytic
leukemia cell lines (WEHI3, WEHI279), and a multipotent cell line
(EML-C1). In contrast, lymphoma cell lines (BCL1 and EL4) and a
myeloid cell line (M1) were not affected by BGIF-Ig. While most of
the cell lines did not die in response to this substance, three of
five LTBMC-derived pre-B cell clones (1A9, BC7.12, 2E8) lost their
viability. It is noteworthy that F10 cells derived from a BCL-2
transgenic mice were resistant to the cell death elicited by
BGIF-Ig.
[0131] Counts of viable WEHI3 and BC7.12 cells treated with BGIF-Ig
or CD44-Ig were performed every day (FIG. 6A). The doubling time of
WEHI3 cells was prolonged from 12 h with the control CD44-Ig fusion
protein to 24 h by exposure to BGIF-Ig.
[0132] DNA nuclear content in the cellular nucleus was also
analyzed. After stimulation, cells (1.times.10.sup.6) were washed
and resuspended in 100:1 of PBS, and then fixed by addition of
900:1 of cold ethanol. The fixed cells were incubated with 300:1 of
staining buffer (1 mg/ml RNase, 20 :g/ml propidium iodide, and
0.01% NP-40 in PBS) at 37.degree. C. for 10 mim. DNA contents were
then evaluated with FACSort (Becton Dickinson, Mountain View,
Calif.) using Cell Quest software.
[0133] As shown in FIG. 6b, analysis of DNA nuclear content
revealed that BGIF reduced S-phase population and increased
G0/G1-phase population in WEHI3 cells (Go/G1-phase; 70.4% with
BGIF-Ig, 50.1% with CD44-Ig, S-phase; 14.7% with BGIF-Ig, 35.1%
with CD44-Ig). In contrast, BC7.12 cells lost their viability when
treated with BGIF-Ig (FIG. 6A). The death of BC7.12 was due to
apoptosis, because a subdiploid peak of DNA appeared within 24 h
after the treatment with BGIF-Ig (FIG. 6B; 43.7% with BGIF-Ig, 0.3%
with CD44-Ig).
[0134] DNA fragmentation in the cell nucleus was examined. DNA
fragmentation was assayed as described in Oritani, K. et al., Blood
93, 1346-1354 (1999). After treatment with BGIF-Ig or CD44-Ig,
cells (1.times.10.sup.7) were lysed in 0.4 ml lysis buffer
containing 200 mM Tris-HCl, 100 mM EDTA, 1% SDS, and 50 :g/ml
proteinase K, and incubated for 4 h at 37.degree. C. DNAs were
extracted with phenol, and then with chloroform/isoamylalcohol. An
aqueous phase was collected and precipitated with NaCl and ethanol.
DNA pellets were suspended in 0.4 ml TE buffer, and treated with 50
:g/ml RNase for 5 h and then with 200 :g/ml proteinase K for 5 h.
DNAs were extracted twice and precipitated as above. DNA pellets
were resuspended in TE buffer, separated by electrophoresis in 1%
agarose gel (1 :g DNA per lane), and stained with 0.5 :g/ml
ethidium bromide, and visualized under ultraviolet light.
[0135] DNAs obtained from BC7.12 cells cultured with BGIF-Ig for 24
h showed extensive degradation with oligonucleosomal fragments
(FIG. 6C). Therefore, BGIF induced Go/G1-arrest or G1-prolongation
in WEHI3 cells and apoptosis in BC7.12 cells.
EXAMPLE 5
Influence of BGIF on Normal Lympho-hematopoietic Cells
[0136] Colony assays were used to evaluate the influence of BGIF on
normal lympho-hematopoiesis.
[0137] Bone marrow or spleen cells were prepared and suspended in 1
ml assay medium as described in Medina, K. L., Smithson, G. &
Kincade, P. W., J. Exp. Med. 178, 1507-1515 (1995) and Yokota, T.
et al., Blood 91, 3263-3272 (1998). The semisolid agar
colony-forming unit assay for B lymphocyte precursors (CFU-IL-7)
was performed with 1 ng recombinant mouse IL-7. Clonable B cells
(CFU-B) were enumerated in semisolid agar containing 25 :g
lipopolysaccharide. The progenitor assay for myeloid cells (CFU-GM)
and erythroid cells (BFU-E) was performed in methylcellulose media
(Veritas, Vancouver, Canada) consisting of IMDM, 0.9%
methylcellulose, 10.sup.-4 M 2-mercaptoethanol, 2mM L-glutamine,
30% FCS, 1% deionized crystallized bovine serum albumin, 3 U/ml
erythropoietin, 100 ng/ml stem cell factor, 3 ng/ml IL-3, and 10
ng/ml IL-6. All colony assays were performed in 35-mm dishes and
incubated at 37.degree. C. for 6 days.
[0138] As shown in FIG. 7, the cloning efficiency of mature mitogen
responsive B cells (CFU-B:B lymphocyte colony-forming units) was
only slightly decreased by addition of BGIF-Ig (43.3.+-.2.49 with
BGIF-Ig, 53.0.+-.2.94 with CD44-Ig per 2.5.times.10.sup.4 spleen
cells). However, interleukin (IL)-7 responding pre-B cells
(CFU-IL-7:IL-7 responding colony-forming units) were dramatically
influenced and their clonal proliferation was decreased
approximately 50% (66.0.+-.4.90 with BGIF-Ig, 121.0.+-.3.74 with
CD44-Ig per 5.times.10.sup.4 bone marrow cells). This inhibition
was highly specific and BGIF had no influence on the responsiveness
of myeloid progenitors to colony stimulating factors (CFU-GM:
granulocyte-macrophage colony-forming units) or that of erythroid
progenitors to erythropoietin (BFU-E: erythroid burst
colony-forming units). We then used the OP42 stromal cell clone
along with IL-7 to selectively support the production of B lineage
lymphocytes in short term cultures. Addition of BGIF-Ig
substantially reduced the yield of B lymphocytes in this model
(data not shown). More complex LTBMC were then exploited to
investigate the influence of BGIF on lympho-hematopoiesis.
[0139] LTBMC of lymphoid cells (W/W cultures) were initiated and
maintained according to published methods (Whitlock, C. A.,
Robertson, D. & Witte, O. N., J. Immunol. Methods 6, 7353-7569
(1984)). Briefly, 6.times.10.sup.6 bone marrow cells were cultured
in 25-cm.sup.2 flasks in 5% CO.sub.2 at 37.degree. C. The medium
consisted of RPMI 1640 supplemented with 5.times.10.sup.-5 M
2-mercaptoethanol and 5% FCS. LTBMC of myeloid cells (Dexter
cultures) were initiated and maintained by methods originally
described by Dexter et al. (Dexter, T. M. & Testa, N. G.,
Methods Cell Biol. 14, 387-405 (1976)). Briefly, 9.times.10.sup.6
bone marrow cells were cultured in 25-cm.sup.2 flasks in 5%
CO.sub.2 at 33.degree. C. The culture medium consisted of
.A-inverted.-MEM supplemented with 10.sup.-7 M hydrocortisone and
20% horse serum (HyClone, Logan, Utah). In both types of cultures,
half the medium was replaced weekly with fresh medium.
[0140] Adherent layers of Whitlock-Witte (W/W) cultures typically
contains macrophages, endothelial and fat cells, in addition to
stromal cells, while the latter is thought to be sufficient to
support lymphocyte formation from early progenitors (Whitlock, C.
A., Robertson, D. & Witte, O. N., J. Immunol. Methods 6,
7353-7569 (1984)). Adherent layers formed normally in the presence
of the control CD44-Ig fusion protein and the cultures produced
foci of lymphoid cells (FIG. 8). In contrast, adherent cells were
sparse in cultures containing BGIF-Ig and they did not support the
formation of lymphoid cells. In Dexter cultures, BGIF-Ig did not
affect either the formation of adherent layers or the production of
myeloid cells. These findings open the possibility that BGIF may
influence the lympho-hematopoietic microenvironment, in addition to
its direct effect on lymphoid progenitors.
EXAMPLE 6
BGIF Utilizes the IFN-.A-inverted./ Receptors and Induces IFN
Regulatory Factor (IRF)-1
[0141] BGIF has weak homology with IFN-.A-inverted., IFN-, and
IFN-.omega.. We analyzed whether BGIF displays its biological
effects via the IFN-.A-inverted./ receptors. A CFU-IL-7 colony
assay was performed using IFN-.A-inverted./ receptor knock out mice
(IFN-.A-inverted./R.sup.0- /0) (Muller, U. et al., Science 264,
1918-1921 (1994)). In wild type 129Sv mice (WT), BGIF-Ig reduced
CFU-IL-7 colony formation (FIG. 9A). However, BGIF-Ig did not
inhibit the IL-7 dependent clonal expansion of lymphocyte
precursors derived from mice whose IFN-.A-inverted./ receptors were
destroyed. IRF-1 is known to be a downstream effector of the IFN
receptors and inducible by the IFN receptor ligation. As shown in
FIG. 9B, BGIF-Ig induced expression of the IRF-1 gene, while IRF-2
was expressed constitutively and was not affected by BGIF-Ig. These
findings indicate that BGIF influences B lymphocyte precursors via
the IFN-.A-inverted./ receptors and induces at least one mediator
of IFN action.
EXAMPLE 7
Signal Transduction Pathway Utilized by BGIF
[0142] Cytokine functions are mainly mediated by the Janus kinase
(Jak) family of protein tyrosine kinases along with signal
transducers and activators of transcription (Stat) (Ihle, J. N.,
Cell 84, 331-334 (1996)). Tyrosine phosphorylation of Jak-Stat
proteins were analyzed after WEHI3 and BC7.12 cells were exposed to
BGIF-Ig or CD44-Ig respectively.
[0143] Immunoprecipitation, gel electrophoresis, and immunoblotting
were performed according to methods described previously
(Matsumura, I. et al., Mol. Cell. Biol. 17, 2933-2943 (1997)).
Briefly, cells were serum-starved, stimulated with BGIF-Ig, and
then lysed in lysis buffer. After insoluble material was removed by
centrifugation, the lysates obtained from 1.times.10.sup.7 cells
were incubated with 1 :g of the indicated antibodies, followed by
the addition of protein G sepharose beads (Amersham). The
immunoprecipitates were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The proteins were
electrophoretically transferred onto a polyvinylidene difluoride
membrane (Immobilion; Millipore Corp., Bedford, Mass.). After
blocking of residual binding sites on the filter, immunoblotting
was performed with the appropriate antibodies. Immunoreactive
proteins were visualized with the enhanced chemiluminescence
detection system (DuPont NEN, Boston, Mass.).
[0144] In WEHI3 cells, BGIF-Ig induced tyrosine phosphorylation of
Jak1, Jak2, Tyk2, Stat 1, and Stat3, but not that of Jak3 (FIG.
10A). Stat5 molecule was constitutively phosphorylated and was not
affected by BGIF-Ig. Activation of Stat1 and Stat3 was also
indicated by the fact that treatment WEHI3 cells with BGIF-Ig
induced expression of the Stat1-dependent gene, IRF-1 (FIG. 9B).
Jun-B is a Stat3-dependent gene and it was also induced (data not
shown). In BC7.12 cells, BGIF-Ig induced tyrosine phosphorylation
of Jak1, Tyk2, Stat1, and Stat5, but not that of Jak2, Jak3, or
Stat3 (FIG. 10B). CD44-Ig did not induce tyrosine phosphorylation
of any examined Jak-Stat proteins in both of cell lines. Therefore,
BGIF activates Jak1, Tyk2, and Stat1 in several cell types, as well
Jak2, Stat3, and Stat5 in certain cellular environments.
Sequence CWU 1
1
17 1 997 DNA Mus musculus CDS (161)..(706) 1 agaagtcgcg tccagcgtcc
agcgcagcgc aggcagtcag cgagcaagag cccgaagctc 60 cgagtgaact
attaaagcag caaactccag gctcaatggg aaggcggcct tgccctcgcg 120
ctccccctgc aggccagccc cgcaatcgtc aagcttcagc atg ctg cct gtg cat 175
Met Leu Pro Val His 1 5 cta ttc ctg gtg gga ggg gtg atg ctg agc tgc
agc cca gcc agc tca 223 Leu Phe Leu Val Gly Gly Val Met Leu Ser Cys
Ser Pro Ala Ser Ser 10 15 20 ctt gat tct ggt aaa tct ggg agc ctg
cac ctg gag cgc agc gaa acc 271 Leu Asp Ser Gly Lys Ser Gly Ser Leu
His Leu Glu Arg Ser Glu Thr 25 30 35 gcg cgc ttc cta gca gag ctc
cga agc gtg ccg ggt cac cag tgc ctg 319 Ala Arg Phe Leu Ala Glu Leu
Arg Ser Val Pro Gly His Gln Cys Leu 40 45 50 cgg gac agg acc gat
ttc cca tgt ccc tgg aag gaa gga act aac atc 367 Arg Asp Arg Thr Asp
Phe Pro Cys Pro Trp Lys Glu Gly Thr Asn Ile 55 60 65 aca cag atg
act ctg gga gaa acc acc agt tgc tac tcc cag acc ctc 415 Thr Gln Met
Thr Leu Gly Glu Thr Thr Ser Cys Tyr Ser Gln Thr Leu 70 75 80 85 agg
cag gtc ctc cac ctc ttt gac aca gag gcc agc aga gct gcc tgg 463 Arg
Gln Val Leu His Leu Phe Asp Thr Glu Ala Ser Arg Ala Ala Trp 90 95
100 cac gag agg gcg ctg gac cag cta cta tct agc ctg tgg cgt gag ctg
511 His Glu Arg Ala Leu Asp Gln Leu Leu Ser Ser Leu Trp Arg Glu Leu
105 110 115 caa gtg ctg aag agc cca aga gag cag ggc cag tcc tgt cca
ctg cct 559 Gln Val Leu Lys Ser Pro Arg Glu Gln Gly Gln Ser Cys Pro
Leu Pro 120 125 130 ttt gcc ctg gcc atc cgc acc tac ttc cga ggg ttc
ttc cgc tat ctg 607 Phe Ala Leu Ala Ile Arg Thr Tyr Phe Arg Gly Phe
Phe Arg Tyr Leu 135 140 145 aag gca aag gca cac agc gct tgc tcc tgg
gag atc gtc aga gtc caa 655 Lys Ala Lys Ala His Ser Ala Cys Ser Trp
Glu Ile Val Arg Val Gln 150 155 160 165 ttg caa gtg gac ctt cca gcg
ttc cca ctg tct gcg aga aga ggc cca 703 Leu Gln Val Asp Leu Pro Ala
Phe Pro Leu Ser Ala Arg Arg Gly Pro 170 175 180 aga tgaggagaag
ccccgtgcag gaatctctct gctctcgtga caccacgctc 756 Arg cctctctcca
ttcaaagcag acgcacggat tcggattcag caccaacagg cgaaatgggc 816
atgcatcgac caagaacatc gagttcttta tgtcttccct gccagaggcc ccgaagcatc
876 ctactgtaca tcatacactg cgaaagatgt ttgaaagaaa acctgtgctc
ttgcatttga 936 ggtggcttct gaataaattg atgatctcgg ttaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 996 a 997 2 33 PRT Mus musculus 2 Met Gly Arg
Arg Pro Cys Pro Arg Ala Pro Pro Ala Gly Gln Pro Arg 1 5 10 15 Asn
Arg Gln Ala Ser Ala Cys Cys Leu Cys Ile Tyr Ser Trp Trp Glu 20 25
30 Gly 3 182 PRT Mus musculus 3 Met Leu Pro Val His Leu Phe Leu Val
Gly Gly Val Met Leu Ser Cys 1 5 10 15 Ser Pro Ala Ser Ser Leu Asp
Ser Gly Lys Ser Gly Ser Leu His Leu 20 25 30 Glu Arg Ser Glu Thr
Ala Arg Phe Leu Ala Glu Leu Arg Ser Val Pro 35 40 45 Gly His Gln
Cys Leu Arg Asp Arg Thr Asp Phe Pro Cys Pro Trp Lys 50 55 60 Glu
Gly Thr Asn Ile Thr Gln Met Thr Leu Gly Glu Thr Thr Ser Cys 65 70
75 80 Tyr Ser Gln Thr Leu Arg Gln Val Leu His Leu Phe Asp Thr Glu
Ala 85 90 95 Ser Arg Ala Ala Trp His Glu Arg Ala Leu Asp Gln Leu
Leu Ser Ser 100 105 110 Leu Trp Arg Glu Leu Gln Val Leu Lys Ser Pro
Arg Glu Gln Gly Gln 115 120 125 Ser Cys Pro Leu Pro Phe Ala Leu Ala
Ile Arg Thr Tyr Phe Arg Gly 130 135 140 Phe Phe Arg Tyr Leu Lys Ala
Lys Ala His Ser Ala Cys Ser Trp Glu 145 150 155 160 Ile Val Arg Val
Gln Leu Gln Val Asp Leu Pro Ala Phe Pro Leu Ser 165 170 175 Ala Arg
Arg Gly Pro Arg 180 4 21 PRT Mus musculus 4 Met Leu Pro Val His Leu
Phe Leu Val Gly Gly Val Met Leu Ser Cys 1 5 10 15 Ser Pro Ala Ser
Ser 20 5 30 DNA Artificial Sequence Description of Artificial
SequenceArtificially Synthesized Primer Sequence 5 gggctgcagt
cagcgagcaa gagcccgaag 30 6 33 DNA Artificial Sequence Description
of Artificial SequenceArtificially Synthesized Primer Sequence 6
ggggctagcc acaggcagca tgctgaagct tga 33 7 32 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 7 ggggctagca caggcagcat gctgaagctt ga 32 8 30 DNA
Artificial Sequence Description of Artificial SequenceArtificially
Synthesized Primer Sequence 8 gggctcgagt cagcgagcaa gagcccgaag 30 9
27 DNA Artificial Sequence Description of Artificial
SequenceArtificially Synthesized Primer Sequence 9 gggctcgagc
tgggctgcag ctcagca 27 10 27 DNA Artificial Sequence Description of
Artificial SequenceArtificially Synthesized Primer Sequence 10
gggctcgaga atcgtcaagc ttcagca 27 11 27 DNA Artificial Sequence
Description of Artificial SequenceArtificially Synthesized Primer
Sequence 11 gggctcgagc ttctcctcat cttgggc 27 12 18 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 12 tccagcgtcc agcgcagc 18 13 18 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 13 agcacttgca gctcacgc 18 14 21 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 14 cctaaggcca accgtgaaaa g 21 15 21 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 15 tcttcatggt gctaggagcc a 21 16 29 DNA Artificial
Sequence Description of Artificial SequenceArtificially Synthesized
Primer Sequence 16 ggggcggccg ccgcaatcgt caagcttca 29 17 29 DNA
Artificial Sequence Description of Artificial SequenceArtificially
Synthesized Primer Sequence 17 gggctcgagc ttgggcctct tctcgcaga
29
* * * * *