EP1501920A2

EP1501920A2 - Method for the production of cells with increased development potential

Info

Publication number: EP1501920A2
Application number: EP03724890A
Authority: EP
Inventors: Ulf R. Rapp; Bernd Neufeld
Original assignee: Individual
Current assignee: RAPP, R. ULF, PROF. DR.
Priority date: 2002-05-07
Filing date: 2003-05-07
Publication date: 2005-02-02
Also published as: US20060094115A1; WO2003095633A2; WO2003095633A3; AU2003227513A1; DE10220480C1

Abstract

The invention relates to a method for the production of cells with increased development potential from cells which are taken from an organism. The cells thus taken are cultivated in vitro and a native nuclear non-activated MAPKAP kinase of the cultivated cells is activated or an activated MAPKAP kinase is introduced into the nucleus of the cells.

Description

Process for the production of cells with increased development potential.

Field of the Invention.

The invention relates to a method for producing cells with increased development potential, cells obtainable with such a method and use of such cells.

Background of the Invention and Prior Art.

In the embryonic and fetal phase, an organism is formed by differentiating effector cells from stem cells. These embryonic or fetal stem cells have an increased development potential compared to somatic stem cells from adult tissues. One speaks of pluripotency of the embryonic or fetal stem cells, since these can differentiate to all tissues, organs or cell types.

However, the production of embryonic stem cells for purposes other than in-vitro fertilization is opposed to serious ethical concerns. Therefore, a search for alternative sources for pluripotent or totipotent cells focuses on the generation of an increased development potential of somatic cells, in particular somatic stem cells from adult organisms, compared to the natural state. These research directions are encouraged by the realization that transdifferentiation of somatic stem cells is possible, for example neural ones Stem cells form blood cells, while blood stem cells can form brain and muscle cells. For this purpose, reference is made, for example, to the literature references US-6, 087, 168, US-6,093,531 and US-6,093,531. Increasing the plasticity of the somatic stem cells is therefore desirable in order to enable a wide variety of organ repairs to be carried out by means of somatic stem cells removed from an organism, treatment thereof and reimplantation. Plasticity plays a particularly important role here because, with increased plasticity, those with good availability (quantity and / or easy removal), for example hematopoietic stem cells, can be used as starting cells.

3pK belongs to a kinase family (MAPKAP kinases) that are activated by one or more members of the MAPK family (mitogen-activated protein kinase). 3pK is also known as MAPKAP kinase 3 (MAPK activated protein kinase 3). The mechanisms for activating 3pK via the mitogenic kinase cascade are relatively well elucidated, for which reference is made, for example, to the literature references G. Sithanandam et al. ; Mol. Cell Biol. 16 (3), 868-876, and S. Ludwig et al. , Mol. Cell Biol. 16 (12), 6687-6697. 3pK can also be via stress kinase cascades via p38 (S. Ludwig et al., Mol Cell Biol 16: 6687-6697 (1996); MM McLaughlin et al., J Biol Che 271: 8448-8492 (1996)) be activated, ie by induction of Rac (CDC42), for example by heat shock or proapoptotic substances such as TNF-alpha. For this, reference is made, for example, to the references JM Kyriakis et al., Nature 369: 199-211 (1994), J. Raingeaud et al. , J Biol Chem 279: 7420-7426 (1995), A. Minden et al. , Cell 81: 1147-1157 (1995), S. Zhang et al. , J Biol Chem 279: 23934-23936, J. Han et al. , Science 265: 808-811 (1994), S. Kumar et al. , Biochem Biophys Res Commun 235: 533-538 (1997), G. ang et al. , Cytogenet Cell Genet 78: 50-55 (1997) and CJ Lee et al. , Nature 372: 739-746 (1994).

Another MAPKAP kinase is MK2, which is primarily activated via p38 as a cellular response to stress, for example by heat shock or cytokines such as TNF-alpha (J. Rouse et al., Cell 78: 1027-1037 (1994); K. Engel et al., J Cell Biochem 57: 321-330 (1995)).

3pK and MK2 have a sequence homology of 75% at the amino acid level. Both are arranged in the non-activated state in the cell nucleus and leave it after activation or phosphorylation. Other homologues are MAPKAP-Kl, RSK2, RSK3, MK4 and PRAK.

Very little is known about the physiological substrates with regard to the physiological functions of MAPKAP kinases such as 3pK or MK2.

The differential expression of homeotic genes determines the formation of different parts of the body along the longitudinal axis of an organism. In the early embryonic phase, homeotic genes are activated in the right areas by transcription factors. However, these factors are soon no longer active and the correct expression of homeotic genes in the course of the further development of the organism is inter alia from proteins of the so-called Polycomb-group (Pc-G) controlled. Pc-G proteins keep genes that are no longer expressed after a certain developmental state suppressed. This property is also passed on to daughter cells. They form part of a cellular memory system, so they are part of an epigenetic expression control. The Pc-G proteins are believed to produce a chromatin configuration that is inaccessible to transcription factors and thus inhibit the transcription of homeopathic genes. For the function of the Pc-G proteins, reference is made in detail to the following review article: JJL Jacobs et al. , Cell & Developmental Biology, Vol. 10, 1999, pp. 227-235. Members of the Pc-G family include BMI1 and HPH1 and HPH2 (human polyho eotic).

In general it can be said that some knowledge about the mechanisms of action of the Pc-G proteins has already been gained. However, it is hardly known how the Pc-G proteins are regulated.

Technical problem of the invention.

The invention is based on the technical problem of specifying a method for increasing the plasticity of cells.

Basics of the invention.

To solve this technical problem, the invention teaches a method for producing cells increased development potential from cells, wherein the removed cells are cultivated in vitro and a native nuclear non-activated MAPKAP kinase of the cultivated cells is activated or an activated MAPKAP kinase is transported into the cell nucleus. The term nuclear denotes a localization of the MAPKAP kinase in the cell nucleus, in particular in the chromate jelly. A MAPKAP kinase can be activated if it can be phosphorylated with the result that the phosphorylated MAPKAP kinase can phosphorylate its substrates and is exported from the cell nucleus. However, within the scope of the invention, the said MAPKAP kinase can also be activated directly in vivo, the cells according to the invention then being formed directly in vivo.

The invention is based on the knowledge that nuclear MAPKAP kinases bind both with HPH2 and with BMI1. The invention is based on the further finding that the activated, phosphorylated MAPKAP kinase in turn phosphorylates BMI1 before it is exported from the cell nucleus, which is also mobilized thereby, which ultimately makes a locus represented by the Pc-G complex accessible and transcribed. This reactivates genes that are normally inactive at a certain stage of development, which leads the cells back to a status similar to that of an early embryonic stem cell, ie the plasticity of the cells used is increased. The invention can in principle be used with all cell types, but is particularly suitable when using somatic stem cells. In detail, the following findings are based. 3pK is a BMI1 kinase in vitro and develops a suppressive effect similar to other Pc-G proteins in an artificial repression assay. 3pK binds to putative full-length HPH2 and has the highest affinity for a 73 amino acid C-terminal fragment of HPH2, which encompasses the HDII / SEP domain. This region is part of the HPH2 dimerization domain, ie it comprises the alpha-helical HDII / SEP domain required for hetero- / homodimerization and for Bmil binding and overlaps with the domain for 3pK binding. The stronger binding to the said fragment compared to the full length can be due to a folding of full-length HPH2 over the HDII / SEP domain. The status of the phosphorylation of HPH2 is unclear and a phosphorylation of the putative full-length HPH2 by 3pK could not be observed in vitro. However, phosphorylation is not excluded either, since a mouse homolog of HPH2 (mPh.2, NCBI Accession U81491) with a long N-terminal attachment contains three potential 3pK phosphorylation sites. Since HPH1 and mouse mPhl proteins have the same length, it can be assumed that HPH2 also has the same length as mPh2.

The above findings are also supported by the fact that BMI1 coprecipitates with 3pK. 3pK is a kinase that phosphorylates BMI1. This plays an important role in that on the one hand the dissociation of Pc-G complexes and the BMI1 phosphorylation are interrelated and on the other hand hypophosphorylated BMI1 specifically in the Chromatin-associated protein fraction is kept out of the cell nucleus, while phosphorylated BMIl is not chromatin-bound. In summary, it was found that 3pK forms part of the Pc-G complex and is a BMIl kinase. 3pK should therefore regulate the phosphylation-dependent Pc-G complex / chromatin interaction, namely by binding to HPH2, which provides 3pK in the Pc-G complex BMIl, with the result of its phosphorylation and release of the complex from the chromatin. Similar relationships were found in the case of MK2.

Interestingly, the phosphorylation status of 3pK does not appear to be relevant for binding to HPH2. Comparative experiments with known phosphorylation sites of the 3pK mutated 3pK variants (T313E, T313A, TT201 / 313EE) showed no changes in the binding to HPH2. 3pK also coprecipitated after treatment of the cells with arsentite (a powerful agent to induce 3pK phosphorylation and activation) with PcG complexes. Incidentally, coprecipitation also takes place with endogenous 3pK and PcG complexes.

The invention further relates to cells obtainable by a method according to the invention and the use of cells according to the invention for the production of a pharmaceutical composition, in particular for the treatment of degenerative nerve diseases. In such diseases, the increase in plasticity ensures colonization of the target tissue and differentiation of the introduced cells according to the invention into the desired cell type. A particular aspect of the findings according to the invention of independent importance also lies in the use of a substance which promotes the expression or activation of a MAPKAP kinase, in particular 3pK, for the production of a pharmaceutical composition for the prophylaxis or treatment of cancer. Alternatively, the tumor cells can be brought to expression of activated 3pK by genetic engineering measures. This aspect makes use of the fact that BMIl has oncogenic functions and its modulation can thus control the proliferation of cells. It is known, for example, that reduced proliferation can be based on overexpression of the tumor suppressors plβ and pl9ARF, both of which are encoded by the INK4a tumor suppressor locus (see JJ Jacobs et al., Nature 397: 164-168 (1999)). A release of this locus in tumor cells would consequently induce the tumor suppressors in question, with the result of the inhibition or reduction of the proliferation, which was excessive due to the disease. Otherwise, the above and following explanations apply analogously. Substances that induce or activate 3pK in tumor cells are therefore suitable for the production of pharmaceutical compositions for cancer treatment. It is recommended to either apply the substances locally or to couple them to tumor-cell-specific substances, for example interaction partners of tumor markers or ligands for tumor-specific receptors.

Preferred embodiments of the invention. The MAPKAP kinase can be 3pK, MNK1, MNK2, MSK1, MK2, MK4 or PRAK, preferably 3pK. In the case of activation of native MAPKAP kinase, it is preferably a wild-type MAPKAP kinase.

Activated MAPKAP kinase can be induced in the cell nucleus in various ways. So it is possible that the cells are transformed to overexpress an activated MAPKAP kinase. Activation by induction of p38 is also possible, the induction of p38 again being possible by induction of Rac and / or Ras.

The activation of 3pK can be carried out in detail by induction of the mitogenic kinase cascade, in particular induction of Raf, MEK and / or ERK, for example by incubation with a serum growth factor or with TPA. However, the activation can also be carried out by inducing a stress kinase cascade, for example i) by treating the cells with conditions inducing stress kinase, in particular a heat shock, osmolarity shock or UV, or ii) by incubation in a physiologically effective dose with at least one Stress kinase cascade inducing substance, in particular cytokines such as IL-1, TNF-alpha, anisomycin, arsenite and / or alkylating agents.

Of particular importance is the observation that the presence of B-raf in cells, naturally present or artificially induced, is important for increasing the development potential. It was observed that, in the absence of B-raf, formation of or there was no increase in the plasticity of neural stem cells in the mouse. A preferred aspect of the invention is therefore the generation or induction and / or maintenance of B-raf in target cells whose development potential is to be increased.

It is preferred if the cells used are atic stem cells, for example hematopoietic or neural stem cells. For reasons of better immune tolerance, the cells used should be autologous, which avoids undesirable immune reactions when administered or reimplated.

The invention is explained in more detail below with the aid of examples and experiments.

methods:

Plasmid construction:

Human PKRSPA-3pK, which carries the gene under the control of the Rous Sarcoma Virus (RSV), pEBG-3pK, which expresses the gene as a GST fusion protein under the control of the human EFla promoter, and the mutation of the lysine in the area of the putative ATP binding region of the 3pK (3pK K73M) are known from the literature reference S. Ludwig et al (see above). Another mutation at 73 or derivatization can also be used, which renders 3pK inactive. pcDNA3HA-3pk was generated by inserting an Nhel-Spel fragment of pPC97-3pK into pcDNA3HA. pPCH-3pK K73M was obtained by inserting the Sall-Notl fragment of pPC97-3pK into pPCH (available from C. Hagemann, MSZ, Würzburg), which is a pPC97 derivative in which the LEU nutrient cassette is replaced by the TRP marker of pPC86 (PM Chevray et al., Proc Natl Acad Sei USA 89 (13), 1992, 5789-5793). The BamHI-Ba HI insert of pGEXKG-3pK was ligated into the yeast two hybrid vector PÄS2.1 (Clontech) to obtain pAS2.1-3pK K73M. pMT2SM-HA-bmi (mouse), ppuro GAL4 DB-Bmil and ppuro GAL4 DB were obtained from Mv Lohuizen (Amsterdam). PPuro GAL4 DB 3pK was cloned by cutting pGEXKG-3pK with EcoRI and subsequently inserting this fragment in ppuro GAL4 DB. pBEVY-GU-Xb i was by ligation of the EcoRI fragment from pPC97-Xbmi (available from A. Otte, Amsterdam) into pBEVY-GU (available from Ch.A. Miller, New Orleans). pGAD10-HPH2 (137-432 aa), hereinafter referred to as pGAD10-HPH2 (C-295 aa), and pGAD10-HPH2 (432 aa) are available from A. Otte and described in MJ Gunster et al., Mol Cell Biol. 17 (4), 2326-2335.

Yeast two hybrid system:

The yeast two hybrid screens were carried out using a human heart MATCHMAKER cDNA library, which is cloned into the yeast two hybrid vector pGADIO (Clontech). In the first screen, the kinase-active 3pK K73M was used in the single copy plasmid pPCH. The yeast strain CG-1945 was manipulated according to the MATCHMAKER Library User Manual (PT1020-1, Clontech) using the sequential transformation protocol. Positive clones were identified by growth on SD / -TRP / -LEU / -HIS plates and activity determination of the lacZ reporter gene in filter assays. DNA sequence analysis showed that the two independently isolated cDNA library plasmids contained the same 879 bp of HPH2 encoding a 73 aa (220 bp) and part of the 3UTR (659 bp). An independent screen of the same two hybrid libraries using pAS2.1-3pK K73M as bait and yeast strain Y190, which has a higher lacZ reporter gene expression, resulted in 128 on SD / -TRP / -LEU / -HIS / + 25mM 3 -AT (3-amino-1,2,4,4-triazole) grown ß-gal positive clones, of which 17 encoded C-terminal fragments of HPH2: five clones contained the fragment of the first screen, four cDNAs encoded 145, three 198 and five 209 C-terminal amino acids. Direct two hybrid tests with these interaction partners and pAS2.1-3pK K73M were also carried out in accordance with the standard protocols. The liquid culture ß-galactosidase assay with ONPG (o-nitrophenyl-ß-D-galactopyranoside) as a substrate was carried out using the strain Y190 according to the MATCHMAKER instructions for use. To reduce the spread, five different transformers of each cotransfection were examined. pAS2.1-3pK K73M was co-transformed as bait with the following six different (length) constructs: GAL4 AD-HPH2 (C-73 aa), GAL4 AD-HPH2 (C-145 aa), GAL4 AD-HPH2 (C -198 aa), GAL4 AD-HPH2 (C-209 aa), GAL4 AD-HPH2 (C-295 aa) and GAL4 AD-HPH2 (C-432 aa). In a further series of experiments, pAS2.1-3pK K73M and pGAD10-HPH2 (C-73 aa) were co-expressed with a third yeast expression vector, either pBEVY-GU without insert or PbEVY-GU-Xbmil, and on SD / - TRP / -LEU / -URA plates are cultivated and then subjected to the liquid culture β-galactosidase assay.

Cell cultures and antibodies: The human embryonic kidney cell line HEK293, which was used for immune complex kinase assays and coimmune precipitation experiments, was added to Dulbecco's modified Eagle's Medium (DMEM), supplemented with 10% (v / v) FCS (at 56 ° C for 30 min. Heat inactivated) 37 ° C cultivated in humidified air with 6% C02. The human osteosarcoma cell line U20S GAL4-TKluc, which is stably transfected with a TK-luciferase reporter plasmid with five GAL4 binding sites above the TK promoter and was used for the repression assay, was cultivated in DMEM with 10% FCS (v / v). Polyclonal rabbit antiserum against bacterially expressed 3pK was obtained according to G. Sithananda (see above). Anti-glutathione S-transferase (GST) antisera were obtained from rabbits which were immunized with bacterially expressed and purified GST. 1: 750 dilutions of both sera were used for immunoblots. The monoclonal anti-HA tags (12CA5) were used in a concentration of 1 μg / ml for Western blots.

Transfection and 3pK activation:

For the transfection of both cell lines HEK293 and U-20S, 5 x 10 ^A 5 cells were sown in a 10 cm dish and cultured for 24 hours in DMEM with 10% FCS before the transfection. The transfections were carried out using the calcium phosphate coprecipitation method using 5-10 μg DNA for HEK293 or 15 μg DNA for U-20S according to a modified Stratagene protocol (according to S. Ludwig et al., See above). HEK293 cells were starved 48 hours before transfection in DMEM with 0.3% FCS. To activate 3pK, HEK293 cells with 0.5 M sodium metaarsenite were used for 30 min. before the Harvest or with 20% FCS in combination with 100 ng tetradecanoylphorbol acetate (TPA) per ml 60 min. stimulated before harvest.

Immun precipitation and immunoblotting:

Transfected HEK293 cells were in Tritonlysis buffer (TLB, 20 mM Tris (pH 7.4), 50 mM sodium β-glycerophosphate, 20 mM sodium pyrophosphate, 137 mM NaCl, 10% (v / v) glycerol, 1% ( v / v) Triton X-100, 2 mM EDTA, 1 mM Pefabloc, 1 mM sodium orthovanadate, 5 M benzamidine, 5 mg per ml apritinin, 5 mg per ml leupeptin) on ice for 10 min. lysed. Cell solids were removed by centrifugation at 15,000 rpm for 10 min. away. Supernatants were incubated with various antisera for 2 h at 4 ° C. Untagged 3pK was immunoprecipitated with anti-3pK antiserum. Tagged variants of the proteins were immune-purified with corresponding anti-tag antibodies. The immune complexes were precipitated with 25 μl Protein A agarose and washed extensively with either TLB or RIPA (IM NaCl) for immune precipitation or immune complex kinase assays. To detect the proteins in Western blots, the immune complexes were suspended in electrophoresis sample buffer and for 3 min. heated at 100 ° C. After SDS PAGE, gels were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) and subjected to immunodetection using the corresponding primary antibodies. Proteins were visualized using horseradish peroxidase-conjugated Protein A agarose (Amersham) and a standardized enhanced chemiluminescence reaction (Amerscham).

In-complex complex kinase assay with 3pK: Separately immunoprecipitated GST-3pK and HA-BMIl were combined and twice with a high NaCl concentration (25 mM Tris (pH 8), 1 M NaCl, 10% (v / v) glycerol, 0.1% SDS, 0.5% Sodium deoxycholate, 1% NP40, 2 mM EDTA (pH 8), 1 mM Pefabloc, 1 mM sodium orthova nadate, 5 mM benzamidine, 5 mg per ml aproptin, 5 mg per ml leupeptin) and specific kinase buffer (10 mM MgCl2, 25 mM β-glycerophosphate, 25 HEPES (pH 7.5), 5 mM benzamidine, 0.5 mM dithiothreitol, 1 mM sodium vanadate). The kinase assays were carried out in the same buffers, supplemented with 5 mCi [gamma32-P] -ATP, 0.1 mM ATP, at 30 ° C. and the reaction after 30 min. ended by adding Laemmli buffer and 3 min. at 100 ° C. After gel electrophoresis and blotting on nitrocellulose membrane, the BMIl phosphorylation was analyzed using an X-ray film (Amersham).

Repression Assay: These experiments were carried out essentially according to MJ Alke a et al., J Mol Biol 173, 1997, 993-1003. U-2 OS were cultured in 10 cm dishes up to 40-60% confluence and transiently transfected using calcium phosphate. Each 10 cm dish was transfected with either 15 μg ppuro GAL4 DB, ppuro GAL4 DB-BMI1, ppuro GAL4 DB-3pK or PKRSPA and 2 μg of a ß-GAL reporter gene. After 20-24 h, the transfection medium was replaced with DMEM with 10% FCS after several washes with PBS to remove specimens. After 8-10 hours, this medium was replaced by DMEM with 10% FCS and 10 μg / l puromycin, in which the cells were cultured for a further 38-62 h for removal of all non-transfected cells. Cells transfected with the non-puromycin-resistant vector pKRSPA served as a control for the complete elimination. The remaining cells were then cultured for 6 hours in DMEM with 10% FCS, after which the cells were examined for luciferase and β-gal activity. U20S were harvested in 100 ml lysis buffer (50 mM Na-2 (N-morpholino) ethanesulfonic acid, pH 7.8, 50 mM Tris HC1, pH 7.8, 10 mM dithiothreitol, 2% Triton X-100). The untreated cell lysates were clarified by centrifugation and 50 ml of the pre-clarified cell extract was added to 50 ml luciferase assay buffer (125 mM Na-2 (N-morpholino) ethanesulfonic acid, pH 7.8, 125 mM Tris HCl, pH 7.8, 25 mM magnesium acetate, 2 mg / ml ATP) added. Immediately after the injection of 50 ml of 1 mM D-luciferins (Applichen) for each sample, the luminescence was measured for 5 s in a luminometer (MicroLumat LB96P, EG&G Berthold). The β-galactosidase assay was performed with 20 μl cell lysate according to a standard protocol (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. 1989, Cold Spring Harbor Laboratory Press, New York). The ratio of the background-corrected luciferase values and ß-gal values of dishes transfected independently of one another were used to correct the transfection effectiveness and related to empty vector transfected controls. All transfections were carried out at least twice and standard deviations were calculated.

Results: FIG. 1 shows as a result that 3pK binds in vivo in the yeast two-hybrid system to the C-terminal part, including the homology domain II (HDII), of HPH2. Figure 1A is a schematic representation of the 3pK interacting HPH2 clones of different lengths. Interestingly, both the domain required for homo- or heterodimerization with HPH2 or HPH1 (104 C-terminal amino acids) and the binding domain for BMIl (295 C-terminal amino acids) overlap with the interaction domain for 3pK binding. The minimal 3pK binding domain of HPH2 (73 C-terminal amino acids) contains a 67 amino acid long homology domain II (called HDII, SAM or SEP), which is identical or similar to corresponding domains of other polyhomeotic proteins (eg Mph2, Mphl / Rae-28 or HPH1). The strongest interaction 3pK / HPH2 was determined with the smallest HPH2 fragment, as can be seen from the X-Gal test (FIG. 1B) and the quantitative two-hybrid test with ONPG as the substrate (FIG. IC).

In the experiment in FIG. 1B, Y190 yeast was cotransformed with pAS2.1 / 3pK K73M and the specified pGAD10 / HPH2 constructs and cultivated on SD / -TRP / -Leu plates. The colonies obtained were streaked on filters on SD / -TRP / -LEU / -HIS and cultured for 12-24 at 30 ° C before the ß-Gal assay was performed. A liquid culture ß-galactosidase assay with ONPG as substrate was carried out to quantify the binding affinities. The activity for 3pK-HPH2 (C-73 aa) was set to 100% for comparison purposes. Figure 2 shows that 3pK interacts with HPH2 in vivo, even in the case of mammalian cells. 3pK should be part of the Polycomb complex, which contains HPH2. Coimmune precipitation experiments were carried out to analyze whether 3pK is present in a Pc-G together with HPH2. In a first series of experiments, HEK293 cells were co-transfected with GST tagged HPH2 (C-73 aa) and 3pK wt. As a positive control, polyclonal anti-GST serum was used for the immunoprecipitation and immunoblotting of GST-HPH2 (C-73 aa) (FIG. 2A, lane 5). 3pK was precipitated with a 3pK-specific antiserum and coimmunoprecipitated GST-HPH2 (C-73 aa) was detected in Western blots with the anti-GST antiserum (FIG. 2A). Lane 1 shows that GST-HPH2 (C-73 aa) precipitated with 3pK, while no coprecipitation was found in the control samples with GST alone, or in the absence of 3pK or GST-HPH2 (C-73 aa) ( Lanes 2 to 4).

In the experiments in FIG. 2B, GST-HPH2 fragments of various lengths (as indicated) were cotransfected with a HA-tagged variant of 3pK. Immunoprecipitation and immunoblotting with anti-GST antiserum confirms the expression of the various GST-HPH fusion proteins (lanes 1 to 4 and 9 to 12). Co-immunoprecipitation with HA-3pK using HA mab was observed in the case of GST-HPH2 (C-73 aa), lane 6, and GST-HPH2 (C-198 aa), lane 8. (C-145 aa) and (C-295) were not observed due to presumably insufficient binding affinity. This also confirms the bond strengths according to the figure IC. The bands at 55 kDa are the result of a cross reaction with heavy chains of the antibodies in the samples and have nothing to do with the above relationships. The procedure was as follows. HEK 293 cells were transfected with the plasmids indicated. 3pK was expressed either untagged by an RSV promoter (FIG. 2A) or as an HA-tagged version by a CMV promoter (FIG. 2B). The proteins were immunoprecipitated, as indicated, with either anti-3pK (A), anti-GST (A, B) or anti-HA (12CA5) (B) antibodies, washed twice in TLB buffer, separated on SDS-PAGE gels ( 10%) and blotted in nitrocellulose filters. Immunoblots were sampled with the appropriate antiserum and detected with ECL.

Experiments not shown in the figures examined whether 3pK is associated with chromatin. Differential nuclear extracts from cells that overexpress human 3pK constructs showed that only a part of the wild-type 3pK is associated with chromatin. In contrast, constitutively active or kinase-inactive 3pK mutants did not, or significantly less, associated with chromatin, in accordance with other studies, according to which such mutants are mainly present in the cytoplasm.

FIG. 3 shows results that demonstrate an in vivo interaction between 3pK and BMIl. The detailed procedure was as follows. The cells were transfected with HA-BMI1 or pEBG-3pK DNA. Immunoprecipitation was performed with either anti-GST or anti-HA (12CA5) antibodies, followed by two washes with TLB buffer. The proteins were then on separated on SDS-PAGE gels (10%) and blotted in nitrocellulose filters. Immunoblots were sampled with the appropriate antiserum and detected with ECL. The bands at 55 kDa have the above cause.

It can be seen that OHMI1 is specifically coprecipitated with GST-3pK (lane 1) and vice versa (lane 3). Lanes 2 and 4 show no interactions with HA-BMIl or GST alone.

Figure 4 shows that 3pK BMIl phosphorylates. HEK 293 cells were transfected with HA-BMIl or pEBG-3pK DNA. The kinase was stimulated with arsenite or serum / TPA for 60 min. activated. The substrate and kinase were immunoprecipitated with anti-HA (12CA5) or anti-GST antibodies. Immune complexes were combined, washed twice under stringent conditions with RIPA buffer containing 1 mM NaCl and subjected to an in vitro kinase reaction in the presence of [gamma32P] ATP. Lanes 2 and 3 of the upper panel show that a strong phosphorylation occurs when 3pK is activated. In contrast, no or hardly any phosphorylation takes place in the absence of 3pK or in the absence of activation (lanes 1 and 4). The lower panel shows a positive control reaction using Hsp27 as a substrate for immunoprecipitated 3pK. This was found in HeLa and 293T cells, but also in primary human TIG3 fibroblasts, which shows that the invention is not restricted to certain cell types or is bound to specific cellular phenotypes. FIG. 5 shows that BMI1 weakens the interaction 3pK / HPH2. An assay similar to that of Figure IC was performed for the experiments. In addition to the transformation of pAS2.1 / 3pK K73M and pGAD10 / HPH2 (C-73 aa), either pBEVY-GU or pBEVY-GU / XBMIl were co-transformed. XBMI1 (Xenopus BMIl) is 90% identical and 95% similar to human and mouse BMIlDa since an ONPG ß-galactosidase assay is 6 orders of magnitude less sensitive than with X-Gal, only the (C-73 aa) fragment was used. Each bar represents 5 parallel experiments and the data shown are representative of 5 independent assays. It can be seen that XBMI1 reduces the bond strength from 100% to 44%.

The results of a repression assay are shown in FIG. FIG. 6A shows a schematic representation of the stably integrated luciferase reporter construct. FIG. 6B shows the fusion proteins used with the GAL4 DNA-binding domain (Gal4 DB). For the results in FIG. 6C, the procedure was as described under "Methods".

Expression of Gal4 DB-BMI1 was used as a positive control and exactly four-fold inhibition was used, as described by Alkema et al. reported (see above) found. The same result was also found for Gal4 DB-3pK, which shows that non-activated and Pc-G associated 3pK is able to recruit Pc-G proteins such as BMIl or HPH2 to the chromatin-rearranged promoter, where an inhibitory complex is formed ,

Claims

Claims:

1. Method for the in vitro production of cells with increased development potential from cells which have been removed from an organism, the removed cells being cultivated in vitro and wherein a native nuclear non-activated MAPKAP kinase of the cultivated cells is activated or an activated MAPKAP kinase is the nucleus of the cell Cells is supplied.

2. The method of claim 1, wherein the MAPKAP kinase is 3pK, MNK1, MNK2, MSK1, MK2, MK4 or PRÄK, and is preferably a wild-type MAPKAP kinase.

3. The method according to claim 1 or 2, wherein the cells are transformed to overexpress an activated MAPKAP kinase.

4. The method according to any one of claims 1 to 3, wherein the activation takes place by induction of p38.

5. The method according to claim 4, wherein the induction of p38 takes place by induction of Rac and / or Ras.

6. The method according to any one of claims 1 to 4, wherein the activation of 3pK by induction of the mitogenic kinase cascade, in particular induction of Raf, MEK and / or ERK.

7. The method of claim 6, wherein the cells are incubated with a serum growth factor or with TPA.

10. The method according to any one of claims 1 to 5, wherein the activation takes place by induction of a stress kinase cascade.

9. The method of claim 8, wherein the activation i) by treating the cells with stress kinase cascade-inducing conditions, in particular a heat shock, osmolarity shock or UV, or ii) by incubation in a physiologically effective dose

20 with at least one substance inducing the stress kinase cascade, in particular cytokines such as IL-1, TNF-alpha, anisomycin, arsenite and / or alkylating agents.

25

10. The method according to any one of claims 1 to 9, wherein the cells are atic stem cells.

11. The method according to claim 1, wherein the cells are taken from a non-embryonic human organism.

12. Cells obtainable by a method according to one of claims 1 to 11.

13. Use of cells according to claim 12 for the production of a pharmaceutical composition, in particular for the treatment of degenerative nerve diseases.

14. Use according to claim 13, wherein the cells are autologous.

15. A method for the treatment of degenerative nerve diseases, wherein a diseased organism cells according to claim 12 are administered in a suitable galenic preparation or wherein a diseased organism is administered a MAPKAP kinase activating substance in a suitable galenical preparation.

16. Use of a substance activating a MAPKAP kinase for the manufacture of a pharmaceutical composition for the treatment of degenerative nerve diseases.