WO1994013800A2 - Preparation a base de proteine mk et son utilisation dans les cultures cellulaires - Google Patents

Preparation a base de proteine mk et son utilisation dans les cultures cellulaires Download PDF

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Publication number
WO1994013800A2
WO1994013800A2 PCT/GB1993/002527 GB9302527W WO9413800A2 WO 1994013800 A2 WO1994013800 A2 WO 1994013800A2 GB 9302527 W GB9302527 W GB 9302527W WO 9413800 A2 WO9413800 A2 WO 9413800A2
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protein
cells
cell
heparin
treatment
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WO1994013800A3 (fr
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John Kaye Heath
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Cancer Research Campaign Technology Ltd
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Cancer Research Campaign Technology Ltd
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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
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a pluripotential embryonic stem cell-derived neuroregulatory factor, the MK protein, and more particularly to its use in the regulation of a variety of cell types.
  • MK protein was first described as a retinoic acid-induced protein in HM-1 embryonal carcinoma (EC) cells (Kadomatsu et al (1988) Biochem. Biophys. Res. Commun. 151, 1312- 1318).
  • the amino acid sequence of human MK is shown in Seq. ID No. 1.
  • the murine homologue of this protein has about 85% homology at the amino acid level and is shown as Seq. ID No. 2.
  • the function of MK was unknown although in situ hybridisation studies showed that the MK gene is transcribed in a number of embryonic cell types including the egg cylinder, nervous system, lung and kidney ([Kadomatsu et al (1990) J. Cell. Biol. 110, 607-616).
  • MK has about a 65% homology at the amino acid level to the 18 kD protein isolated by heparin affinity chromatography from adult brain as neurite outgrowth- promoting factor for foetal rat CNS neurons.
  • the protein has been termed B-GAM (Rauvala et al (1989)EMBO J. 8, 2933-2941), pleiotropin (Li et al (1990) Science 250, 1690-1694) or heparin-binding neurotrophic factor (Bohlen et al (1990) Growth Factors 41, 97-107).
  • the gene encoding MK protein is desirably carried by a vector adapted to be compatible with the host cell for replication of the vector and for expression of the MK gene.
  • the cells may be stably transformed or transfected with a construct designed to transiently express the MK protein.
  • Nerve cells may also be damaged through mechanical injuries.
  • one method of therapy proposed is the regeneration of neuronal cells to combat the degeneration of such cells associated with the disease or the treatment of neuronal cells in damaged tissues.
  • Such treatment may involve either the implantation of cells into the affected area of the body (ie. where cell degeneration is occurring) or the provision of a factor to such an area.
  • the implant may be allogenic, autogenic or homotypic.
  • An allogenic implant comprises cells or tissue which have a different genetic constitution than the host tissue, this is because they are obtained from a subject other than the recipient (eg. a foetus).
  • An autogenic implant comprises cells or tissue originating within the body of the recipient (with the same genetic constitution).
  • a homotypic implant comprises cells of the same genetic constitution and the same type and function.
  • the invention provides a method of treating diseases of the human or animal body which involve neurodegeneration or other damage to nerve tissue which method comprises treating neurectodermal cells or their immature precursors in vitro with an effective amount of MK protein to provide for the growth and/or differentiation of such cells, and implanting such treated cells into the human or animal subject.
  • the invention also provides cells grown in vitro in the presence of MK protein for use in a method of treatment or therapy of the human or animal body.
  • Such treatment includes the treatment of the conditions mentioned above.
  • neuronal cells within the human or animal body may be treated directly by the introduction of MK protejn into the human or animal body at a location where the MK protein can act upon the cells, in order to induce neuronal growth.
  • MK protein for use in a method of treatment or therapy of the human or animal body.
  • the present invention also provides pharmaceutical compositions comprising MK protein together with a pharmaceutically acceptable carrier or diluent.
  • the invention further provides such compositions for use in a method of treatment or therapy of the human or animal body.
  • the invention further comprises a method of treatment of the human or animal body which comprises administration to a subject in need of treatment an effective amount of MK protein or a pharmaceutical composition comprising MK protein.
  • the effective amount of protein and the route of administration will ultimately be at the discretion of the physician, taking account of the state of the patient and the nature of the disease being treated. However, an effective dose will typically be in the range of from 1 to 100 ⁇ g of protein per kg of body weight of the patient, for example about lO ⁇ g/kg.
  • the protein may be administered by any convenient route, for example parenterally by injection or orally. If by injection, it may be into the blood or directly at the site of the body where the action of the MK protein is required, eg in the brain, spinal column or site of tissue damage.
  • the treatment may be performed in accordance with the discussion above in connection with the transplant of cells grown in vitro for reimplantation into a patient. Similar doses of cells will be required, and the cells may also be administered by injection or other suitable route. Methods of in situ retroviral-mediated gene transfer are disclosed by Ram et al, (1993) Cancer Research 53; 83-88.
  • the invention also provides a vector adapted to express MK protein in target cells within the human or animal body, and such a vector for use in a method of treatment of the human or animal body.
  • Fig. 1 Northern blot analysis of MK expression in murine EC and ES cells.
  • Total cytoplasmic RNA was probed with 32 P-labelled MK cDNA (MK) and the blot reprobed with a murine glyceraldehyde phosphate dehydrogenase cDNA (mGAP) as a loading control.
  • MK MK cDNA
  • mGAP murine glyceraldehyde phosphate dehydrogenase cDNA
  • Fig. 7 E12 sympathetic neuron survival on ECM derived from MK-transfected 10T1/2 clones.
  • 10 3 E12 neurons were plated onto ECM preparations from 10T1/2 cell clones which expressed MK from a transfected plasmid (clones 1, 6, 10 and 11) or from a clone recovered in the same experiment in which the MK expression plasmid had not integrated (clone 15).
  • C non-transfected 10T1/2 cells. Cell numbers were determined after 3 hours and 24 hours.
  • Cell culture medium comprised DME.F12 (50:50 vol:vol; Gibco) supplemented with foetal calf serum (10% by volume, selected batches). All cells were cultured in a humidified incubator at 37 °C in an atmosphere of 5% CO 2 in air. ES cell culture medium was additionally supplemented (unless otherwise indicated) with 10 ng/ml recombinant leukemia inhibitory factor (Smith et al., 1988) and 10 ⁇ M betamercaptoethanol (Sigma, tissue culture grade). In some experiments the FCS used for cell culture was charcoal stripped to remove endogenous RA by the method described by VanderBurg et al., (1988).
  • E14TG2a ES cell differentiation was achieved either by exposure to 10" 7 M RA (all trans: Sigma, added from a 10 "2 M stock dissolved in tissue culture trade DMSO) or by culture of cells at low density in the absence of leukemia inhibitory factor (Smith et al., 1988).
  • 1009 EC cell differentiation was achieved by plating 1009 cells at 10" cells/ well into 5 mm 6-well cluster dishes in 4 ml of medium (DME;F12 10%FCS).
  • RA was added from a 10 "2 M stock (dissolved in DMSO) after 24 hours to a final concentration of 5xl0" 7 M.
  • 6x10° 10T1/2 fibroblasts were co-transfected with pXMT2-MK by the calcium phosphate method of Chen and Okyama (1987) using 10 ⁇ g of pXMT2-MK and 10 ⁇ g of PGK-Neo/3. 5 The day after transfection the cells were plated into 10x10 cm. tissue culture dishes and cultured in the presence of 300 ⁇ g/ml G418 (Sigma) for 14 days. A random sample of G418 resistant colonies were picked under a dissecting microscope and expanded in mass culture for further analysis. A matrix preparation was obtained from selected clones by release of cells with EDTA as described by Rathjen et al., (1990). 10
  • 15 rMK was purified from media conditioned by COS cells transfected with the MK expression plasmid pXMT2MK.
  • 5xl0 7 COS cells were transfected with 50 ⁇ g of the PXMT2MK by electroporation (Biorad gene pulser, 330 V, 500 ⁇ F) and plated into 175 cm 2 tissue-culture flasks in 75 ml of culture medium. The following day the medium was changed to 100 ml DME:F12 supplemented with 10 ⁇ g/ml transferrin (Sigma) and 10 ⁇ g/ml Heparin (BDH).
  • COS-cell conditioned medium 200 ml was pumped onto a 1 ml Hitrap heparin affinity column (Pharmacia) at a flow rate of 200 ⁇ l/minute at 4°C.
  • the column was washed with 25 10 ml of 50 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and the rMK eluted by washing the column with 5 ml of phosphate buffer (pH 7.4) containing 2 M NaCl.
  • the 5 ml heparin affinity eluate was then desalted (pharmacia PD-10 desalting column) and used for biological assays.
  • Tissue culture substrata were coated where indicated with a solution containing 0.1 mg/ml poly-L-lysine in distilled water for 5 minutes. Substrata were rinsed three times in water and allowed to dry. In some experiments the substrata were further coated with either EHS laminin or fibronectin (Sigma 10 ⁇ g/ml in PBS, 2 hours at room temperature) and rinsed with DMEM immediately before use, or heparin (10 ⁇ g/ml (BDH) in PBS, 2 hours at room temperature), or a mixture of both. In some experiments, MK was coated by incubation for 2 hours, at room temperature, over prepared substrata at designated concentrations to a maximum of 100 ng/ml. Growth factors were added to the culture medium at the nominated concentrations.
  • the sympathetic ganglia from embryonic day-12 (E12) chicks were dissected out and dissociated into single cells using standard techniques ( Wakade et al., 1982). Briefly, the ganglia were trypsinised for 30 minutes in Ca 2+ and Mg 2+ -free phosphate-buffered saline, triturated, and the resultant single-cell suspension pre-plated over tissue culture plastic in DMEM: 10% FCS for a period of 45 minutes to enrich for neurons. They were plated at a concentration of 2.0X10 3 cells per 16 mm well of a 24-well cluster under the appropriate experimental conditions. After incubation at 37°C for 2 days in the presence or absence of MK, the resulting cell survival, and the proportion of neuronal cell bodies with neurites longer than 2 cell diameters were monitored under phase contrast microscopy.
  • Cell pellets (10 7 -10 8 cells) were washed in phosphate-buffered saline (PBS) and lysed in 5 ml of 30 mM Tris pH 7.5, 150 mM NaCl, 15mM MgCl 2 , 0.4% NP40. Nuclei were removed by centrifugation for 15 minutes at 3000g. An equal volume of TUNES (10 mM Tris, pH 7.5, 7 M urea, 0.35 M NaCl, 1 mM EDTA, 2% SDS) was added to the supernatant. The cytoplasmic RNA was extracted twice with methanol/chloroform and once with chloroform before ethanol precipitation and recovery by centrifugation. The pellet was washed with 70% ethanol before being dissolved in DEPC-treated water.
  • PBS phosphate-buffered saline
  • cytoplasmic RNA in 40 ⁇ l DEPC-treated water was heated at 65°C for 3 minutes, quenched on ice and added to 5 ⁇ l 10X RTC buffer (BRL), 2.5 ⁇ l 10 mM dNTP mix, 1 ⁇ l (40 units) of RNasin (Promega), 0.5 ⁇ l (0.5 ⁇ g) oligo dT 12-18 (Pharmacia) and 1 ⁇ l (200 units) cloned MuLV-1 reverse transcriptase (BRL). The reaction was heated at 37°C for one hour and terminated by heating at 95 °C for 5 minutes.
  • Oligonucleotides were synthesised on an applied biosystems 380A DNA synthesiser.
  • 5'MK had the sequence 5'-GCAATTCATGAGCACCGAGGTTCTT-3'.
  • the ATG underlined represents the initiation codon of murine MK (Matsubara et al., 1990, Tomomura et al 1990b).
  • the preceding nucleotides constitute an EcoRI site used to facilitate cloning of the amplified material.
  • 3 'MK had the sequence 5'-AAGTCGACGGCCTCCTGACTTAGTCCTT- 3'.
  • the first 8 residues constitute a Sail site used to facilitate cloning the amplified material.
  • the rest of the oligonucleotide represents residues 415-434 of murine MK (Kadomatsu et al 1988). 8. Polvmerase chain reaction and cloning
  • 1 ⁇ l of cDNA was amplified in a 50 ⁇ l reaction volume containing 50 mM KC1, 10 mM Tris- HC1 pH 8.3, 1.5 mM MgCl 2 , 0.01 % (w/v) gelatin, 200 ⁇ M each dNTP, 0.5 ⁇ M 5'MK and 3'MK oligonucleotide primers, 2 units Taq polymerase (Cetus).
  • PCR was performed according to the following protocol: an initial denaturation for 5minutes at 95 °C was followed by 30 cycles of 1.5 mins denaturation, annealing at 64°C for 1.5 minutes and extension for 1.5 minutes at 72 °C.
  • reaction was increased to 100 ul by addition of reaction buffer to which was added 5 ⁇ l 10% SDS and 1 ⁇ l of proteinase K (1 mg/ml). The reaction was heated at 55 °C for 30minutes before extraction with phenol/chloroform and ethanol precipitation.
  • the amplified material was digested with EcoRI and Sail and subjected to electrophoresis in a 1.5% agarose gel. For cloning, the amplified band was excised from the gel and purified by means of a gene clean kit. The purified insert was cloned into EcoRI/Sall cut pBS KS+ (Stratagene).
  • MK was cloned into the expression plasmid pXMT2 in both orientations.
  • the pBSMK plasmid was cut with Sail, the ends were blunted by Klenow fill in and the insert released by digestion with Pstl.
  • the pXMT2 vector was prepared by cutting with EcoRI followed by blunting with Klenow and digestion with Pstl.
  • the insert was released from pBS/MK as an EcoRI/Sall fragment and cloned into EcoRI/XhoI cut pXMT2.
  • MK and B-GAM transcripts in ES cells were examined by polymerase chain reaction (PCR) amplification of cDNA derived from E14TG2a ES cell polyA-f mRNA using primers corresponding to the nucleotide sequence of the predicted amino terminus and carboxy terminus of both proteins.
  • PCR polymerase chain reaction
  • a strong MK amplification product was obtained after 30 cycles of amplification.
  • Significant amplification of an B-GAM product was also observed although at lower levels.
  • the PCR-amplified cDNAs were cloned into plasmid vectors for further analysis of MK gene expression.
  • MK transcripts were reported to be present at very low levels in HM-1 EC cells (Kadomatsu et al., 1988, Huang et al., 1990) and increased significantly upon exposure of the cells to retinoic acid.
  • Northern hybridisation of P19 and E14TG2a cells differentiated by exposure to RA revealed that MK mRNA expression was not significantly altered by either exposure to 10 "7 M retinoic acid or differentiation (by withdrawal of LIF) of ES cells in the absence of retinoic acid.
  • MK expression is induced in these cells by trace levels of retinoids present in FCS.
  • a second protein species detected by SDS PAGE of rpHPLC fractionated material had an apparent Mr of 65x10 s and was devoid of both biological activity and anti-MK immunoreactivity. This species was also detected in conditioned media derived from COS cells transfected with the PXMT2 expression plasmid cloned in the antisense orientation. In addition, no biological activity could be detected in high salt eluates of heparin affinity chromatography of media conditioned by cells expressing the antisense MK expression construct.
  • rpHPLC as a purification step resulted in a substantial loss of MK bioactivity resulting, at least in part, from exposure to acid solvents. Similar findings have been reported for HBNF (Bohlen et al., 1990). It was also noted in the course of these experiments that biological activity present in either COS-cell-conditioned media or heparin affinity-purified samples was very unstable upon storage between -20° and +4°C. In order to circumvent this problem, MK preparations for biological assays described below were used within 5 days of purification and not subjected to the rpHPLC purification step (which was thereafter employed for quantitation of rMK protein concentration present in the heparin affinity eluates).
  • FGF-4 does not account for the totality of heparin-binding mitogens present in EC cell conditioned medium (Heath et al., 1989)
  • rMK protein did not account for the totality of heparin-binding mitogens present in EC cell conditioned medium (Heath et al., 1989)
  • MK was completely inactive as a mitogen for Swiss 3T3 cells.
  • COS cell-derived rMK is not detectably contaminated with FGF-like biological activities (such as bFGF) which might have been expected to co-purify with MK.
  • rMK was, however, able to induce DNA synthesis in quiescent 10T1/2 cells (Fig 3).
  • RA-treated 1009 cells therefore represent a population of neuronal cells which, in view of their EC derivation, resemble the neurectodermal cell types of the early embryo.
  • rMK is a significant growth factor for neurectodermal cells derived by RA treatment of 1009 EC cells (Fig. 4) for 4 days.
  • the induction of DNA synthesis observed was comparable to that found for rFGF-4, with a half maximal response at approximately 1 nM.
  • No mitogenic effect of rMK was observed on undifferentiated 1009 EC cells.
  • MK can be physically associated with matrix components in a biologically active form when expressed continuously in stably transformed fibroblast cell lines.
  • MK is involved in the control of neurectodermal proliferation in the early development of the in vivo nervous system, and may function as a transforming gene in neuroectodermal tumours and cell lines.
  • MK is multifunctional regulator of neuronal cell function with distinct actions on different cell types within the developing nervous system.
  • MK and heparin presumably in the form of HSPGs
  • maximal activity could reflect either a stabilisation of the three dimensional structure of MK in a form available for receptor interaction, or a direct participation of HSPGs in the process of MK/receptor interaction.
  • a retinoic acid responsive gene MK found in the teratocarcinoma system is expressed in spatially and temporally controlled manner during mouse embryogenesis. J.Cell.Biol. 110, 607-16
  • Matsubara S Tomomura M, Kadomatsu K, Muramatsu T. (1990). Structure of a retinoic acid-responsive gene, MK, which is transiently activated during the differentiation of embryonal carcinoma cells and the mid-gestation period of mouse embryogenesis. J.Biol.Chem. 265, 9441-9443.

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Abstract

L'invention se rapporte à un procédé de développement de cellules neurectodermiques qui consiste à mettre en culture ces cellules in vitro en présence de la protéine MK. Une forme appropriée de la protéine MK est la protéine de la Seq. ID. No. 1. Les cellules développées en présence de la protéine MK, ou de la protéine elle-même, peuvent être utilisées pour traiter des états où les cellules nerveuses ont été endommagées.
PCT/GB1993/002527 1992-12-11 1993-12-10 Preparation a base de proteine mk et son utilisation dans les cultures cellulaires Ceased WO1994013800A2 (fr)

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Application Number Priority Date Filing Date Title
AU56566/94A AU5656694A (en) 1992-12-11 1993-12-10 Mk protein preparation and use in cell culture

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GB9225928.2 1992-12-11
GB929225928A GB9225928D0 (en) 1992-12-11 1992-12-11 Growth factor

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WO1994013800A2 true WO1994013800A2 (fr) 1994-06-23
WO1994013800A3 WO1994013800A3 (fr) 1994-09-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036635A3 (fr) * 1999-11-15 2002-03-21 Curagen Corp Nouveaux polypeptides de facteur de croissance et acides nucleiques codant pour ceux-ci
WO2004085642A1 (fr) * 2003-03-27 2004-10-07 Takashi Muramatsu Gene lie a l'arthrite et son utilisation dans l'examen de l'arthrite

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5210026A (en) * 1990-08-20 1993-05-11 American Cyanamid Company Human mk gene and method of expression

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036635A3 (fr) * 1999-11-15 2002-03-21 Curagen Corp Nouveaux polypeptides de facteur de croissance et acides nucleiques codant pour ceux-ci
WO2004085642A1 (fr) * 2003-03-27 2004-10-07 Takashi Muramatsu Gene lie a l'arthrite et son utilisation dans l'examen de l'arthrite
JPWO2004085642A1 (ja) * 2003-03-27 2006-06-29 村松　喬 関節炎関連遺伝子及びこれの関節炎検査等への利用
JP4646070B2 (ja) * 2003-03-27 2011-03-09 メディカル セラピーズ リミテッド 関節炎関連遺伝子及びこれの関節炎検査等への利用

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GB9225928D0 (en) 1993-02-03
WO1994013800A3 (fr) 1994-09-29

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