DE10106829A1 - Medicines with a DNA sequence coding for the RNA-binding KOC protein, a KOC protein or a DNA sequence from the KOC promoter - Google Patents

Abstract

The invention relates to a medicament containing an RNA-binding protein KOC or a DNA sequence that codes this protein. The invention also relates to a diagnostic method with regard to tumors associated with expression of KOC, and to different applications of the inventive medicament, preferably for artificially inducing the pluripotentiality of body cells. The invention additionally relates to a DNA sequence of the KOC promoter, whereby the KOC promoter is characterized in that it is only activated in embryonic tissues or malignant tumors, and, finally, relates to different applications of the KOC promoter.

Description

The present invention relates to a medicament that binds an RNA Protein KOC or a DNA sequence coding for this protein, preferably wisely advertised on a vector, contains. The present invention relates to also a diagnostic method with regard to the expression of KOC associated malignant tumors as well as various uses of the inven drug according to the invention, preferably for artificially inducing the Pluripotency of body cells. Finally, the present invention also relates to a DNA sequence of the KOC promoter, the KOC promoter thereby is characterized to be found only in embryonic tissues or malignant tumors ren is activated, as well as various uses of the KOC promoter.

The failure of individual organs or differentiated cells within an organ is the cause of many diseases, which to this day hardly amenable to therapy are. Despite medical-technical advances in transplant medicine is about the lack of donor organs reason for the annual death of Thousands of patients. In addition, the functionality of the trans planted organs only through continuous postoperative immunosup pression, the z. T. is associated with serious side effects Afford.

For these reasons, new regeneration technologies are developing diseased organs or for the reconstitution of organs to focus on of medical research. Use is the body's own cellular development and differentiation potentials for therapeutic An set goal of these new treatment strategies. This is after matching Methods sought to help achieve cell differentiation and proliferation while maintaining the function of the individual cell type, without a transformie to have great potential. This could make it possible in the future, from z. B. healthy cell material from patients obtained through biopsies to generate targeted manipulation and cultivation of stem cell populations, from which, due to their proliferation potential, specific and dif Fermented cells can develop. This could eventually lead to bottlenecks solid replacement organs in transplant medicine.

The coordinated process of a complex cellular differentiation program, which ultimately leads to the development of a functional organ is a hitherto largely unsolved problem (Lanza et al., Nature Medicine 5, No. 9: 975-977 (1999); Vogel, G. (1999) Science 283: 1432-1434; Solter et al., Science 283: 1468-1470 (1999)). A realization of this concept he seems only tangible in cases where a differentiated single cell is ultimately "Organ function" determines how z. B. the insulin-producing beta cell of the Pancreas or, in the case of antibody-producing B cells, as well as progenitor cells of the bone marrow, which are produced by specific wax factors can be differentiated.

The subject of intensive research is therefore the development of new methods for Development of functional organs, tissues and stem cells too Transplant purposes. For this purpose, individual genes have already been identified that are essential in the control of development processes. Furthermore it is succeeded in producing non-differentiated cells capable of dividing (embryonic stem cells, It cells), which are targeted by a transfer of certain genes can be differentiated. For example, by treating with the "bone morphogenetic protein 4" (bmp4) to mesenchymal ES cells Differentiate cells. In addition, these cells can be defined by Culture conditions are also manipulated in such a way that three-dimensional nal, tissue-like structures with organ-specific expression patterns to be developed. Another example of the pluripotency of stem cells shows that it has been possible to use neural stem cells in mice to implant destroyed bone marrow, which there differentiate into blood cells ren.

So far, however, there is still no controlled reprogramming of adults, differentiated cells to ES cells so succeeded in doing this medically is justifiable because, for example, mouse ES cells are per se tumorigenic and if injected into adult mice, teratocarcinomas can develop. on Transferring the human realm, this procedure is also not only technically very complex, but also touches on ethical aspects, as for this procedure requires human blastocysts or mammalian oocytes ("Therapeutic Cloning").

Thus, the invention is essentially based on the technical problem To provide means by which a dedifferentiation of a mature Cell type into an embryonic phenotype that is capable of dividing, but still Merk male of the respective differentiated cell is made possible.

The solution to this technical problem was provided by the provision of the in the claims characterized embodiments achieved.

As part of an investigation of the differential gene expression in pancreatic cancer, a gene was originally cloned which codes for an RNA-binding protein of the KH family, hence the name KOC (KH domain containing protein overexpressed in cancer) (Müller-Pillasch et al., Oncogene 14: 2729-2733 (1997)). KOC is only expressed in pancreatic carcinoma, but not in normal pancreatic tissue or in chronic pancreatitis. The gene codes for a 69 kDa protein with four KH domains ("hnRNP K homologous"); see Fig. 1. The sequence is in the NCBI database under the accession no. U97188 deposited.

KH domains are highly conserved, functionally significant structures. binding proteins responsible for direct mRNA binding. Studies have shown that RNA-binding proteins have a central role Play a role in the post-transcriptional regulation of gene expression. In RNA-binding proteins are essential for the regulation of this function Growth and development. Examples are pattern formation and cellular Differentiation during embryonic development.

Two mechanisms of post-transcriptional gene regulation through Proteins containing KH domains are controls of mRNA stability and the specific subcellular localization of the mRNA. Although already a Number of proteins containing KH domains identified, So far it has not been possible to show for any KH protein that it is capable of the phenotype of differentiated cells into a phenotype of immature, dedifferentiated ter, divisible cells to revert and thus as a possible Thera peutikum for the production of syngeneic stem cell populations that large numbers of divisible, specifically differentiable cells are available place.

It could be shown in the present invention that with the help of the Gene encoding the RNA-binding protein KOC described above be problems can be solved. With the RNA-binding protein KOC is a dedifferentiation of a mature cell type into an embryona len phenotype, which still bears the characteristics of the differentiated cell, possible so that a generation of stem cells of any cell type can be generated in the same organism. In doing so, the KOC gene is reversed the phenotype of differentiated cells into a phenotype of immature, dedifferentiated ter, divisible cells. The RNA-binding protein KOC is therefore suitable as a possible therapeutic agent for the production of syngeneic stem cell populations functions that produce large numbers of divisible, specifically differentiable cells Make available, which then z. B. in the field of transplant medicine and Oncology, through to the development of biologically functional replacement organs, for Can be used. Furthermore, the RNA-binding protein KOC can be used for Influence on physiological or induced by physical noxae Aging processes, e.g. B. human skin.

The studies were carried out on pre-B cells and carried out by Genmani pulation of these differentiated cells with KOC could result in an undifferentiation of these cells denoted state, that of an earlier, embryonic precursor or stem cells of these cells corresponds to be transferred. So on pre B-cell model can be shown that, for example, the DNA synthesis of the KOC / p210 cells is significantly stronger in comparison to that of the p210 cells and that the KOC / p210 cells compared to the p210 cells have a large number of upregulate embryonic markers, or markers that adult Differentiate pre-B cells from fetal pre-B cells, downregulate them. With the KOC gene "back differentiated" cells are against by radiation and Dexamethasone induced cell death much less sensitive than untreated Cells. In addition, these cells re-enter the cell cycle and proliferate. KOC differs fundamentally from previously used th conventional growth factors, which only ever increase the proliferation of one differentiated cell type or stimulate a cell type to a malignant phenotype transform. Numerous properties result from the properties mentioned potential uses for KOC in transplant medicine and cancer treatment. The use of KOC is for the development of new techniques Restoration and regeneration of damaged organs of importance (Organ replacement), especially at the generation level, type-specific syngeneic stem cells and artificial tissue, such as in diseases such as Alzheimer's (neural stem cells), diabetes (pre-beta cells), heart failure ciency (precursors of myocardial cells), liver (precursors of hepatocytes). Furthermore, the use of KOC in skin injuries, such as. B. Burn in the future by the induction of rapid extracorporeal multiplication of Skin cells for transplant purposes may be of concern.

In summary, the following areas of application arise for KOC (either as part of gene therapy or as protein administration):

• 1. Achieving resistance of differentiated cells to different ones Toxins induced cell death (e.g. radiation), and
• 2. Transfer of lines from a differentiated, non-proliferative into an undifferentiated, proliferative precursor state, which is still bears clear features of the differentiated phenotype, and thus according to Expan sion of the cell population by switching off the KOC expression in the parent cell type. This becomes syngeneic, i.e. in the same Unrestricted production of stem cells of any organism Cell type and thus also the unlimited production of differentiated, z. T. not or only very poorly divisible cells possible at any age.

Thus, for the purposes described above, the administration of KOC itself or the gene coding for this protein is useful. on the other hand may also include the possibility of inactivation of KOC in diseases that are associated with too high activity of KOC or too high expression of the gene go hand in hand, be therapeutically useful. Such inactivation can occur take place at different levels, for example at the genetic level ("Knock out ", inhibition of translation by antisense RNAs, ribozymes or aptams re) or protein level (via inhibiting ligands, e.g. antibodies or antagonists etc).

In addition, within the framework of the leading to the present invention Investigations also cloned the DNA sequence of the KOC promoter and sequenced, which it was found that this only in embryonic tissue or malignant tumors is activated.

The present invention thus relates to a DNA sequence of the KOC promoter which comprises the nucleic acid sequence shown in FIG. 9 or a nucleic acid sequence deviating therefrom, the biological function of which essentially corresponds to that of the KOC promoter.

The term "a different nucleic acid sequence" used in connection with the KOC promoter in the present invention relates to any nucleic acid sequence which differs from the nucleic acid sequence shown in FIG biological function of the promoter is retained, ie to be able to control the transcription of a gene, the promoter being activated essentially only in embryonic tissue or malignant tumors. The differing nucleic acid sequences are preferably nucleic acid sequences which hybridize with the nucleic acid sequence shown in FIG. 9, reference being made to the definition below with regard to the term "hybridize", or fragments of the nucleic acid sequence shown in FIG. The “differing nucleic acid sequences” are preferably 70%, more preferably 80% and most preferably 90% identical to the DNA sequence of FIG. 9.

The present invention also relates to a medicament which contains a DNA sequence which codes for an RNA-binding protein KOC with the amino acid sequence shown in FIG. 1, or a DNA sequence which contains the nucleic acid sequence shown in FIG. The DNA sequence is preferably cDNA.

The present invention also relates to a medicament which contains a DNA sequence of the KOC promoter according to the invention or a DNA sequence which codes for a protein with the biological properties of an RNA-binding protein KOC, this DNA sequence being from of the DNA sequence of FIG. 1 differs in the codon sequence due to the degeneracy of the genetic code, hybridized with the above DNA sequences or which is a fragment, an allelic variant or another variant of the above DNA sequences.

The term "hybridized" as used in the present invention relates on conventional hybridization conditions, preferably on hybridization conditions in which 6 × SSC, 1% SDS, 1 × Denhardts Solution is used and the hybridization temperatures between 35 ° C and 70 ° C, preferably 45 ° C. After hybridization is preferred assign with 2 × SSC, 1% SDS or with 0.2 × SSC (stringent conditions) Temperatures between 40 ° C and 75 ° C, preferably washed at 50 ° C (for For a definition of SSC and Denhardt's solution, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Stringent ones are particularly preferred Hybridization conditions, as described, for example, in Sambrook et al., Supra, or Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6 are described.

The terms “variant” or “fragment” used in the present invention encompass DNA sequences which differ from the sequences indicated in FIG. 1 by deletion (s), insertion (s), exchange (s) and / or others Re distinguish modifications known in the prior art or comprise a fragment of the original nucleic acid molecule, the protein encoded by these DNA sequences still having the biological properties of the RNA-binding protein and being biologically active in mammals. This also includes allele variants. Methods for generating the above changes in the DNA sequence are known to the person skilled in the art and are described in standard works of molecular biology, for example in Sambrook et al., Supra. The person skilled in the art is also able to determine whether a protein encoded by a DNA sequence modified in this way still has the biological properties of the RNA-binding protein KOC. In the following, all of these DNA sequences are generally referred to by the term "KOG DNA sequences".

By lowering or inhibiting the expression of the above DNA sequences described, the synthesis of the encoded by these Proteins are reduced or eliminated, for example in certain Disease states is desirable. Therefore concerns another preferred Embodiment of the present invention Antisense RNA that thereby is characterized as being complementary to the above DNA sequences and the synthesis of the protein encoded by these DNA sequences can reduce or inhibit and a ribozyme, which is characterized that it belongs to a part of the above DNA sequences and to those of these DNA sequences can specifically bind transcribed RNA and cleave them, thereby facilitating the synthesis of the protein encoded by these DNA sequences is reduced or inhibited. Preferably these are antisense RNAs and Ribozymes complementary to a coding region of the mRNA. The subject man is able, starting from the disclosed DNA sequences, ge to produce and use suitable antisense RNAs. Appropriate approach Ways are described in EB-B1 0 223 399 or EP-A1 0 458, for example. Ribozymes are RNA enzymes and consist of a single strand of RNA. These can cleave other RNAs intermolecularly, for example those from the DNA sequences according to the invention transcribed mRNAs. These ribozymes must in principle have two domains, (1) a catalytic domain and, (2) a domain that is complementary to and attached to the target RNA can bind, which is the prerequisite for cleavage of the target RNA. Excellent It is now based on the procedures described in the literature possible to construct specific ribozymes that attach a desired RNA cut a certain, preselected point (see for example Tanner et al., in: Antisense Research and Applications, CRC Press, Inc. (1993), 415- 426).

Those encoding the RNA-binding protein KOC or the related protein DNA sequences or those for the antisense RNAs described above or DNAs encoding ribozymes can also be incorporated into a vector, preferably Expression vector. Thus embracing the present invention also drugs containing these vectors or expression vectors. the The term "vector" refers to a plasmid (pUC18, pBR322, pBlueScript etc.), to a virus or other suitable vehicle. In a preferred embodiment is the DNA molecule according to the invention im Vector functionally linked with regulatory elements that of its Allow expression in prokaryotic or eukaryotic host cells. Such In addition to the regulatory elements, vectors contain, for example, a Promoter, typically an origin of replication and specific genes that allow phenotypic selection of a transformed host cell. To the regulatory elements for expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in Eukaryotes the AOX1 or GAL1 promoter in yeast and the CMV, SV40, RSV, Metallothioneinl and polyhedrin promoter, CMV or SV40 enhancer for the Expression in animal cells. Suitable regulatory sequences are also described in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). More examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable expression vectors for E. coli include, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. Among those for expression in yeast suitable vectors include pY100 and Ycpad1, for expression in animals ger cells pMSXND, pKCR, pEFBOS, cDM8 and pCEV4 as well as from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors. To the Expression vectors of the invention also include baculovirus derived vectors for expression in insect cells, for example pAcSGHisNT-A.

Preferably, the above-described KOC DNA sequences that can also comprise a DNA sequence of the KOC promoter, in a for the gene therapy inserts a suitable vector, for example under control a tissue-specific promoter, and introduced into the cells. In a preferred embodiment is the DNA- Vector containing sequences a virus, for example an adenovirus, Vaccinia virus or retrovirus. Retroviruses are particularly preferred. Examples suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. Vectors suitable for gene therapy are also in WO 93/04701, WO 92/22635, WO 92/20316, WO 92/19749 and WO 92/06180 are disclosed. For For gene therapy purposes, the above-described KOC-DNA- Sequences also in the form of colloidal dispersions to the target cells be transported. These include, for example, liposomes or lipoplexes (Mannino et al. Biotechniques 6: 682 (1988)). Finally, the KOC DNA sequences or the KOC protein locally z. B. in the form of a cream, ointment etc. are administered.

General methods known in the art can be used to construct of expression vectors coding for the RNA-binding protein KOC DNA sequences and / or a DNA sequence of the KOC promoter and suitable control sequences can be used. To this Methods include, for example, in vitro recombinant techniques, synthetic ones Methods, as well as in vivo recombination methods, as described, for example, in Sambrook et al., Supra. The DNA according to the invention Sequences can also be used in conjunction with a for another protein or Peptide-encoding DNA can be inserted, so that KOC, for example, in the form a fusion protein can be expressed. The expert also knows Process for the recombinant production of KOC, d. H. the cultivation suitable host cells under conditions conducive to expression of the protein (or fusion protein) allow (preferably stable expression), and the Obtaining the protein from the culture or from the host cells. Suitable Purification processes (e.g. preparative chromatography, Affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are also well known.

An essential goal in the diagnosis of malignant diseases is specific and sensitive detection of early malignant changes. In addition For example, more and more gene mutations are found in epithelial tumors, which occur in the context of the adenoma-carcinoma sequence are used. The disadvantage of this method is that some gene mutations occur in normal Tissues occur, such as in ki-ras in normal pancreatic tissues or in severely inflamed tissues, what a distinction between chronic, inflammatory or tumorous tissues that are therapeutic of paramount importance makes impossible. The aim of the research is therefore Identify genes that are exclusive to malignant degenerate cells are expressed / upregulated and are therefore used for unequivocal tumor diagnosis suitable.

As already discussed above, KOC is expressed in pancreatic carcinoma, but not in normal pancreatic tissue or in chronic pancreatitis. This finding that KOC is only expressed in malignant tissues (organs or cells) but not in benign tissues has been confirmed many times in a wide variety of tissues or types of cancer. Reference is made to Tab. 1. KOC is therefore suitable for the differential diagnosis between malignancy and benignity, e.g. B. in inflammation, to be able to distinguish a tissue, preferably from the pancreas or colon. KOC is particularly characterized by the fact that its expression can be detected very early in the development of cancer, long before any auto-antibodies against KOC are (can) be formed. Furthermore, KOC makes it possible to differentiate between different stages of malignancy, especially in pancreatic and colon cancer. It has been shown here that the KOC expression increases with the severity of the dysplasia. KOC thus represents a universally applicable tumor indicator. The present invention therefore also relates to diagnostic methods for the detection of a malignant tumor or its precursors associated with the expression of KOC, in which a sample is mixed with a for specific hybridization with that of a KOC-DNA- Sequence transcribed mRNA brings suitable probe or a primer or an antibody against the KOC protein or a fragment thereof and then directly or indirectly determines whether the concentration of KOC and / or the KOC mRNA in the sample compared to a Differentiate control sample. The person skilled in the art knows methods for suitable sampling and also suitable control samples. The control sample is preferably a tissue which corresponds to the same tissue as the tissue affected by the tumor, but comes from a healthy source. An increased KOC expression compared to the control sample is a diagnostic sign of a tumor or a predisposition for a tumor. The probes that can be used for this diagnostic method include primers based on the KOC-DNA sequences, for example for PCR, RT-PCR or aptamer diagnostics (SELEX method). The probes (or primers) are preferably oligonucleotides which comprise a nucleic acid region which hybridizes under stringent conditions with at least 10, preferably at least 15 consecutive nucleotides of the sequence of FIG. 1 or naturally occurring variants thereof. The probe (or the primer) can also carry a label, e.g. B. a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. The person skilled in the art also knows conditions that allow only the KOC mRNA but not the KOC DNA to be amplified in the PCR etc. or a distinction can be made between DNA amplification products and mRNA amplification products, e.g. B. by treating the sample with DNAse or by using primers that also lead to the amplification of intron areas, so a DNA amplicon is longer compared to the mRNA amplicon. Alternatively, an oligo (dT) anchored primer for PCR, RT-PCR etc. can also be used.

The antibodies for the diagnostic method according to the invention can monoclonal, polyclonal or synthetic antibodies or fragments of that. In this context the term "fragment" means all parts of the monoclonal antibody (e.g. Fab, Fv or "single chain Fv" Fragments) that have the same epitope specificity as the full antibody exhibit. The preparation of such fragments is known to the person skilled in the art. The antibodies according to the invention are preferably monoclonal antibodies. These antibodies can according to standard procedures be prepared, preferably the KOC protein or a (synthetic) fragment thereof serve as an immunogen. Procedure for Obtaining monoclonal antibodies are known to the person skilled in the art.

A preferred embodiment of the method according to the invention relates to the differential diagnosis between chronic pancreatitis and Pancreatic cancer.

Furthermore, the present invention relates to a diagnostic kit for Implementation of the diagnostic method according to the invention, the one for specific hybridization with one of a KOC DNA sequence transcribed mRNA suitable probe or a primer and / or an anti- Contains KOC antibodies or a fragment thereof. Depending on the design of the Diagnostic kits can contain the sample or the probe or the primer or the Antibody or the fragment thereof may be immobilized. The antibodies can for example in immunoassays also in the liquid phase. Here you can the antibodies can be labeled in various ways. Suitable markers and labeling methods are known in the art. examples for Immunoassays are ELISA and RIA.

The medicament according to the invention allows the implementation of a number of (therapeutic) measures. Preferably the drug is with a suitable carrier combined. Suitable carriers and the formulation such medicaments are known to the person skilled in the art. To suitable carriers include, for example, phosphate-buffered saline solutions, water, Emulsions, for example oil / water emulsions, wetting agents, sterile solutions etc. The drugs can be administered orally or parenterally. to Methods for parenteral administration include topical, intra arterial, intramuscular, subcutaneous, intramedullary, intrathecal, in traventricular, intravenous, intraperitoneal or intranasal administration.

Thus, the present invention relates to the use of the above described compounds (KOC DNA sequences / KOC protein) for Artificially bringing about the pluripotency of body cells. This is e.g. B. for the Development of replacement organs of importance, especially for the Generation of artificial pancreatic, liver or nerve tissue. Thus be the present invention also applies to the use of the foregoing Compounds for the production of tissues or organs via the Differentiation of stem cell populations.

The present invention also relates to the use of the above described compounds (KOC DNA sequences / KOC protein) for the Enhance ex vivo expansion of hematopoietic stem cells, e.g. B. for autologous bone marrow transplants. In particular, syngeneic Precursor cells can be generated without "purging". This will be autologous Transplants on a much larger scale than previously possible. The too Expanding cells can be tested beforehand in the FACS (fluorescence activated cell Sorting) can be clearly characterized, so that an expansion is malignant Cells is excluded. Immunological problems associated with allogeneic Transplantation occurring frequently are excluded. Problems and Complications of a delayed regeneration of the marrow due to an low number of reinfused stem cells, such as B. in sepsis or le Threatening yeast infections, are eliminated, as the amount of stem cells that are available, can be determined as required. But it also becomes a further dose increase in chemotherapy is possible because the endogenous Stem cell population no longer exclusively for the regeneration of the marrow is needed. This means that the method can be used in high-dose Chemotherapy with repeated administration of chemotherapy and reinfusion of autologous bone marrow stem cells.

Thus, the present invention also relates to the use of the foregoing Connections to high dose chemotherapy.

Another therapeutic effect consists in the fact that KOC stem cells, as long as the protein is expressed, less sensitive to Are chemotherapy or radiation therapy. I. E. under therapy when expressing KOC is expanding the number of reinfusions required Stem cells lower. The subsequent shutdown of KOC can then after completion of chemotherapy or radiation therapy the normal Sensitivity of cells to noxae / apoptotic stimuli again produce.

Thus, the present invention also relates to the use of the foregoing Compounds for producing a prophylactic effect in chemo- or Radiotherapy.

As it was shown in expression analyzes that not only the mRNA of KOC at the nucleic acid level but also the protein by means of a specific Antibody can be detected accordingly, can of it it can be assumed that the protein as such has its specific effect unfolded on the respective cells. That is why the application of KOC is also on Skin cells, preferably through protein dissolved in creams, are advantageous. Da ins especially with topical application of KOC in creams or lotions only superficial layers of skin are reached is not by one sustained proliferation-promoting (side) effect on the entire dermis to go out. Rather, it will have a lasting effect only in the top Skin layers obtained by exposure to environmental influences (UV) can be particularly damaged and malignant degenerate (spinalioma, Basalioma). This is a therapeutic-prophylactic effect of KOC radiation-exposed skin - also in the context of therapeutic irradiation of Body regions with gamma emitters - given.

Thus, the present invention also relates to the use of the foregoing Compounds to produce a prophylactic effect radiation-exposed skin.

Furthermore, this can be the case with an allogeneic bone marrow transplant Donormark can be transfected with KOC. It is then after the transplant expect a faster and less complication engraftment. this also follows from the results from first Bone marrow transplants performed in mice as part of this invention were carried out. Mark transfected with KOC showed a clear faster engraftment.

Thus, the present invention also relates to the use of the foregoing Compounds for Enhancing Engraftment in Allogeneic Bone marrow transplants.

In addition, the use of KOC can also lead to slowing down or slowing down. Contribute to the reversal of aging processes, with the Aging processes preferably around aging processes in the area of the Vascular endothelium, neurons, keratinocytes and myocytes.

Thus, the present invention also relates to the use of the foregoing Connections for slowing down and / or reversing through physiological or physical noxae induced aging processes, preferably aging processes, which are caused, for example, by noxae such as UV Radiation and other environmental influences. KOC (Gen or protein), preferably topically via a suitable carrier (e.g. as in Cream dissolved liposomes, lotions, plasters, bandages, injections, etc.) applied to or administered into the skin. This use includes also a cosmetic application of KOC (gene or protein) on the skin to influence aging skin / wrinkles.

The present invention further relates to the use of the foregoing Compounds for the regeneration of skin defects or for accelerated Wound healing. This also includes the formation of granulation tissue, e.g. B. after Trauma, burns or various leg ulcers Genesis.

Furthermore, it has been shown that an individual using KOC against with the Expression of KOC-associated malignant tumors and their precursors can be immunized.

Thus, the present invention relates to the use of the compounds described above (KOC DNA sequences / KOC protein) for immunizing an individual, e.g. B. an animal or a human, against malignant tumors associated with the expression of KOC and their precursors. In particular, the present invention relates to a medicament suitable for this purpose, which contains these compounds in an amount suitable for the immunization of an individual. The KOC protein can be in wild-type or modified form. The latter form includes changes in the amino acid sequence, such as additions, deletions, substitutions and / or inversions of one or more amino acids. Fragments of the KOC proteins can also be present as such or in connection with carriers, it being possible for the fragments to have a wild-type or an altered amino acid sequence. It is beneficial if the carriers do not act as immunogenic in the individual. Such carriers can be proteins or fragments thereof that are intrinsic or foreign to the individual. Carriers such as serum albumin, fibrinogen or transferrin or a fragment thereof are preferred. It is particularly favorable if the fragments of the KOC proteins contain epitopes which are produced by cytotoxic T cells, e.g. B. CD8 ⁺ T cells are recognized and can induce a cytotoxic immune response. Such epitopes of the KOC proteins can be determined by methods known to the person skilled in the art, in particular by using a software system from the NIH (NIH bioinformation service http: /bimas.dcrt.nih.gov.cgi-bin/molbio/ken_parker_comboform). It can also be advantageous if different of the KOC proteins or fragments thereof, for which the above statements apply accordingly, are present at the same time. For the production of the KOC proteins or their fragments, in addition to the above statements, reference is made to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).

When a KOC DNA sequence is used to immunize an individual, it must be in an expressible form; d. H. it can as such together with or in conjunction with elements suitable for their expression with a vector. Reference is made to the statements above.

The term "malignant tumors and their precursors" includes malignant tumors of any kind and origin associated with the expression of KOC, and their preliminary stages. For example, carcinomas of epithelial origin, in particular pancreatic, colon, breast, bronchial and anogenital carcinomas be.

The expression "amount suitable for immunizing an individual" includes any amount of the KOC protein to which the above statements apply accordingly, or an expressible KOC-DNA sequence, to which the above statements apply accordingly, with which an individual can be immunized . The amount depends on whether the KOC protein or the expressible KOC DNA sequence is used. The amount also depends on whether the immunization of the individual is based more on induction of antibodies directed against the KOC protein or on stimulation of cytotoxic T cells directed against the KOC protein, e.g. B. CD8 ⁺ T cells. Both possibilities of immunization can be achieved by the present invention. Furthermore, the amount depends on whether the immunization is intended as a prophylactic or therapeutic treatment. In addition, the age, sex and weight of the individual play a role in determining the amount. It is favorable if the individual is injected with 100 µg - 1 g of the KOC protein or 10 ⁶ - 10 ¹² MOI of a recombinant virus containing an expressible KOC DNA sequence. The injection can be made intramuscularly, subcutaneously, intradermally or in any other form of administration at several points of the individual. It can also be beneficial to carry out one or more “booster injections” with approximately the same amount, it being particularly beneficial to use different fragments of the KOC protein in the individual injections. Furthermore, it is advantageous if the drug contains immunization adjuvants, such as GM-CSF or Freund's adjuvant, for immunizing the individual.

Furthermore, a non-human mammal ("knock-out") can be provided by customary methods whose KOC gene is modified. A method that comprises the following steps is favorable:

• a) Production of a DNA fragment, in particular a vector, containing an altered KOC gene, the gene by insertion a heterologous sequence, in particular a selectable marker, has been changed;
• b) Preparation of embryonic stem cells from a non-human Mammals (preferably mice);
• c) Transformation of the embryonic stem cells from step (b) with the DNA fragment from step (a), showing the KOC gene in the embryonic Stem cells by homologous recombination with the DNA fragment is changed by (a),
• d) culturing the cells from step (c),
• e) Selection of the cultured cells from step (d) for the presence the heterologous sequence, especially the selectable marker,
• f) generating chimeric non-human mammals from the cells of step
• g) by injecting these cells into mammalian blastocysts (preferred Mouse blastocysts), transferring the blastocysts to pseudo-pregnant women female mammals (preferably mice) and analysis of the obtained Offspring for a change in the KOC gene.

In step (c) the mechanism of homologous recombination (cf. R. M. Torres, R. Kühn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) to trans fify. The homologous recombination between those in one chromosome existing DNA sequences and new added cloned DNA-Se quenzen enables a cloned gene to be inserted into the genome of a living cell instead of the original gene. With this method you can when using embryonic germ cells are obtained via chimeric animals, those for the desired gene or the desired gene or the desired mutation are homozygous.

The term "embryonic stem cells" refers to any embryonic stem cells of a non-human mammal that mutate the KOC- Own gens. Preferably the embryonic stem cells are from the mouse, especially cells E14 / 1 or 129 / SV.

The term "vector" encompasses any vector which is produced by recombination with embryonic stem cell DNA is a change in the KOC gene possible. The vector preferably has a marker with which on dene stem cells can be selected in which the desired Rekom bination has taken place. Such a marker is e.g. B. the IoxP / tk neo-Cassette, which comes with Can be removed from the genome again using the Cre / IoxP system.

Furthermore, the person skilled in the art knows the conditions and materials for the steps (a) - (f) to perform.

The present invention provides a non-human mammal whose KOC gene is changed. This change can turn it off of function. With such a mammal or cells from it can the function of the KOC protein can be investigated selectively. Furthermore, it is herewith possible to find substances, drugs and therapeutic approaches with which can be influenced selectively on the function. Hence the present Invention of a basis to act on a wide variety of diseases. Such diseases are e.g. B. the induction of cancer by failure in the Control of cell proliferation. It also allows the mammal to be modified KOC function Investigations whether you can apply various Cancerogens show a weakened, if not even no tumor development observed in these animals. The animal represents a new disease model for the Development of new therapeutic approaches.

Finally, the present invention also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence deviating therefrom, the biological function of which essentially corresponds to that of the KOC promoter, for the isolation and / or enrichment and / or selective reproduction of Stem cells, preferably non-human embryonic stem cells.

Stem cells are the original cells from which the individual tissues of an organism develop. Because all cell types of an organism arise from only one single starting cell, the zygote (fertilized egg cell), all stem cells have precursors which, depending on the developmental stage of the individual, have different abilities (potencies) to form new tissues. One speaks of toti-, pluri- and multipotent stem cells. The earliest stem cells to appear, embryonic stem cells, are totipotent. Any tissue can arise from them. Later stem cells lose this ability and are then only able to generate individual tissues. If stem cells are required for scientific or medical purposes, careful separation from other differentiated cells must be ensured during stem cell production, since these can overgrow the embryonic stem cells under culture conditions. Furthermore, the continuous presence of certain differentiated cells induces the differentiation or programmed cell death of stem cells. For this reason it is desirable to have a selection of stem cells, e.g. B. to make embryonic stem cells from the "mixed culture". Genetic manipulation makes it possible to introduce genetic selection markers into the cells, the gene products of which guarantee selective survival of the stem cells under certain culture conditions. For example, a gene product can cause resistance to certain antibiotics. All cells that do not express this resistance gene can be eliminated by adding the respective antibiotic to the culture medium. A decisive prerequisite for the selection of stem cells is that the respective selection marker is under the control of a promoter that is activated exclusively in the stem cells. Since the KOC promoter is only activated during this time, it can be used as a control unit for the selection of stem cells. For this purpose, through infection / transfection of a vector in which a selection marker is under the control of the KOC promoter, the stem cells have a selection advantage under conditioned culture conditions (e.g. addition of antibiotics) over cells that are no longer in the embryonic state or . are already differentiated, provides a selection advantage. Methods for the infection / transfection of stem cells, suitable vectors and suitable selection markers, e.g. B. the system neo-Gen / G418, are known to the person skilled in the art; see also the above statements with regard to the description of the non-human mammal. The person skilled in the art also knows suitable culture methods and media in order to obtain stem cells, e.g. B. embryonic stem cells to be able to cultivate; see e.g. B. DE 196 08 813 C2, EP-B1 0 695 351. The "basic medium" which z. B. contains the antibiotic in a suitable concentration for selection, can be any medium conventionally used for the cultivation of mammalian cells. The cells are cultured in the above medium under suitable conditions, if necessary with (partial) renewal of the medium at suitable time intervals. Suitable conditions, for example with regard to suitable containers, temperature, relative humidity, O ₂ content and CO ₂ content of the gas phase, are known to the person skilled in the art. The cells are preferably cultivated in the medium under the following conditions: (a) 37 ° C., (b) 100% rel. Humidity, (c) 10% O ₂ and (d) 5% CO ₂ . The person skilled in the art can also monitor the desired inhibition of the differentiation of the cells on the basis of customary criteria (morphological criteria, presence or absence of specific surface proteins, etc.).

The present invention also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence deviating therefrom, the biological function of which essentially corresponds to that of the KOC promoter, for determining the degree of differentiation or dedifferentiation of a cell or tissue. For this purpose, it is advantageous to combine the DNA sequence of FIG. 9 or the nucleic acid sequence deviating therefrom with a reporter gene and to introduce the construct into the cell or tissue and to demonstrate the expression of the reporter gene, which provides information about the degree of differentiation or Dedifferentiation there.

The present invention also relates to a diagnostic kit for carrying out the diagnostic method described above, which kit contains a probe or a primer suitable for specific hybridization with a DNA sequence of the KOC promoter. With regard to the design of the kit, the statements made above with regard to the diagnostic kit relating to the KOC protein or KOC gene apply accordingly. The inventors have characterized primer pairs which are suitable for the purposes mentioned above, e.g. B. as a tumor indicator, ideal. These can be provided as part of the kit. The preferred primers are:

Forward primer:
ATG 14: 5'-ATATCGGAAACCTCAGCGAG-3 '
ATG 651: 5'-CATTCGGAACATCACCAAAC-3 '
ATG 778: 5'-GGGGTATTAAACGTGCATTT-3 '
ATG 1428: 5'-CACTGTCATGAGCCTCACTA-3 '

Reverse primer:
ATG 98: 5'-GTAGCCAGTCTTCACCAGGA-3 '
ATG 341: 5'-TGCAGTTTCCGAGTCAGTGT-3 '
ATG 757: 5'-GACTTACAAGCCGCAGAGGT-3 '
ATG -217: 5'-TGACAGAATTTAAACCAGCA-3 '

The following primer pair combinations are very particularly preferred:
ATG 14 - ATG 98 (1)
ATG 651 - ATG 757 (2)
ATG 14 - ATG 341 (6)

With the conventional tumor markers used in the clinic up to now it is usually an acute phase protein, which, although in the course of a Tumor disease increase in their measurable value, but also with numerous pathophysiological conditions, e.g. B. renal insufficiency, Inflammations, etc., are found in the serum higher than normal. These tumor markers only have their clinical significance when they firstly, they are significantly increased and secondly in the course of an illness or therapy change in value. That is, most of the common ones Tumor markers are only suitable for assessing the course of the disease or Therapy evaluation. However, they have primarily no diagnostic Informative value. By using KOC as a tumor indicator, it becomes the first time possible, from incidental findings, e.g. B. in imaging, with excellent Specificity and sensitivity predict whether a lesion is a malignant or benign mass. This is particularly of Interest, because KOC provides this information regarding any tissue and thus a universally applicable tumor indicator to confirm the diagnosis of Space claims is. It is also noteworthy that KOC is supported by others pathophysiological conditions such as B. Inflammation, in his Expression behavior is not influenced and thus a 100% sensitivity and specificity guaranteed. The importance of KOC as a tumor indicator shows is also evident from the fact that examiner-dependent influences are absolute are to be excluded. Thus, KOC can be used as a surrogate parameter for the cytological Significantly improve diagnostic reliability. In addition, refer to the above Comments on the use of KOC as a tumor indicator referenced.

It has also long been known that certain chemotherapeutic agents when People do well at first, but gradually become less and less effective are. The cause of this "resistance" is a mechanism that the tumor cells themselves Get going because chemotherapy drugs are called multi-drug resistance Activate the (mdr-1) gene. The gene product itself is on the Cell membrane and is responsible for transporting foreign substances out of the cell responsible. This way the drugs become ineffective. These Resistance can e.g. B. via the tumor necrosis factor (TNF alpha) prevented will. Supplied from outside or transferred as a gene, TNF alpha inhibits the expression of the MDR1 gene and thus the development of resistance. Packed up for example, the TNF-alpha gene with the promoter sequence of the mdr-1- Gens into a vector and treats an animal with this vector construct and with chemotherapeutic agents that target the promoter sequences of the mdr-1 gene act, so the production of TNF alpha switches on, prevents Formation of resistance and promotes the destruction of the tumor cell through apoptosis. A major disadvantage of this method, however, is that one for the Patients with stressful chemotherapy must also be carried out. Another one An example is the therapy with cisplatin (cis-diaminedichloroplatinum), which is called active agent in a variety of malignancies as Chemotherapeutic drug is used. Since the KOC promoter during the malignant transformation of a cell is activated (see the following Example 6), this is excellently suited as a control unit for a so-called. "gene-directed enzyme pro drug (suicide gene) therapy" (GDEPT) for Treatment of a wide variety of malignancies. Preferably is for this gene therapy cancer treatment the HSV-TK gene (in combination with the Application of ganciclovir, which is phosphorylated via the HSV-TK becomes toxic), a tumor suppressor gene, e.g. B. p53, p16, p21, DPC4, APC, etc., or an apoptosis inducing gene, e.g. B. bad, caspases, etc., with the Functionally linked KOC promoters.

The present invention thus also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence deviating therefrom, the biological function of which essentially corresponds to that of the KOC promoter, for the gene therapeutic treatment of cancer. With regard to the implementation of gene therapy and the vectors suitable for this purpose, the above statements with regard to the KOC-DNA sequences apply mutatis mutandis.

Furthermore, the present invention relates to a method for identifying compounds which bind to the DNA sequence of the KOC promoter according to the invention and modulate its activity, the method comprising the following steps:

• a) Bringing a DNA sequence of the KOC according to the invention into contact Promoters with a test compound in a cellular assay; and
• b) Determination of the modulation of the activity of the KOC promoter, wherein a modulation indicates that the test compound is effective.

The test connections can be very different connections act, both naturally occurring and synthetic, organic and inorganic compounds, as well as polymers, e.g. B. oligopeptides, polypeptides, Oligonucleotides and polynucleotides, as well as small molecules, antibodies, sugars, Fatty acids, nucleotides and nucleotide analogs, analogs of natural occurring structures, e.g. B. peptide "imitators", nucleic acid analogs, etc., and numerous other connections. Are particularly suitable as Test compounds transcription factors.

When performing the cellular assay it must be ensured that the Test compound containing the DNA sequence of the KOC promoter that contains the Transcription of a reporter gene controls, in such contact conditions is brought about that specific binding is made possible. After that, will preferably in the same test system determines whether a modulation of the Transcription of the promoter functionally linked to the reporter gene Presence of the test compound has taken place. Examples of suitable Reporter genes are GFP, Lacz, luciferase or CAT. The found values with regard to the transcription rate preferably with the Values in a second test system compared that differed from the first only differs by the absence of the test compound. Basically appropriate assay formats for the identification of compounds that contain the Modulation of the activity of the KOC promoter are in the Well known and used for the biotechnological and pharmaceutical industries Those skilled in the art are additional assays and variations of the above for Illustrative assays provided obviously.

Changes in promoter activity can be made by any suitable Procedure to be measured. Changes in the expression level can be found under Can be investigated using methods well known to those skilled in the art. This includes monitoring the mRNA concentration, e.g. B. using suitable probes or primers, immunoassays with regard to the Protein concentration, RNAse protection assays, amplification assays, or any other suitable means of detection known in the art is. Further detection methods with regard to the activation of the promoter are based on the specific properties of the respective reporter gene.

Looking for compounds that modulate the activity of the KOC Promoters are effective, can also be done on a large scale, for example by the fact that a very high number of candidate Compounds in substance libraries is screened, with the Substance libraries synthetic or natural molecules, e.g. B. cDNAs Expression libraries. Thus, in a preferred Embodiment of the method according to the invention the test compound Part of a substance library.

The above method according to the invention can be based on protocols those in the scientific literature and the patent literature and are known in the art. For example, a large number of potentially useful molecules in a single test to be screened. For example, if a field of 1000 Connections should be screened, in principle every 1000 connections in one Microplate well and tested at the same time. When a Promoter modulator is discovered, then the pool of 1000 can be divided into 10 pools divided by 100 and the process repeated until one individual modulator is identified. Anyway, the manufacture and the simultaneous screening of large banks of synthetic molecules carried out using well known methods of combinatorial chemistry see for example von Breemen, Anal. Chem. 69: 2159-2164 (1997) and Lam, Anticancer Drug Des. 1997, 12: 145-167.

The inventive method can also be used as a screening with high Throughput ("high throughput screening") are greatly accelerated. This one Assays described in terms of promoter modulation can be used for Use in such a process can be modified accordingly. It It will be apparent to those skilled in the art that numerous methods can be used for this purpose be available.

In addition, a large number of may be useful Promoter Activity Modifying Compounds in Extracts of Natural Products are screened as raw material. Such extracts can come from a large number of sources, such as the species Fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. the Extracts that show modulation can then be used to isolate the active Molecule to be analyzed. See, for example, Turner, J. Ethnopharmacol. 51 (1-3): 39-43 (1996) and Suh, Anticancer Res. 15 (1995) 233-239. the found compounds that modulate the activity of the KOC promoter can often be used therapeutically, e.g. B. in the context of tumor therapy used will.

Brief description of the figures

Fig. 1 DNA sequence of the gene encoding the RNA binding protein KOC gene and deduced amino acid sequence

Figure 2 Proliferation of cells expressing KOC and p210 and cells expressing only p210
Cells expressing KOC and p210 proliferate 10-fold more than cells expressing p210 only.

Figure 3 Growth of cells expressing KOC and p210 and cells expressing only p210
Cells expressing KOC and p210 grow independently of the stroma compared to cells expressing only p210

Fig. 4 resistance of cells which express p210 and KOC, and cells that express only p210, to dexamethasone
Cells expressing KOC and p210 are resistant to dexamethasone compared to cells expressing only p210

Fig. 5 Overexpression of the embryonic marker IGF-II in transgenic mice

Fig. 6 Application of KOC as molecular biological indicator tumor

Fig. 7 Detection of the activation of the embryonalspezifischen KOC promoter via Northern blot analyzes
Northern blot analysis of KOC expression in developing murine embryos. Each lane contains 2 µg poly (A) ⁺ RNA from mouse embryos from different stages of development (days 7-17 postcoitum). ( Fig. 7a)
KOC expression during human embryonic pancreas development. Each lane contains 2 µg poly (A) ⁺ RNA from human embryonic pancreas at different stages of development. The hybridization signals appear as a smear due to the slight degradation of the mRNA. ( Fig. 7b)

Fig. 8 Results of the RT-PCR of different tissues
Result of an RT-PCR analysis with primer pair 6 . The total RNA was used from six different human healthy pancreatic tissues, six different chronic pancreatitis tissues, two healthy colon tissues, one colon carcinoma tissue ( 1 ), one soft tissue sarcoma tissue ( 2 ), one gastric carcinoma tissue (3 ) and eleven pancreatic carcinoma tissues.

Figure 9 DNA sequence of the KOC promoter

The following examples illustrate the invention.

example 1 The overexpression of KOC leads to pre-B cells in adult too Apoptosis resistance

A well-studied model system to study developmental processes are murine pre B cells. Murine pre B cells were isolated from either adult bone marrow or fetal liver. To isolate the adult bone marrow, the tibiae and femura were isolated from mice. They were then sprayed out with medium (DMEM, Life Technologies, Karlsruhe, Germany). To isolate the fetal progenitor cells, the liver of day 15 was isolated from mouse embryos. This was then ground through a sterile cell culture sieve, releasing the individual blood precursor cells. ^{1 × 10 7} cells per 6 cm each were cultured from adult bone marrow and fetal liver. Both the adult cells and the fetal cells could be cultivated comparatively easily in vitro and thus analyzed for differences in gene expression and for functional differences. Various in vitro culture systems were available for both cell populations.

One system consisted in culturing the cells on a stromal cell line in the presence of the growth factor Interleukin 7 (R Systems, Wiesbaden, Germany). The ST2 stromal cell line was used in the present case. The cells were expanded until they were approximately 80% confluent. They were then irradiated at 3000 rad. Cells treated in this way were growth inactive, but still had a metabolism for a few days. Bone marrow or fetal liver cells were plated on this cell line with a cell density of 2 × 10 ⁶ cells at a concentration of 10 ng IL7 per ml. Only pre-B cells grew under these conditions. The cells were plated onto fresh stromal cells twice a week.

In a second system, the bone marrow or the fetal liver cells were transformed with the comparatively low tumorigenic oncogene p210 BCR-ABL. This is a fusion protein that was created by a chromosomal translocation, with parts of the BCR (break point cluster region) gene from chromosome 22 fused with c-ABL (tyrosine kinase) on chromosome 9 (Philadelphia chromosome), whereby the kinase c-ABL is present is activated. The mentioned translocation is associated with chronic myeloid leukemia. Retroviral infection was performed. For this purpose, p210 BCR-ABL was cloned into the retroviral vector pBMNZ (available from Garry Nolan, Stanford; vector folder: http: / / www.stanford.edu/group/nolan/pmaps.htmp). In each case 10 ⁷ cells were incubated with 3 ml retroviral supernatant in the presence of 8 mg / ml Polybrene in 50 ml conical tubes for at least 3 hours. The cells were then plated on stroma for 48 hours with IL7. After 48 hours, IL7 was washed out and cells were plated without IL7. Since BCR-ABL made the pre-B cells IL7 independent, only cells infected with the retrovirus coding for BCR-ABL grew out ( FIG. 3). A selection for z. B. G418 could therefore be omitted. As mentioned, the BCR-ABL transformed cells were initially IL7-independent and later also stroma-independent. The cells transformed in this way, however, were still able to differentiate into mature B cells in vivo.

It was previously known that adult pre B cells from fetal cells can be caused by the Ex differentiate between the pression of two genes: the terminal deoxynucleotide transferase (TdT) and the "myosin light chain 2" (PLRLC) are in adult pre B cells expressed, but not in fetal. There are known functional differences that fetal pre B cells are resistant to steroids while adults Cells become apoptotic after steroid administration. Furthermore, fetal pre B cells are kept in culture indefinitely in vitro, while the adult cells die after a few months of passage. We could show that KOC is expressed in fetal pre B cells but not in adults. In order to KOC is a marker available to detect fetal pre-B cells from adults discriminate.

To study the functional consequences of KOC expression, adult pre B cells that had been transformed with p210 BCR-ABL were infected with a retrovirus (pBMZ) that encoded for KOC. The infection was carried out analogously as described above for the retrovirus coding for the BCR-ABL be. In this way, KOC overexpression was achieved in the adult pre B cells. Compared to non-KOC expressing cells, the expressing cells were significantly more resistant to apoptosis, which was triggered by different stimuli. Most impressive was the resistance to steroids. While the KOC-expressing cells proliferated uninhibitedly at a concentration of 10-6 M dexamethasone, all control cells died. It was also possible to show that the DNA synthesis of the KOC / p210 cells in the tymidine incorporation assay is significantly stronger than that of the p210 cells ( FIG. 4).

Example 2 Through the overexpression of KOC the fetal Genexpres sion reinduced

After the clear phenotypic sub differences in the two cell populations could be observed, it appeared useful to establish a gene expression profile of these cells using the "gene Chip "® system from Affymetrix. This is a Oligonucleotide array with differently fluorescently labeled RNA of the respective cell population is hybridized. The specificity of the hybridization will due to the presence of 20 nucleotide probes per gene with perfect Sequence match and 20 nucleotide probes with a base mismatch ensured. Using this method, numerous genes could be found identified that are differentially expressed. So were for example Cyclin DI and IGF-II only detectable in cells that contain KOC in addition to p210 expressed. In the context described here is particularly mentioned It is worth noting that the marker genes for the adult preB cell phenotype TdT and PLRLC were completely downregulated. Since, as described above, these two markers are the only ones described, which can distinguish adult press cells from fetal ones that overexpression of KOC leads to fetal gene expression is reinduced. It should be emphasized that, firstly, the Degree of differential change in the pancreas of transgenic mice clearly depends on the level of expression of KOC and, secondly, the embryonic Markers of infected press cells are upregulated analogously to KOC expression. This fact makes it possible to give different KOCs Quantities to achieve appropriate dose effects in the respective organs.

As a result of the above experiments, a selection of genes is given below which are differentially expressed by KOC in the cells expressing BCR-ABL:

Example 3 Transgenic mouse model

The redifferentiation of haematopoietic cells shown in vitro also in vivo to confirm on other cell types (e.g. on epithelial cells) the Effect of overexpression of KOC in a transgenic mouse model examined.

For a controllable and ubiquitous overexpression, a mouse line was con structured in which the expression of KOC is under the control of Metallothio no promoter (Palmiter et al., Mol. Cell. Biol. 13 (9) (1993), 5266-5275). The induction of KOC transcription could thus be achieved by zinc application in the drink activated and controlled by water.

To produce the transgenic mice, the coding cDNA of the human KOC gene was cloned in sense orientation directly behind the metallothionein promoter of the expression vector MT-LCR 2999B4 (Palmiter et al., 1993, Mol Cell Biol, 13, 5266-5275). The DNA construct (Dral fragment, which contains the complete open reading frame of KOC, was cloned into the NruI cleavage site of the MT-LCR2999B4) was cut with the restriction endonuclease SalI (e.g. Roche, Penzberg). The ge cut construct was using a microinjection system in the female pronucleus to standard protocol injected (standard protocol described in:. "The Molecular Biology of the Gene," James D. Watson et al, 4 ^th Ed, Trans genic Mice. Mice with new germ line genes, p. 814 ff.). The cells were then transferred to a pseudopregnant female. The animals which had integrated the transgenic construct were identified by means of PCR and Southern blot analysis of the genomic DNA from the tips of the tails of the 1-2 month old mice. These founders were crossed with B6 mice. By renewed PCR analysis of the genomic DNA of the offspring of this F1 generation, the lines that inherit the transgene were identified.

The genomic DNA was isolated from mouse tails with the "Qiagen tissue kit" and was carried out according to the manufacturer's protocol (Qiagen GmbH, Hilden).

The PCR reactions took place under the following conditions:

PCR reaction:
Starting material: 500 ng genomic DNA or 100 ng cDNA
Primer: 100 ng (20 µmol) each
5 × Mg ²⁺ free buffer, pH 9.5: 10 µl
10mM dNTPs: 5 µl
HotWax ™ beads, 2.5 mM MgCl: 1 piece
Taq polymerase (5 U / µl): 0.5 µl
ddH ₂ O: ad 50 µl

PCR conditions:

Primer: Koc2117for: 5'-GAGGACCAGGCAACTTTG-3 '(KOC-specific) hGH1rev: 5'-ACTGGAGTGGCAACTTCC-3' (from the sequence section of the human growth hormone, used for the stability of the mRNA)
HotWax ™, Invitrogen, Groningen, Netherlands

Histological examinations of the pancreas of the mice showed clear changes in the organ structure. Tumors were also not observed in animals that express KOC very strongly in a one-year observation period so far (with an overall age of the animals of about two years). The ectopic KOC overexpression also had no influence on the general condition / behavior of the mice. Finally, it was examined immunohistologically which embryonic markers are upregulated in the pancreas of transgenic mice. The RT-PCR data showed that, in addition to other genes, IGF-II, which is normally only expressed in mice in the embryonic stage, is also overexpressed again in adult transgenic animals ( FIG. 5).

The characterization of transgenic mice was carried out in Western blot by Analy se different organs to identify the transgenic mice that KOC am express strongest. Two of these lines were used for breeding.

Production of Western blots: The proteins were in an SDS-Polyacryl midgel separated and in the electroblot process (Sammy dry Schleicher & Schüll, Dassel) on a nitrocellulose membrane according to the manufacturer's instructions (OPTITRAN BA-S83, Schleicher & Schüll, Dassel) transferred. To unspezi To prevent fish binding of the antibodies, the blots with 5% were lean milk powder incubated in TBS for 30 min at 37 ° C and then three times with TBS washed. Incubation with the anti-KOC rabbit serum was carried out for 2 hours at RT in TBS with 0.2% BSA and 0.01% Tween 20. After three times After washing with TBS, the blots were linked to peroxidase with the second ten anti-rabbit antibodies (from Amersham) incubated for 1 hour at RT. Of the Detection was made after washing three times with TBS using the ECL Western blotting detection reagents (Amersham).

Example 4 Differential diagnosis of malignant tumors versus normal ones or inflammatory tissue

Tissue samples were used, which were obtained by means of fine needle punctures from tissue ben / tumors of unclear dignity in the liver and lymph nodes were obtained. Control samples were taken from inflammatory areas of the colon. the Biopsy specimens were in lysis buffer (either standard guanidinium buffer or RLT Buffer from Qiagen, Hilden, Germany) added and mechanically zer diminishes.

The total RNA was extracted using the “RNAeasy Kit” from Qiagen, Hilden. A DNase digest was then carried out. An RT-PCR was carried out directly with the "One-Step RT-PCR Kit" (from Qiagen, Hilden):

Primers used:
KOC primer: 5'-TGCAGTTTCCGAGTCAGTGT-3 '
5'-ATATCGGAAACCTCAGCGAG-3 '
Annealing temperature: 55 ° C, 35 cycles

5 ml of the respective PCR mixture were applied to a 1% agarose gel. The result is shown in FIG. 6. It should be noted that in lane 1 no band can be seen which would indicate a tumorous event, but rather represents a "smear" due to overloading of the lane. Corresponding KOC bands are only visible in lanes 3 and 4, so that the presence of a tumor can be assumed here.

Example 5 Identification of the DNA sequence for the KOC promoter

The KOC gene is located on chromosome 7 in region 7p11.5. In this respect it was a chromosome 7 specific genomic library (vector Lawrist 4) with screened with a KOC probe. The DNA of positive clones was each with under different restriction enzymes digested and separated by gel electrophoresis. The gel was blotted using standard procedures. The one on the nylon membrane Fixed DNA was hybridized with a radioactively labeled 5 'KOC probe.

A positive 7kb fragment was then cloned into the vector Bluescript KS (Pharmacia). The clone was deposited on November 1 at the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig) under the accession number DSM 13810. The clone was also sequenced. The first 1946 bp upstream from the ATG are shown in FIG . The binding sites of transcription factors such as SP1, SRF, TFIID / TBF, AP1, AP2, BPV-E2, c-Myb, c / EBP, E1A-F, GATA-1, HNF-5, IBP- are located on this DNA sequence. 1, NF-IL6, NF-kB, SP1, T-antigen, TGF-1, v-Myb, etc.

A sequence comparison of the DNA sequence of FIG. 9 with an international genomic sequence database (NCBI, National Center for Biotechnology Information) revealed that sequence homologies of approximately 98% are located on chromosomes 4 and 7. It can be assumed that the KOC promoter is located on chromosome 7 as a duplicate on chromosome 4, with numerous chromosomal in situ hybridizations and the presence of classic exon-intron structures showing that the functional KOC gene is located on chromosome 7. Tests with restriction endonucleases or targeted deletions (e.g. with the Erase-a-base kit from Promega) and subsequent subcloning or the connection of the subfragments obtained with reporter genes, cf. the above explanations, also contributed to this to clarify the structure of the entire deposited clone. Here, too, DNA sequences were determined which had the smallest possible KOC promoter area.

Example 6 The KOC promoter is used exclusively in malignant tissues and expressed tumor cell lines

It could be shown that the KOC promoter exclusively in malignant Tissues and tumor cell lines (e.g. colon and pancreas) is expressed. In human and also murine adult tissues could not produce any KOC transcripts be detected.

Due to the fact that a large number of KH proteins play an essential role during embryogenesis, it was assumed that KOC could also play a role during embryogenesis. Northern blot analysis with poly A ⁺ RNA from mouse embryos from various stages of development showed that KOC is most strongly expressed on day 11 of murine development ( FIG. 7a). It was found that KOC expression decreased rapidly during advanced stages of development. In order to obtain first indications for the function of KOC in organogenesis, in situ hybridizations were also carried out on mouse embryos of different stages. It could be shown that KOC is most strongly expressed at the time of organogenesis, namely in the CNS, intestine, pancreas, thymus, kidney and haematopoietic organs. During later stages of development in the mouse, KOC expression is restricted to the thymus and the intestinal epithelium, with KOC mainly being detected in the intestinal crypts. Two days after birth, KOC expression is completely switched off. Due to the differential gene expression of KOC in pancreatic cancer, the expression of the protein in human pancreatic development was studied. For this purpose, poly (A) ⁺ RNA from human fetal pancreatic tissues from "The Genetics Institute", Boston, USA was used. This was separated by gel electrophoresis and blotted using standard methods. The Northern blot was then hybridized with a radioactively labeled human KOC probe. It was found that KOC is clearly expressed during the 12th week of pregnancy. The strongest KOC expression was observed in the 18th week of pregnancy ( FIG. 7b).

Finally, the transcript was verified by RT-PCR, it was shown that the KOC promoter is exclusively in pancreatic carcinoma tissue, however, not in the pancreas in chronic pancreatitis or in healthy the pancreas is activated. For this purpose samples from different tissues were used removed and immediately after surgical removal in liquid nitrogen shock frozen and stored at -70 ° C. For the extraction of the total RNA the tissue was pulverized in a sterile mortar in liquid nitrogen. The RNA was extracted from this human pancreatic tissue using the RNAeasy Midi Kit according to the manufacturer's protocol (Qiagen). After a DNase I. After digestion, the mRNA was transcribed directly into cDNA. These were comer Targeted reverse transcriptases (e.g. Superscript II RT, Gibco BRL) is used according to the manufacturer's protocol. To set the to be used cDNA quantities were the primers of the acidic for an internal control PCR Use ribosomal phosphoprotein (GenBank (NCB) xs13, Acc.Nr .: Z36785 det. This protein is known to be found in normal, inflammatory and malignant pancreatic tissues is expressed to the same extent. (Forward primer: 5'CTGGCTAAGTTGGT TGCTTT-3 '; Reverse primer: 5'-GCAGCT- GATCAAGACTGGA-3 '; Annealing temperature 58 ° C; 18 cycles).

The following KOC primers were used for the actual KOC RT-PCR:
Forward Primer: 5'-TGCAGTTTCCGAGTCAGTGT-3 '; Reverse Primer: 5'-ATATCGGAAACCTCAGCGAG-3 '; Annealing temperature 55 ° C; 35 cycles). The amplified DNA fragments were made visible by gel electrophoretic separation in an ethidium bromide-stained agarose gel under UV light. The results are shown in FIG . The following results were obtained for the individual tissue samples:

Normal pancreatic tissue: 0/6 positive;
Chronic pancreatitis: 0/6 positive;
Healthy colon tissue: 0/2 positive;
Various tumors: 2/2 positive;
Pancreatic cancer tissue: 7/7 positive.

Example 7 Regression of a tumor by administering an adeno virus with a ste cDNA encoding TNF-alpha

To investigate the possible suitability of the KOC promoter for genthera For the therapeutic treatment of a tumor, an adenovirus was used as the vector det. For this purpose, the KOC promoter sequence was functionally replaced by homologous Re combination in the adenoviral vector adenovirus type 5 (ATCC, VR-5) klo so that the E1A gene (which is essential for the replication of the adenovirus ist) is selectively expressed only in KOC-positive cells (Ad.KOC-E1) In addition the TNF alpha cDNA was under the control of the KOC promoter in this Construct cloned (Ad.KOC-E1 / KOC-TNF); this practice is in Kurihara et al. (J. Clin. Invest. 106: 763-771, 2000). The virus was caused by infection in the human embryonic kidney cell line (HEK293, ATCC). For the first investigations, tumor cells (Pan pancreatic cancer cells PancI or MiaPaCa2; available from the American Type Culture Collection (ATCC)) subcutaneously in the flanks of athymic nude mice (strain NMR nu / nu I). The tumors were down to one Allowed to grow in size by 1 cm. After intratumoral injection of the Kon strukts Ad.KOC-E1 / KOC-TNF was a significant regression of the tumor observed. The anti-tumor effect of this adenoviral construct is confirmed resulted in the histopathological analysis.

These results could also be confirmed in vitro. Cells where the KOC promoter is demonstrably active (human pancreatic carcinoma cell line PancI and MiaPaCa2 (American Type Culture Collection, ATTC)) were with infected with the vector construct described above. These tumor cells went in Compared to cell lines that do not express KOC (NIH 3T3 fibroblasts, ATCC), in apoptosis.

An adenovirus, in which the herpes simplex thymidine kinase (HSV-tk) is under the control of the KOC promoter (Ad.KOC-tk, adenovirus Type 5) is used to ensure that tumor cells expressing in KOC (cf. standing) after infection with this virus the HSV-tk was formed and at simultaneous administration of ganciclovir with this non-toxic substance Phosphorylation was converted into toxic, causing the tumor cells were destroyed.

Example 8 Use of KOC as a diagnostic tool for discrimination benig ner and malignant lesions from biopsy material

Different biopsy materials (tissue / cells) were in RNA lysis buffer (50 g guanidinium thiocyanate, 2.5 ml 1M sodium citrate pH 7.0, 0.5 g N-lauryl sarco sin, 0.7 ml 2-mercaptoethanol in 100 ml H ₂ O ) recorded directly. The total RNA was isolated from the biopsies using the “RNAeasy Mini Kit” (Qiagen). The following RT-PCR reaction was carried out with the “One-Step RT PCR Kit” (Qiagen). The PCR conditions were as follows:

Used KOC primers:
reverse: 5'-TGCAGTTTCCGAGTCAGTGT-3 '
forward: 5'-ATATCGGAAACCTCAGCGAG-3'-
Attachment temperature: 55 ° C, 35 cycles

The amplified DNA fragments are purified by gel electrophoresis separation visible in an ethidium bromide-stained agarose gel under UV light made.

The results are summarized in Table 1.

Example 9 Stimulation of CD8 ⁺ T cells against the KOC protein and lysis of carcinoma cells expressing the KOC protein (A) Stimulation of CD8 ⁺ T cells against the KOC protein

Peripheral mononuclear cells are obtained from a healthy donor and subjected to a so-called ELISPOT analysis. The principle of this experi Mentes is that lymphocytes in culture vessels with specific antigens be stimulated. If the lymphocytes are activated, because they are the When they recognize antigens, the activated lymphocytes release cytokines, theirs on the one hand bind to specific antibodies which are present on the bottom surface of the culture vessels are immobilized. After washing out the lymphocytes you can then the bound cytokines in the culture vessels with the help of a second Antibody that is made visible in a subsequent color reaction, be detected.

Peripheral blood lymphocytes (PBL) were purified from an HLA-A0201 positive healthy volunteer by density centrifugation over a Ficoll Paque® gradient. T lymphocytes were obtained by separating the B lymphocytes or monocytes with the aid of antibody-coupled magnetobeads (CD11, CD16, CD19, CD36 and CD56) (Pant T cell isolation Kit®, Milteny, Bergisch Gladbach, Germany). ^{Approximately 2 × 10 7} T cells were obtained from 30 ml of blood.

HLA-A0201 restricted peptides of the KOC protein were determined using a Soft waresystem of the NIH (NIH bioinformation service [http: /bimas.dcrt.nih.gov.cgi bin / molbio / ken_pa rker_comboform). It was about the after standing peptides:

9mer peptides: 10mer peptides: 199 RLLVPTQFV 33 FLVKtGYAFV 280 KILAHNNFV 142 FQLEnFTLKV 552 KIQEILTQV 26 KIPVsGPFLV

The isolated T cells were incubated with T2 cells which (a) had been loaded with a mixture of the above 9-mer peptides (10 ug / peptide) and (b) with a mixture of the above 10-mer peptides (10 ug / peptide) was. The T cells were restimulated weekly for 6 weeks. In each case 10 ⁷ T cells were co ^{-cultivated with 2 × 10 6} peptide-loaded T2 cells in 24 well plates.

The reactivity to the peptide-loaded T2 cells was weekly determined starting on day 0 of the experiment by using an IFN-γ Elispot analysis was carried out. On day 28, reactivity was determined by the Mixture of (a) (400 specific cells per million cells) observed. the The main reactivity was against peptide 199 (1000 specific cells / 1,000,000 cells). Weaker activity was observed against the Ge mixed of (b) (150 specific cells / 1,000,000 cells) observed. here peptide 33 showed the highest reactivity (600 specific cells / 1,000,000 Cells).

It thus became clear that CD8 ⁺ T cells activated against the KOC protein can be stimulated.

(B) Lysis of carcinoma cells expressing the KOC protein

After a further restimulation, the activated CD8 ⁺ T cells were incubated with the HLA A0201 + pancreatic carcinoma cells PancI, which express the KOC protein. The kidney cells HEK293, which do not express the KOC protein, were used as controls. 10 ⁶ PancI cells were labeled with ⁵¹ Cr (100 μCi) for 1 h at 37 ° C. and ^{cocultivated with increasing numbers of activated CD8 +} T cells for 3 hours. The specific lysis of the PancI cells was determined by the amount of radioactivity released in the supernatant.

It was found that PancI cells ^{are lysed by the activating CD8 +} T cells, but not by the control cells HEK293.

Table 1

Claims (28)

1. DNA sequence of the KOC promoter, comprising the nucleic acid sequence shown in Fig. 9 or a nucleic acid sequence deviating therefrom, the biological function of which corresponds essentially to that of the KOC promoter. 2. Medicament containing the DNA sequence according to claim 1 or a DNA sequence which codes for an RNA-binding protein KOC with the amino acid sequence shown in FIG. 3. Medicament according to claim 2, wherein the DNA sequence contains the nucleic acid sequence shown in FIG. 4. Medicinal product containing a DNA sequence which codes for a protein with the biological properties of an RNA-binding protein KOC encoded by the DNA sequences according to claim 2 or 3:

• a) which differs from a DNA sequence of claim 3 in the Codonse sequence due to the degeneration of the genetic code;
• b) which hybridizes with a DNA sequence of claim 3 or 4 (a); or
• c) which is a fragment, an allelic variant or another variant of the DNA sequence of claim 3, 4 (a) or 4 (b).

5. Medicament containing an expression vector containing a DNA sequence according to any one of claims 1 to 4. 6. Medicaments containing the RNA-binding protein KOC or a protein with its biological activity, which is determined by the DNA sequence after a of claims 2 to 4 is coded. 7. Medicament according to one of claims 2-6, wherein the protein and / or the DNA sequence in one for immunization of an individual the appropriate amount. 8. Diagnostic method for detecting one with the expression of KOC associated tumor, in which a sample with a specific Hybridization with one of the DNA sequence according to one of the An Proverbs 2 to 4 transcribed mRNA suitable probe or a Primer or a KOC protein according to claim 6 or an antibody against the KOC protein defined in claim 6 or a fragment brings into contact with it and then directly or indirectly determines whether the concentration of KOC, KOC mRNA, KOC protein and / or anti-KOC antibodies in the sample compared to a control sample differentiate. 9. Diagnostic method according to claim 8 for differential diagnosis between chronic pancreatitis and pancreatic cancer. 10. Diagnostic method according to claim 8 for diagnosing premalignant lesions with unclear spatial demands. 11. Diagnostic method according to claim 8 for risk stratification in prema ligamentous lesions. 12. Diagnostic method according to claim 8 for evaluating therapeutic success gene. 13. Diagnostic kit for performing the method according to claim 7 or 8, the one for specific hybridization with one of the DNA Sequence according to one of claims 2 to 4 transcribed mRNA A suitable probe or a primer or a KOC protein according to claim 6 and / or an anti-KOC antibody or a fragment thereof. 14. Use of a compound defined in one of claims 2 to 6 for the artificial induction of the pluripotency of body cells. 15. Use of a compound defined in one of claims 2 to 6 dung for the production of tissues or organs via the differen of stem cell populations. 16. Use of a compound defined in one of claims 2 to 6 application for high-dose chemotherapy. 17. Use of a compound defined in one of claims 2 to 6 application for the improvement of ex vivo expansion of hemotopoietic Stem cells. 18. Use of a compound defined in one of claims 2 to 6 for the improvement of engraftment in allogeneic bones market transplants. 19. Use of a compound defined in one of claims 2 to 6 dung for slowing down and / or reversing by physiologi cal or physical noxae induced aging processes. 20. Use of a compound defined in one of claims 2 to 6 dung to produce a prophylactic effect in chemo- or Radiotherapy. 21. Use according to claim 20 for producing a prophylactic Effect on radiation-exposed skin. 22. Use of a compound defined in one of claims 2 to 6 dung for the regeneration of skin defects, skin cosmetics or for accelerated wound healing 23. Use of a compound defined in one of claims 2 to 6 application for immunizing an individual against malignant tumors and their preliminary stages. 24. Use of the DNA sequence of the KOC promoter or one of them different nucleic acid sequence according to claim 1 for isolation and / or enrichment and / or selective propagation of strain cells. 25. Use of the DNA sequence of the KOC promoter or one of them different sequence according to claim 1 for determining the difference degree of sophistication or dedifferentiation of a cell or tissue esp. 26. Use of the DNA sequence of the KOC promoter or one of them different nucleic acid sequence according to claim 1 for gene therapy cancer treatment. 27. Diagnostic kit for use according to claim 25, the one for specific hybridization with a DNA sequence according to claim 1 suitable probe or primer contains. 28. A method for identifying compounds which bind to the DNA sequence of the KOC promoter according to claim 1 and which modulate its activity, the method comprising the following steps:

• a) bringing a DNA sequence according to claim 1 into contact with a test compound in a cellular assay; and
• b) Determination of the modulation of the activity of the KOC promoter, a modulation indicating that the test compound is active.