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WO1999015560A1 - Polypeptide soluble comportant un domaine delta-serrate-lag2 de l'homologue delta et utilisations de ce polypeptide - Google Patents

Polypeptide soluble comportant un domaine delta-serrate-lag2 de l'homologue delta et utilisations de ce polypeptide Download PDF

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Publication number
WO1999015560A1
WO1999015560A1 PCT/US1998/020190 US9820190W WO9915560A1 WO 1999015560 A1 WO1999015560 A1 WO 1999015560A1 US 9820190 W US9820190 W US 9820190W WO 9915560 A1 WO9915560 A1 WO 9915560A1
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delta
del
serrate
cells
homologue
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Wei Han
Malcolm A. S. Moore
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • HGFs hematopoietic growth factors
  • Dill 25 murine Delta homologue, Del ta-like 1 ⁇ Dill ) , in regulation of hematopoiesis in response to HGF stimulation.
  • Dill protein Dill
  • BM adult mouse bone marrow
  • BM cells were isolated from a Dlll la ⁇ z mouse .
  • ⁇ -galactosidase staining of the isolated cells showed that stromal/endothelial cells of BM expressed Dill.
  • stromal/endothelial cells of BM expressed Dill Furthermore, we have demonstrated a role of Dill in regulation of hematopoiesis using a soluble recombinant form of the extracellular DSL domain of Dill produced in Escherichia coli (E. coli . ) .
  • DSL enhanced the expansion of primitive hematopoietic precursors (high proliferation potential colony-forming cells - HPP-CFCs) when combined with HGFs including interleukin-3 (IL-3), granulocyte co lony- s t imul a t ing factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) .
  • IL-3 interleukin-3
  • G-CSF granulocyte co lony- s t imul a t ing factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • DSL promoted the expansion of primitive HPP-CFC responsive to IL-3, interleukin-l ⁇ (IL-l ⁇ ) and macrophage colony-stimulating factor (M-CSF) , and at the same time inhibited the differentiation of primitive precursors into more mature precursor cells responsive to IL-3 alone.
  • Notch encodes a receptor protein with 36 epidermal growth factor (EGF) -like repeats in the extracellular domain, a single transmembrane domain, and 6 cdclO/ankyrin repeats in the intracellular domain (1, 2).
  • EGF epidermal growth factor
  • Delta (3) and Serrate (4) are two Notch ligands with multiple EGF-like repeats in their extracellular domain, a single transmembrane domain, and a very short intracellular domain.
  • Notch homologues have been cloned in vertebrates (5) . The most studied vertebrate Notch homologues is the mouse Notchl (mNotchl) (6-9) .
  • Notch homologue TAN-1 has been implicated in the pathophysiology of a subclass of human T cell leukemia (13,
  • TAN-1 and mNotchl were detected in human and mouse hematopoietic stem/progenitor cells (HSC/HPC) respectively
  • Notchl into a mouse myeloid cell line 32D showed that the transfected cells proliferated, but failed to differentiate into granulocytes in response to G-CSF (16) .
  • Notch ligands are defined by their unique DSL structural domain
  • EGF-like repeats e.g. DLK/Pref-1 (18, 19) .
  • This highly evolutionary conserved DSL domain has been experimentally demonstrated to be essential in mediating the ligand/receptor interaction. Deletion of the DSL domain of Delta abolished its interaction with the extracellular domain of its receptor Notch, with loss of the function of the ligand (20, 21) .
  • most HGFs induce both mitogenic and differentiative signals (22-25) . Mitogenic signals stimulate entry of quiescent cells into cell cycle, and cell proliferation, while differentiation signals facilitate the coordinated expression of differentiation-specific genes along appropriate pathways.
  • HGFs The role of HGFs is thus permissive, necessary for the expression of a differentiation program but not instructive in that the HGFs do not initiate the lineage commitment.
  • CFC assay semi-solid culture systems
  • HPP-CFC High proliferation potential colony-forming cells
  • HPP-CFCs exist in BM at extremely low frequency. However, they can be efficiently enriched by in vivo purging of BM with 5-Fu.
  • a single dose of 5-Fu can reduce the numbers of more mature CFC population by more than 99% while enriching the BM for more primitive HPP-CFCs (26) .
  • HPP-CFCs According to their HGF requirements, subpopulation of HPP-CFCs have been described: primitive HPP-CFC requires IL-3, IL-1 and M-CSF; mature HPP-CFC needs only IL-3 to form colony in CFC assay (27-29) .
  • Dill and Notchl play essential roles in embryonic development (30-32) .
  • vertebrate homologues of Notch and its ligands also mediate inductive signaling between HSC/HPC and BM stromal cells by which the hematopoietic microenvironment influences the competence of HSC/HPC to respond to further instructive signals.
  • Dill protein was detected in adult mouse BM.
  • the soluble DSL domain of this protein affected the proliferation and differentiation of hematopoietic precursors in response to HGFs using the HPP-CFC assay for detection of primitive murine hematopoietic precursors closely related to the HSCs, together with assays for their more committed progeny (24, 27, 28, 33) .
  • DSL had no effect on its own, but in combination with certain HGFs it increased the expansion of the most primitive hematopoietic precursors assayed (HPP-CFC stimulated by IL-3, IL-1 and M-CSF) and decreased the generation of later stages (e.g. HPP-CFC responding to IL-3 alone) .
  • DSL also in combination with IL-3, enhanced the generation of promonocytes over many weeks of culture and blocked their terminal differentiation to macrophages.
  • IL-3 also in combination with IL-3, enhanced the generation of promonocytes over many weeks of culture and blocked their terminal differentiation to macrophages.
  • DSL appears to alter the pattern of response of primitive hematopoietic precursors as well as certain highly differentiated macrophage precursors to HGF treatment.
  • we present evidence indicating that DSL reduces apoptosis of hematopoietic cells in HGF stimulated short-term suspension cultures.
  • This invention provides a method for inducing proliferation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of said hematopoietic cells.
  • This invention also provides a method for inducing proliferation of hematopoietic cells in a subject comprising administering to the subject an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of said hematopoietic cells in the subject.
  • This invention further provides a method for blocking hematopoietic growth factor differentiation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to block the hematopoietic growth factor differentiation of said hematopoietic cells.
  • This invention provides a method for blocking hematopoietic growth factor differentiation of hematopoietic cells in a subject comprising administering to the subject an amount of a soluble polypeptide having sequence of the Delta- Serrate-lag2 domain of a Del ta homologue effective to block the hematopoietic growth factor differentiation of said hematopoietic cells in the subject.
  • This invention also provides a method for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of hematopoietic and to block the hematopoietic growth factor differentiation of hematopoietic cells.
  • This invention provides a method for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of hematopoietic cells in a subject administering to the subject an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of hematopoietic and to block the hematopoietic growth factor differentiation of hematopoietic cells in the subject.
  • hematopoietic growth factors include, but not limited to the hematopoietic growth factor is interleukin-3, granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor.
  • This invention also provides a composition comprising a purified soluble polypeptide having sequence of the Delta- Serrate-lag2 domain of a Delta homologue and an acceptable carrier.
  • This invention provides a pharmaceutical composition for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of said hematopoietic cells comprising a purified soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue and a pharmaceutically acceptable carrier.
  • This invention also provides a method to producing soluble polypeptide having the sequence of the Delta-Serrate-lag2 domain of a Del ta homologue comprising steps of: (a) linking the nucleic acids which encodes the Delta- Serrate-lag2 domain of a Del ta homologue to the nucleic acids which encodes a thioredoxin such that the linked nucleic acids will express a polypeptide having the Delta- Serrate-lag2 domain with the thioredoxin fused to the carboxyl terminus of the Delta-Serrate-lag2 domain; (b) linking the nucleic acids resulting from step (a) to an expression vector; (c) introducing the expression vector resulting from step (b) into an appropriate host such that the polypeptide may be expressed; (d) placing the vector in suitable condition such that polypeptide will be expressed in a soluble form, thereby producing a soluble polypeptide having the sequence of the Delta-Serrate-lag2 domain of a Del ta homologue.
  • FIG. 1 Mouse Dill is expressed in a subset of adult BM cells.
  • A Western blot showing Dill expression in BM. Dill expressed in C0S7 cells is present at ⁇ 80 Kd. An similar major band at ⁇ 80 Kd was detected in adult mouse BM.
  • B ⁇ -galactosidase staining of BM from a DLLl Lac ⁇ z mouse. A subpopulation of BM cells are blue-stained positive cells.
  • A Coomassie-Blue staining of protein fractions purified with a thio-bond affinity chromatography. A major band of 39.3 Kd indicated by an arrow was purified. Lanes: 1, sample of -257. coli extract for purification;
  • IL-3 plus IL-1 plus M-CSF DSL at 250 ng/ml promotes the expansion of HPP-CFCs in combination with IL-3 + IL-1, and IL + KL. However, it does not synergyze with other HGF combinations: IL-1 + IL-6 + KL, IL-1 + KL, IL-6
  • DSL blocks the differentiation of promonocytes to macrophages. The accumulative fold expansions in cell numbers of three independent experiments after culture for various days are shown.
  • 5-Fu BM were cells freshly isolated (first two columns) . 24 hours post 5-Fu BM cells were cultured with different combinations of HGFs ;+ DSL (the rest of columns) . After culture for a week, the cells were fixed and stained with propidium iodide. Their DNA profiles were analyzed by the fluorescence-activated cell sorting. The results are reported as the percentage of cells in the sub-Go/Gl stage (apoptotic cells) over the total cells analyzed.
  • This invention provides a method for inducing proliferation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of said hematopoietic cells.
  • the Delta-Serrate-lag2 domain is defined as a region of approximately 45 amino acids with conserved spacing of six cystine among Notch ligands Delta and Serrate in Drosophila and their homologues identified in other species .
  • Notch ligands and their homologues include but are limited to: human Delta-like 1 (Genebank Acession Number AF003522 and Jagged (59); mouse Delta-like 1 (60), Delta-like 3 (61), Serrate-l/Jagged-1 (62) ; rat Jagged (12), Jagged2 (63); chicken, C-Delta-1 (64), C-Serrate-1 (65); Xenopus, X-Delta-1 (66), X-Delta-2 (67); C. elegans, Lag-2 (17), Apx-1 (68); and Drosophila, Delta (3) and Serrate (14).
  • This invention also provides a method for inducing proliferation of hematopoietic cells in a subject comprising administering to the subject an amount of a soluble polypeptide having sequence of the Delta-Serrate- lag2 domain of a Del ta homologue effective to induce proliferation of said hematopoietic cells in the subject.
  • This invention further provides a method for blocking hematopoietic growth factor differentiation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to block the hematopoietic growth factor differentiation of said hematopoietic cells.
  • This invention provides a method for blocking hematopoietic growth factor differentiation of hematopoietic cells in a subject comprising administering to the subject an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to block the hematopoietic growth factor differentiation of said hematopoietic cells in the subject.
  • This invention also provides a method for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of hematopoietic cells comprising contacting the said cells with an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of hematopoietic and to block the hematopoietic growth factor differentiation of hematopoietic cells.
  • This invention provides a method for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of hematopoietic cells in a subject administering to the subject an amount of a soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue effective to induce proliferation of hematopoietic and to block the hematopoietic growth factor differentiation of hematopoietic cells in the subject.
  • the Del ta homologue is Del ta -like 1, Del ta-like 2 or Del ta-like 3.
  • the Del ta homologue is human Delta- like 1, human Jagged, mouse Delta-like 1, mouse Delta-like
  • hematopoietic growth factors include, but not limited to the hematopoietic growth factor is interleukin-3, granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor.
  • This invention also provides a composition comprising a purified soluble polypeptide having sequence of the Delta- Serrate-lag2 domain of a Del ta homologue and an acceptable carrier.
  • This invention provides a pharmaceutical composition for inducing proliferation of hematopoietic cells and blocking hematopoietic growth factor differentiation of said hematopoietic cells comprising a purified soluble polypeptide having sequence of the Delta-Serrate-lag2 domain of a Del ta homologue and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers means any of the standard pharmaceutical carriers.
  • suitable carriers are well known in the art and may include, but not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solutions, phosphate buffered saline containing Polysorb 80, water, emulsions such as oil/water emulsion, and various type of wetting agents.
  • Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.
  • Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium sterate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients.
  • Compositions comprising such carriers are formulated by well known conventional methods.
  • the Del ta homologue is Del ta-like 1, Del ta-like 2 or Del ta-like 3.
  • the Del ta homologue is human Deltalike 1, human Jagged, mouse Delta-like 1, mouse Delta-like 3, mouse Serrate-1/Jagged-1, rat Jagged, rat Jagged2, chicken C-Delta-1, chicken C-Serrate-1, Xenopus X-Delta-1, Xenopus X-Delta-2, C. elegans Lag-2, C. elegans Apx-1, Drosophila Delta or Drosophila Serrate.
  • This invention also provides a method to producing soluble polypeptide having the sequence of the Delta-Serrate-lag2 domain of a Del ta homologue comprising steps of: (a) linking the nucleic acids which encodes the Delta- Serrate-lag2 domain of a Del ta homologue to the nucleic acids which encodes a thioredoxin such that the linked nucleic acids will express a polypeptide having the Delta- Serrate-lag2 domain with the thioredoxin fused to the carboxyl terminus of the Delta-Serrate-lag2 domain; (b) linking the nucleic acids resulting from step (a) to an expression vector; (c) introducing the expression vector resulting from step (b) into an appropriate host such that the polypeptide may be expressed; (d) placing the vector in suitable condition such that polypeptide will be expressed in a soluble form, thereby producing a soluble polypeptide having the sequence of the Delta-Serrate-lag2 domain of a Del ta homologue.
  • This invention also provides production of soluble polypeptide having the sequence of the Delta-Serrate-lag2 domain of a Del ta homologue by direct expression of the polypeptide in an appropriate host.
  • the expressed protein may then be solubilized.
  • the expressed protein is solubilized by detergent.
  • the detergent is SDS.
  • the appropriate host for the production of soluble polypeptide is a prokaryote or a eukaryote.
  • the eukaryotic host is a mammalian cell, an insect cell or a yeast cell.
  • the prokaryotic host is a bacterial cell such as E.coli.
  • the Del ta homologue is Del ta -like
  • the Del ta homologue is human Delta- like 1, human Jagged, mouse Delta-like 1, mouse Delta-like 3, mouse Serrate-1/Jagged-1, rat Jagged, rat Jagged2, chicken C-Delta-1, chicken C-Serrate-1, Xenopus X-Delta-1, Xenopus X-Delta-2, C. elegans Lag-2, C. elegans Apx-1, Drosophila Delta or Drosophila Serrate.
  • COS7 cells were obtained from ATCC and cultured in medium containing Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; HyClone Laboratories, Logan, Utah) and 0.05 mg/ml gentamicin (GIBCO, Grand Island, NY) .
  • IMDM Iscove's modified Dulbecco's medium
  • FCS fetal calf serum
  • FCS HyClone Laboratories
  • GIBCO fetal calf serum
  • GIBCO fetal calf serum
  • Balb/C mice from Jackson laboratory (Bar Harbor, MA) were maintained and bred under laminar-flow conditions and used when they were at least 8 weeks of age. Details of Dill knock-out mice were reported earlier (32) .
  • D-Z-Zl gene was mutated by homologous recombination in embryonic stem cells such that amino acids 2-116 were replaced with an in-frame fusion of the lacZ gene of E. coli .
  • This allele, called Dll l lacZ was introduced into the mouse germ line.
  • HGFs include recombinant mouse IL-3 (IL-3, 10 ng/ml, Intergen, Purchase, NY), c-kit ligand (KL, 20 ng/ml, Immunex, Seattle, WA) , GM-CSF (1000 U/ml , Immunex, Seattle, WA) ; recombinant human M-CSF (M-CSF, 1000 U/ml Cetus, Emeryville, CA) , G-CSF(1000 U/ml, Agen) , IL-l ⁇ (100 U/ml, Syntex, Palo Alto, CA) , and interleukin-6 (IL-6, 20 ng/ml, Imclone, New York, NY) .
  • M-CSF human M-CSF
  • M-CSF 1000 U/ml Cetus, Emeryville, CA
  • IL-l ⁇ 100 U/ml, Syntex, Palo Alto, CA
  • the pBos/Dlll plasmid was constructed by subcloning the full-length Dill cDNA into a mammalian expression vector pEF-BOS (34) .
  • the complete coding region of Dill was constructed using the following parts: 1) the first 280 bp Xbal-Sall fragment created by polymerase chain reaction (PCR) ; 2) the middle 900 bp Sall-Celll fragment from a Dil l cDNA subclone 649; 3) the last 617 bp Celll-EcoRI fragment from a Dil l cDNA subclone 617.
  • the plasmid was transfected into the COS7 cells by the calcium phosphate method according to the manufacturer instructions (Promega, Madison, Wl) .
  • cell extracts were prepared by lysing the transfected and mock-transfected cells with RIPA buffer (10 mM sodium phosphate, pH 7.0, 0.15 M NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) , 1% aprotinin) .
  • RIPA buffer 10 mM sodium phosphate, pH 7.0, 0.15 M NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) , 1% aprotinin
  • the pTrx/DSL plasmid was constructed by subcloning the PCR-amplified DSL region of the Dill cDNA into an E. coli expression vector pTrxFus (Invitrogen, San Diego, CA) .
  • pTrxFus E. coli expression vector
  • the sequence of forward primer was GATCTCTAGACCTCCATAC AGACTCT, starting from the position of 390 bp ( Dill data base accession number X80903) ; the sequence of reverse primer was GATCGTCGACAGAT TGGGTCAGTGCA, ending at the position of 688 bp.
  • the template for the PCR-amplification was a plasmid pl5-6 containing the DSL region.
  • PCR-amplification Xbal-Sall DNA fragment was subcloned into the pTrxFus vector.
  • the pTrx/DSL plasmid was amplified in a E. coli strain GI724 as described by the manufacturer (Invitrogen, San Diego, CA) .
  • the expression of DSL/thioredoxin fusion protein from the pTrx/DSL plasmid was induced with tryptophan.
  • the cells were resuspended and sonicated in buffer H (0.1 M Tris/HCl, pH 8, 0.1 M NaCl, 2.5 mM EDTA) containing 1 ⁇ g/ml leupeptin and 0.5 mg/ml lysozyme.
  • buffer H 0.1 M Tris/HCl, pH 8, 0.1 M NaCl, 2.5 mM EDTA
  • a total of 200 mg soluble protein in 20 ml buffer H was recovered by centrifugation of the E. coli lysate for 30 min at 15000 x g.
  • the fusion protein was purified by gel filtration column Sephacryl S-200 HR (Pharmacia Biotech, Piscataway, NJ) .
  • the column size was 2.6 X 100 cm with a running speed of 1 ml/min and a fraction size of 5 ml. Fifty fractions were collected, and analyzed by SDS-PAGE and Western blotting with anti-thioredoxin monoclonal antibody (see results
  • the DSL/thioredoxin protein was used as antigen to raise anti-Dill antibody.
  • the gel filtration purified fusion protein was separated on two preparative 12% SDS gel by electrophoresis .
  • the corresponding fusion protein band was localized by Coomassie Blue staining.
  • the excised gel slices were send to the company to raise the antibody in chicken using the company's standard protocol (Lampire biological laboratories, Inc. Pipersville, PA) .
  • the antibody (IgY) was purified from the egg yolk using a gamma yolk kit as described by the manufacturer (Pharmacia Biotech, Piscataway, NJ) .
  • the crude IgY was further purified by immunoaffinity purification using an antigen column.
  • the purified DSL protein was coupled into CNBr activated Sepharose 4B beads according to the protocol by the manufacturer (Pharmacia Biotech) .
  • the crude IgY was allowed to bind to the antigen gel in a batch absorption fashion overnight.
  • the non-specific proteins bound to the gel were washed by 10 column volumes of 0.5 M NaCl/PBST (phosphate buffered saline, 0.1% Tween 20).
  • the anti-DSL IgY was eluted with 4 M MgCl 2 /0.25 M glycine, pH 3.5.
  • Ten fractions were collected and dialyzed with PBS overnight.
  • the antibody activity in each fractions was determined in a standard Western blotting with the fusion protein as antigen. Fraction 6 to 10 were pooled and used as the affinity purified anti-Dill antibody.
  • the horseradish peroxidase-labeled rabbit anti-chicken IgY (Jackson ImmunoResearch) was then used at 1:5000 dilution.
  • the immunocomplexes were detected by exposure to X-ray film ( Fig . IA) .
  • the blot was then incubated with goat-anti-mouse IgG conjugated by alkaline phosphatase at 1:250 dilution.
  • the protein bands were visualized by incubation in an alkaline phosphatase substrate solution containing 0.03% nitro-blue tetrazolium chloride (NBT) , 0.002% 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) (Fig. 2C) .
  • BM cells were isolated from heterozygous adult Dlll lacZ mice, fixed with 0.05% glutaraldehyde for 15 min. After the cells were washed twice in 0.1 M potassium phosphate buffer (pH 7.4), they were incubated with X-gal solution (10 mM potassium ferricyanide, 10 mM potassium ferrocyanide, 2 mM MgCl 2 , 1 mg/ml Xgal) overnight, and photographed using a Axioplan microscope (Zeiss, Germany) .
  • X-gal solution 10 mM potassium ferricyanide, 10 mM potassium ferrocyanide, 2 mM MgCl 2 , 1 mg/ml Xgal
  • Colony-forming cell (CFC) assay HPCs capable of forming colonies were assayed in 35-mm petri dishes containing a variable number of hematopoietic cells in 1 ml of IMDM/20% FCS and 0.36% agarose (SeaPlaque; FMC, Rockland, Maine) plus HGFs (28) .
  • HGFs in the CFC assay were IL-3, GM-CSF, and KL.
  • Triplicate cultures were incubated for 14 days at 37°C in a fully humidified 5% C0 2 atmosphere.
  • CFC colonies (greater than 50 cells) and HPP-CFC colonies (greater than 0.5 mm in diameter) were scored from triplicate dishes.
  • HGFs used in the CFC assays were: IL-3, IL-1 and M-CSF; or IL-3.
  • DSL promotes the expansion of primitive HPP-CFC at the expense of mature subset.
  • the standard secondary CFC assay was performed with 48 hours post 5-Fu BM cells. Liquid culture of 10 5 cells were carried out in quadruplicate for a week with HGF combinations including IL-3, IL-3 plus IL-1, and IL-3 plus IL-1 plus M-CSF with or without DSL.
  • the starting cells (input HPP-CFC) and cells after 7-day liquid cultures (output HPP-CFC) were quantified for the presence of HPP-CFCs with combinations of HGFs including IL-3, IL3 plus IL-1, and IL-3 plus IL-1 plus M-CSF. Data are the calculated numbers (mean +_ SEM) of HPP-CFC of quadruplicate liquid cultures.
  • mice were treated with 5-Fu by i.v. injection of 150 mg/kg body weight in a volume of 200 ⁇ l .
  • Marrow was obtained 24 to 48 hours post-5-Fu.
  • Primary 5-Fu resistant marrow cells were assayed in the CFC assay, and cultured in quadruplicate in 1 ml of IMDM supplemented with 20% FCS, and multiple HGFs for 7 days at 37°C in a fully humidified 5% C0 2 atmosphere. Suspension cells in 4 cultures were counted manually. The number of CFC of the suspension cells in each culture was determined in the secondary CFC assay (28)
  • mice were flushed from six male B6SJL mice. The low density cells were separated by Nycodenz. The cells were incubated with antibody cocktail for lineage depletion including biotinylated monoclonal antibodies against CD3, CD8, B220, Gr-1, TER 119, and Ly-6C (PharMingen, San Diego, CA) . After washing once, the cells were incubated with second sets of monoclonal antibodies against Sca-1 (PE labeled) , Streptavidin (Cychrome labeled) , and WGA (FITC labeled) . The labeled cells were then sorted by flow cytometer selecting PEZ Cychrome " , and FITC, which are cells with phenotypes of Sca-1% Lin " , WGAZ
  • Suspension culture (Delta) assay We established quadruplicate 1 ml Delta-culture consisting of 5 x 10 5 day 1 5-Fu BM cells in 24 well plates with IMDM/20% FCS medium as described by Moore (24) .
  • the cultures were incubated in the presence of HGFs for 7 days at 37°C in a fully humidified atmosphere containing 5% C0 2 in air.
  • Suspension cells were counted and passaged weekly: 5 x 10" suspension cells from the previous one week culture were transferred into new wells and cultured under the identical conditions as previous week.
  • Suspension cells generated at each week were assayed for presence of CFC in the standard agarose CFC assay with a combination of IL-3, GM-CSF, and KL.
  • the CFC generated at each week were calculated using the formula: ( (CN x ACE) x CFO/CNAP.
  • ACE Cell expansion of the week x cell expansion of last week.
  • Cell expansion input cells/output cells.
  • the cytospin preparations of suspension were stained by the Wright's method with LeukoStat solution I and II according to the manufacturer's instruction (Fisher Scientific, Pittsburgh, PA. ) . Conventional light microscopy of the stained cells was carried out for differential morphological analysis.
  • FACS fluorescence-activated cell sorting
  • BM cells were fixed with 50% ethanol and stained with 200 mg/ml of propidium iodide. Samples were analyzed by a fluorescence-activated flow cytometer (Becton Dickinson,
  • apoptotic cells over the total cells analyzed are presented as percentage of apoptosis.
  • Dill is expressed in adult murine BM tissue. Dill expression in murine BM was analyzed by Western blotting and ⁇ -galactosidase staining of BM from adult Dlll lacZ transgenic mice.
  • the affinity purified chicken anti-Dill antibodies detected a protein of approximately 80 kD in cell lysates from mouse BM (Fig. IA) . The size of the detected protein corresponded well with the predicted size of the Dill, and the size of recombinant Dill expressed in COS7 cells (Fig. IA) , suggesting that the antibody specifically recognized Dill in BM cell extract.
  • ⁇ -galactosidase expression reflects expression of the endogenous Dill gene (32) .
  • ⁇ -galactosidase expression in BM was detected (Fig. IB) .
  • No singular hematopoietic suspension cells were stained positive for the ⁇ -galactosidase.
  • the observed positive cells were confined to the tightly associated cluster of cells indicating of the stromal and endothelial elements of the BM.
  • coli cell extracts were passed through a thio-bond affinity column, and the fusion protein of 39 kD was eluted with 2-mercaptoethanol as shown in Figure 2A.
  • the E. coli cell extract was purified into 50 fractions.
  • protein profiles of representative fraction 2, 10, 15, and 20 were visulized by SDS-PAGE and Coomassie Blue staining (Fig. 2B) .
  • the positive fractions containing the fusion protein were identified by Western blotting with monoclonal anti-thioredoxin antibody (Fig. 2C) .
  • the fusion protein appeared to be partially degraded and formed dimers and oligomers before the purification (Fig. 2B, lane 1) which were also observed after purification (Fig. 2B, Lane 2, 3) .
  • the active fractions from the purification were fractions 1 to 10 which corresponded to the estimated molecular weights of 400 kD to 120 kD.
  • the results suggested that the fusion proteins in solution formed trimers and multimers.
  • the presence of high concentration of reducing agent in the solution prevented multimerisation as seen in Figure 2A.
  • the DSL part of the fusion protein was confirmed at the amino acid sequence level using peptide microsequencing techniques (SKI Protein Core Facility) .
  • the fraction 2 was further purified by polymyxin affinity chromatography to deplete residual endotoxin from the solution.
  • the level of endotoxin in this final preparation was measured by an endotoxin kit to be less than 3 ng/ml, and this preparation was used in the cell cultures described below.
  • DSL promotes expansion of HPP-CFCs when combined with specific HGFs in the secondary CFC assay.
  • DSL alone would stimulate the proliferation of HPC in the CFC assay. No colonies were detected in the CFC assay of normal marrow, or marrow obtained 24 and 48 hours post-5-Fu BM in the presence of DSL alone at concentrations from 50 ng/ml up to 6250 ng/ml (data not shown) . The results suggest that DSL is not a mitogenic factor for the growth of HPCs.
  • HGFs including IL-3, G-CSF, GM-CSF, and M-CSF, were used with DSL at 250 ng/ml in the CFC assay with normal mouse BM. DSL did not change the number of colonies in combination with any HGFs nor the average size of the colonies significantly (data not shown) . Therefore, DSL does not act like other synergistic factors (e.g. KL, IL-1, IL-6) (28) in recruiting and enhancing proliferation of HPCs.
  • KL IL-1, IL-6
  • Notch mediated cell-cell communication inhibits cell differentiation and regulates the cellular competence response to more specific developmental signals (37, 38) .
  • Notchl mediates signals regulating hematopoietic cells in respond to the HGFs.
  • the DSL effect was then measured by the secondary CFC assays using a combination of HGFs including IL3, KL, and GM-CSF. As shown in Figure 3, the generation of HPP-CFC with different HGF and DSL combinations is shown for three independent experiments .
  • DSL functioned in a dose-dependent fashion, with an optimal dosage of 250 ng/ml in synergy with IL-3 to expand the HPP-CFC (Fig. 3A) , the dosage used for the experiments shown in Figure 3B and 3C.
  • DSL did not change the number of HPP-CFCs generated in cultures with M-CSF, IL-1 + IL-6 + KL, IL-1 + KL, IL-6 + IL-3, and IL-1 + IL-6 (Fig. 3A, 3C) .
  • DSL interacts with some but not all HGFs in promoting expansion of HPC in the liquid cultures.
  • DSL increases the production of CFCs in suspension culture but does not extend the duration of CFC production in response to HGFs.
  • GM-CSF Fig. 4C
  • the maximal effect of DSL was observed when it was used in combination with IL-3 for three weeks.
  • the Delta culture was initiated with only 34.7 CFCs (input CFC) but after culture for 3 weeks with IL-3 alone, the CFC number was expanded 530 fold reaching 18,400 CFCs.
  • the expansion was 6,519 fold with CFC number reaching 226,206 which was a 12.3-fold greater expansion over IL-3 alone.
  • Similar calculations applied to the conditions with G-CSF (B) and GM-CSF (C) where the expansion with DSL was 6.4-fold higher than G-CSF alone and 3.6-fold higher than GM-CSF alone.
  • DSL promotes expansion of primitive HPP-CFC responsive to IL-1, IL-3 and M-CSF at the expense of more mature subsets.
  • Growth of primitive hematopoietic cells requires combination of early acting HGFs.
  • IL-1 synergizes with IL-3 to increase the expansion of HPP-CFCs, (28) and combination of IL-1, IL-3, and M-CSF stimulates the formation of the most primitive HPP-CFCs in the culture of 48 hours post-5-Fu BM (29) .
  • BM cells were cultured in liquid with IL-3, IL-3 plus IL-1, and IL-3 plus IL-1 plus M-CSF, with or without DSL (Table 1) . After 7 days, the suspension cells from each culture were assayed for the formation of HPP-CFCs in response to IL-3 alone, IL-3 plus IL-1, and IL-3 plus IL-1 plus M-CSF. DSL together with IL-3, and IL-3 plus IL-1, increased the production of HPP-CFCs responsive to all three combinations of HGFs (Table 1) .
  • DSL blocks the terminal differentiation of promonocytes in response to IL-3 and IL-3 plus KL. Having shown that DSL altered the cellular commitment of primitive progenitor cells, we then tested the effect of DSL and HGFs on later stages of hematopoietic development in vitro.
  • BM cells from 24 hours post-5Fu treated mice with different HGFs alone or in combination with DSL. The expanded cells were passaged weekly for up to nine weeks. In cultures with IL-3, or IL-3 + DSL at 50 ng/ml, cellular expansion ceased by 28 days and the majority of cells generated were morphologically mature non-dividing macrophages (Fig. 5D) .
  • DSL enhances the function of HGFs to suppress apoptosis of hematopoietic cells.
  • Notchl receptor expression in hematopoietic tissue is not only in hematopoietic progenitors, but also in mature blood cells. Mature blood cells have a life-span of a few hours to several months, depending on lineages, and apoptotic death regulates both the life span of mature cells and the size of the progenitor pool.
  • BM cells derived from 24 hours post-5-Fu-treated mice for 7 days with different HGF combinations.
  • BM cells 24 hours after treatment with 5-Fu were almost all viable (Fig. 6A) .
  • Fig. 6A After culture for 7 days without HGFs, 90% of the 5-Fu treated cells died by apoptosis, and addition of DSL did not prevent apoptotic cell death (Fig. 6A) .
  • apoptosis of the BM cells was reduced in cultures with combination of DSL with IL-3, IL-3 plus KL, M-CSF, or GM-CSF compared to the cultures with IL-3, IL-3 plus KL, M-CSF, or GM-CSF (Fig. 6) .
  • DSL did not change apoptosis of BM cells in combination with other HGFs including IL-3 + IL-1, IL-3 + IL-6, G-CSF, IL1 + IL-6, and IL1 + KL + IL-6 (Fig. 6A) .
  • TAN-1 is expressed in a broad range of BM hematopoietic cells
  • mNotchl is expressed in a murine BM derived myeloid precursor cell line FDCP mix A4. It is thus reasonable to expect that mNotchl is expressed in mouse BM hematopoietic cells.
  • mNotchl ligand Dill is expressed in adult mouse BM, and more importantly, only in stromal and endothelial cells of the BM microenvironment (Fig. 1) .
  • lateral specification if the signal originates within a population of equivalent cells, or as inductive if the signal arises outside of the population of equivalent cells (39) .
  • Notch signaling is involved in both type of cellular interactions (5) .
  • Segregation of neural and epidermal' precursor cells in the ectoderm of the Drosophila embryo is an example of lateral specification.
  • Specification of germ cells in C. elegans is an example of inductive specification (40) .
  • detection of Dill in the stromal/endothelial cells and Notchl in hematopoietic cells supports the view that the Notch signal transduction pathway involves inductive specification.
  • Stromal cells have long been shown to support long term stem cell proliferation in mouse (41) and man (42, 43) . More recently BM sinusoidal endothelium has been shown to be equally effective in supporting in vitro stem cell proliferation and differentiation (44).
  • Local microenvironmental/steiri cell signals like DLLl/mNotchl could play an important role in regulating, or modulating, the competence of primitive hematopoietic precursors and their progeny to respond to HGF signals.
  • DSL had profound effects on myelopoiesis in BM suspension culture.
  • DSL inhibited the terminal differentiation of promonocytes (Fig. 5) .
  • DSL promoted expansion of very primitive hematopoietic precursors, at, or close to the stem cell stage of development (primitive HPP-CFC) , and decreased generation of more differentiated progenitors (Table 1) .
  • DSL activates its receptor mNotchl on HPP-CFC responsive to IL-1, IL-3, and M-CSF leading to an increase in the probability of their self-renewal rather than differentiation in response to the stimulus of HGFs.
  • Notch signaling in death, as well as birth, processes in hematopoiesis is suggested by our observations on apoptosis in hematopoietic cell culture.
  • HGF-stimulated BM suspension cultures with DSL exhibited fewer apoptotic bodies than did cultures with HGFs alone.
  • Further analysis by quantitative flow cytometry demonstrated that DSL in combination with IL-3, GM-CSF or M-CSF dramatically reduced the apoptosis of BM cells after seven days of suspension culture.
  • Human Notchl is expressed in peripheral blood monocytes and granulocytes (15) .
  • Notch signaling in modulating the competence of cells has been studied using a myogenesis model.
  • Activation of the mNotchl receptor leads to a part of its intracellular domain translocating to the nucleus.
  • the nuclear mNotchl then interacts with a transcription factor RBP-Jk (homologue of Drosophila Su (H) ) forming a heterodimer, which then binds to the promoter of HES-1 (Homologue of Drosophila E (Spl) ) .
  • HES-1 Homologue of Drosophila E
  • Upregulation of HES-1 a basic helix-loop-helix protein then blocks transcriptional activation by the MyoD protein (47). Therefore, activation of mNotchl leads to generation of an antagonist of myoblast differentiation.
  • HGFs and their receptors have been identified at the molecular level (48) . Recent results have provided evidence for the presence of multiple signaling pathways elicited by a single receptor (49) . Binding of HGFs to their receptors elicits both proliferation and differentiation signals.
  • the receptor functional domains have been mapped by mutational analysis of normal receptors and by studies of disrupted receptor structures in hematopoietic disorders (50) .
  • the membrane-proximal domains of the EPO (51), GM-CSF (52), G-CSF (53), and TPO (54) receptors are sufficient for mediating mitogenic signals, while the membrane-distal domains are required only for generating differentiation signals.
  • HGF-mediated receptor oligomerisation juxtaposes and activates JAK kinases associated with the membrane-proximal box 1 and box 2 motifs of the receptor. Activated JAKs then phosphorylate and induce nuclear translocation of the STAT DNA-binding proteins (55) . STATs appear only to be involved in functional differentiation (as opposed to mitogenic) responses to HGFs (49) .
  • Vassin H, Bremer KA, Knust E, Canpos-Ortega JA The neurogenic gene Del ta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats. EMBO J. 6: 3431, 1987 4. Fleming RJ, Scottgale TN, Diederich RJ, Artavanis-Tsakonas S: The gene Serrate encodes a putative EGF-like transmembrane protein essential for proper ectodermal development in Drosophila melanogaster. Genes Dev 4: 2188, 1990 5.
  • Visser JW, Vries Pd Analysis and sorting of hematopoietic stem cells from mouse bone marrow.

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Abstract

Cette invention se rapporte à un procédé servant à induire la prolifération de cellules hématopoïétiques, à un procédé servant à bloquer la différenciation par facteur de croissance hématopoïétique des cellules hématopoïétiques et à un procédé servant à induire la prolifération des cellules hématopoïétiques tout en bloquant la différenciation par facteur de croissance hématopoïétique de ces cellules hématopoïétiques, en utilisant un polypeptide soluble présentant la séquence du domaine Delta-Serrate-lag2 d'un homologue Delta. Cette invention se rapporte également à une composition contenant un polypeptide soluble purifié présentant la séquence du domaine Delta-Serrate-lag2 d'un homologue Delta et un excipient acceptable. Cette invention se rapporte également à une composition pharmaceutique servant à induire la prolifération de cellules hématopoïétiques et à bloquer la différenciation par le facteur de croissance hématopoïétique de ces cellules hématopoïétiques, cette composition renfermant un polypeptide soluble purifié présentant la séquence du domaine Delta-Serrage-lag2 d'un homologue Delta, ainsi qu'un excipient acceptable sur le plan pharmaceutique. Cette invention propose également un procédé de production d'un polypeptide soluble comportant la séquence du domaine Delta-Serrate-lag2 d'un homologue Delta.
PCT/US1998/020190 1997-09-26 1998-09-25 Polypeptide soluble comportant un domaine delta-serrate-lag2 de l'homologue delta et utilisations de ce polypeptide Ceased WO1999015560A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5270181A (en) * 1991-02-06 1993-12-14 Genetics Institute, Inc. Peptide and protein fusions to thioredoxin and thioredoxin-like molecules
US5631219A (en) * 1994-03-08 1997-05-20 Somatogen, Inc. Method of stimulating hematopoiesis with hemoglobin

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5270181A (en) * 1991-02-06 1993-12-14 Genetics Institute, Inc. Peptide and protein fusions to thioredoxin and thioredoxin-like molecules
US5631219A (en) * 1994-03-08 1997-05-20 Somatogen, Inc. Method of stimulating hematopoiesis with hemoglobin

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Title
BETTENHAUSEN B, ET AL.: "TRANSIENT AND RESTRICTED EXPRESSION DURING MOUSE EMBRYOGENESIS OF DII1, A MURINE GENE CLOSELY RELATED TO DROSOPHILA DELTA", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD., GB, vol. 121, 1 August 1995 (1995-08-01), GB, pages 2407 - 2418, XP002915686, ISSN: 0950-1991 *
FITZGERALD K, GREENWALD I: "INTERCHANGEABILITY OF CAENORHABDITIS ELEGANS DSL PROTEINS AND INTRINSIC SIGNALLING ACTIVITY OF THEIR EXTRACELLULAR DOMAINS IN VIVO", DEVELOPMENT, THE COMPANY OF BIOLOGISTS LTD., GB, vol. 121, 1 December 1995 (1995-12-01), GB, pages 4275 - 4282, XP002915689, ISSN: 0950-1991 *
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MOORE K. A., ET AL.: "HEMATOPOIETIC ACTIVITY OF A STROMAL CELL TRANSMEMBRANE PROTEIN CONTAINING EPIDERMAL GROWTH FACTOR-LIKE REPEAT MOTIFS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 94., 1 April 1997 (1997-04-01), US, pages 4011 - 4016., XP002915979, ISSN: 0027-8424, DOI: 10.1073/pnas.94.8.4011 *
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