ES2213429B1 - USE OF THE GEN SLUG AS A GENETIC MARKER OF FUNCTIONS MEDIATED BY SCF (STEM CELL FACTOR) AND APPLICATIONS. - Google Patents

Abstract

The Slug gene mediates the functions of the SCF ligand and its c-kit receptor, so it can be used both the Slug gene, the cDNA of that gene, the Slug protein as drugs or substances that activate the expression of the Slug gene, as an agent therapeutic in the mobilization of hematopoietic stem cells for transplantation or gene therapy, in the ex vivo expansion of hematopoietic stem cells, and / or in the treatment of male sterility problems.

Description

Use of the Slug gene as a genetic marker of functions mediated by SCF (stem cell factor) and applications.

Field of the Invention

The invention relates to the use of the Slug gene as a genetic marker of functions mediated by SCF (stem cell factor) and as a therapeutic agent, both of the Slug gene, the cDNA of said gene, the Slug protein and of drugs or substances that activate the Slug gene expression, in the mobilization of hematopoietic stem cells for transplantation or gene therapy, in the ex vivo expansion of hematopoietic stem cells, and / or in the treatment of male sterility problems.

Background of the invention

Hematopoiesis is a process that produces replacement of both hematopoietic progenitor cells and of mature blood cells from a reserve of pluripotent stem cells. Daily cell production blood in a normal adult is precisely regulated, partly involving a complex interaction between cytokines stimulants and inhibitors, soluble and membrane bound, and their corresponding receivers. The molecular donation of these hematopoietic growth factors and their receptors has constituted an effective instrument to trace the routes that lead from a single stem cell hematopoietic to various terminal differentiated cells in the system hematopoietic

Although numerous cytokines have effects on progenitor cells and stem, in vivo or in vitro , a cytokine discovered in the early 1990s, identified as c-kit, appears to have unique and non-redundant activities on primitive / progenitor cells (Witte, 1990, Cell, 63: 5-6). The in vivo functions of c-kit are well understood due to the existence of mutant mice in which the genetic code of the receptor (c-kit) and / or its respective ligand (SCF) are defective. Mutations in the c-kit receptor and its ligand (SCF) are well represented by the existence of numerous mutant alleles with white spots (W) and Steel (S1), respectively.

Mice suffering from mutations in the W locus were originally identified by the presence of white spots on pigmented mice (Silvers, 1979, "Dominant spotting, patch, and rump-white", in Silvers WK (eds): The coat colors of mice : a model for mammalian gene action and interacion. New York, NY, Springer-Verlag, p 206). The detailed examination of the mice showed that the mutation was pleitropic. W mice also suffered defects in germ cell development and hematopoiesis (characterized by macrocytic anemia). Subsequently, it was shown that the W locus encoded a tyrosine kinase receptor known as c-kit (Nature, 1988, 335: 88; Cell, 1988, 55: 185).

Prior to the discovery of the W locus , a mutation (S1) was identified in mice that had a phenotype almost identical to that of the W mice (Sarvella and Russell, 1956, J. Hered. 47: 123). Since mutations in two different chromosomes had the same complex phenotype, which affected pigmentation, germ cells and hematopoiesis, it was hypothesized that some relationship between proteins encoded in those two loci should emerge. In 1990, the protein encoded in the S1 locus was identified and referred to as mast cell growth factor, stem cell factor (SCF), and c-kit ligand (Cell, 1990, 63: 203; Cell, 1990, 63: 167; Cell, 1990, 63: 175; Cell, 1990, 63: 213).

Although the primary function of the SCF in the Early hematopoiesis could be to induce the growth of inactive progenitor / stem cells through interactions synergistic with other early acting cytokines, there are also ample evidence that SCF, in the absence of other cytokines, selectively stimulates viability before proliferation of murine progenitor cells. Although the route SCF / c-kit migration and development are fine documented, little is known about molecular mechanisms that provide biological specificity to the signaling path SFC / c-kit in the formation and migration of various cells from the bone marrow.

The biological events controlled by the SCF / c-kit signaling path remind those that take place in epithelial-mesenchymal transitions (EMT) in mammalian development. In fact, the process of mesoderm formation involves the acquisition of migratory properties and the determination of cell fate. These EMTs are controlled by a conserved family of "zinc finger" type proteins, the Snail family, in fact, the Drosophila Snail gene is vital for the formation of the mesoderm and the fate of cell migration. The related murine genes (Snail and Slug) have also been presented as participants in the formation of mesoderm and cell migration.

The use of SCF in the mobilization of hematopoietic stem cells for transplantation or gene therapy, and / or in the ex vivo expansion of hematopoietic stem cells has important side effects and has been limited by its mast cell activating properties.

It has now been discovered that the route of SCF / c-kit signaling specifically induces expression of a member of the Snail gene family, the Slug gene, both in natural c-kit + cells and in artificially created cells.

Compendium of the invention

The invention faces, in general, the problem of finding an alternative method for mobilizing hematopoietic stem cells for transplantation or gene therapy, and / or in ex vivo expansion of hematopoietic stem cells, which lacks the drawbacks previously mentioned in relationship with the use of SCF.

The solution provided by this invention is based on the identification of the Slug gene as responsible for functions of the c-kit receptor and its SCF ligand. Indeed, it has now been discovered that the signaling path SCF / c-kit specifically induces the expression of Slug gene, a member of the Snail gene family, both in cells natural c-kit + as in the created cells artificially As a consequence of gene identification Slug as the gene that mediates the functions of the SCF ligand and its c-kit receptor, said Slug gene can replace the SCF in its functions and applications with the exception that it lacks of the side effects of SCF derived from the activation of mast cells

Therefore, in view of SCF applications, an object of this invention is the use of the Slug gene, the cDNA of said gene, the Slug protein or substances that activate the Slug gene expression, in the elaboration of a composition pharmaceutical for the mobilization of hematopoietic stem cells for transplantation or gene therapy.

A further object of this invention is a method for ex vivo expansion of hematopoietic stem cells.

Another additional object of this invention is constitutes the use of the Slug gene in the elaboration of a pharmaceutical composition for the treatment of problems of male sterility

The pharmaceutical composition comprising the gene Slug, the cDNA of that gene, the Slug protein or substances that activate the expression of the Slug gene constitutes an additional object of this invention.

Brief description of the figures

Figure 1 illustrates the fact that activation of the c-kit receiver by SCF specifically induces Slug's expression. Slug and Snail's expression was analyzed by RT-PCR in LAMA 84 cells (panels A and B) and in Ba / F3 cells designed to express c-kit (panels C and D) in the absence and in the presence of SCF. The products of the PCR were transferred to a nylon membrane and analyzed by hybridization with internal oligonucleotide probes, labeled on the ends, specific for each gene (it was used β-actin to verify integrity and loading of cDNA).

Figure 2 shows the defects in the pigmentation v in the testicles of mice Slug-deficient. Figure 2A is a photograph of a homozygous mutant mouse for Slug with the characteristic mark of a white guide on the forehead. Figure 2B shows the histological analysis results of mouse testicles wild type and mice with the Slug mutation. The sections paired testicles of 6-week-old wild-type mice (Slug  + / +), heterozygous mice (Slug +/-), homozygous mice Slug (- / -), Steel mutant mice (S1 / S1) and W mutant mice (W / W v) were stained with hematoxylin and eosin (H&E). There is a general reduction in the size of the canaliculi seminifers in Slug - / - mice, a feature that also can be verified in Slug +/- mice (Slug +/- center versus Slug  +/- right). In the interstitial space of Slug mice - / - exists a reduced amount of Leydig cells. On the contrary, the interstitial space in the testicles of the W / W v mice and S1 / S1 increases disproportionately and is filled with Leydig cells.

Figure 3 shows the developmental defects of Erythroids in Slug-deficient mice. The figure 3A shows the result of the histological examination of the spleens of non-pregnant mice versus the spleens of pregnant control mice 12-day (Slug + / +), heterozygous Slug mice (Slug +/-) and homozygous Slug mice (Slug - / -). Staining with H&E evidenced, during pregnancy, a huge increase in pulp red of the spleen of Slug + / + mice, in which the white pulp of the spleen, marked with white arrows, was very small. He increase in red pulp of the spleen was much less evident in the Slug +/- and - / - mice. Figure 3B shows the result of representative analysis of c-kit + cells present in the bone marrow (BM) and in the spleen of mice after the induction of hemolytic anemia with phenylhydrazine. The cells isolated from a wild-type control mouse (Slug + / +), a mouse heterozygous mutant (Slug +/-), a homozygous mutant mouse (Slug - / -), a Steel mutant mouse (S1 / S1 d) and a mutant mouse W (W / W v) were stained with the monoclonal antibody PE-CD 117 and analyzed by flow cytometry. Be indicates the percentage of c-kit + cells.

Figure 4 illustrates the deficit development of T cells and apoptosis in the thymus of mice Slug-deficient. A histological examination was performed. in sections of the thymus of mice, 4 weeks old, Steel mutants (S1 / S1 d), W (W / W v) mutants, wild type (control) and homozygous mutant mice (Slug - / - mice). All sections were stained with H&E. The paired sections of Slug - / - mice were subjected to DAPI or TUNEL verification. Increased apoptosis in Slug-deficient animals correlated with thymus atrophy. On the left it shows a representative analysis of the cells present in the thymus of these mice. Cells isolated from a wild type mouse (control), a heterozygous mutant mouse (Slug +/-), a mutant mouse homozygous (Slug - / -), a Steel mutant mouse (S1 / S1) and a mouse mutant W (W / W v) were stained with monoclonal antibodies v se analyzed by flow cytometry. The percentage of cells.

Figure 5 illustrates the development of B cells, Myeloids and mast cells in Slug-mutant mice. Be show the results of the representative analysis of the cells B and myeloids present in the spleen (Figure 5A) and in the bone marrow (BM) (Figure 5B), respectively. Isolated mouse cells wild type (control), a heterozygous mutant mouse (Slug +/-), a homozygous mutant mouse (Slug - / -), a Steel mutant mouse (S1 / S1) and a mutant mouse W (W / W v) were stained with the monoclonal antibodies and analyzed by flow cytometry. Be Indicates the percentage of cells. Figure 5C shows what results of histological examination performed on ear sections of 4-week-old mice of the homozygous mutant type (Slug - / -) and mutant W (c-kit - / -). All sections were Dyed with Giemsa. Arrows indicate the presence of mast cells in Slug-mutant mice - / - but not in mice W. mutants

Figure 6 shows that the defect in mice Slug-mutants is intrinsic to the stem cell. The Figure 6A shows the result of kit a immunoprecipitations from isolated mast cells probed for kit and re-probed for phosphotyrosine. Slug +/-: mice heterozygous, Slug - / -: homozygous mice, Slug + / +, mice wild guy Figure 6B shows the result of the analysis of the hematopoietic system in reconstituted normal recipient mice with Slug - / - HSC by FACS. Marrow, spleen and thymus samples were stained with monoclonal anti-bodies and analyzed by flow cytometry. Hematopoietic composition It is similar to that of Slug - / - mice. On the left you see a representation of the c-kit cells present in the bone marrow (BM) and in the spleen of normal recipient mice reconstituted with Slug - / - HSC after induction of anemia hemolytic with phenylhydrazine.

Figure 7 illustrates the signaling path of SCF / c-kit in development. Figure 7A shows c-kit + cells present in the bone marrow (BM) and in the spleen of wild-type (control) mice, mice Steel (S1 / S1 d) mutants and W (W / W v) mutant mice after induction of hemolytic anemia with phenylhydrazine. For the separation and classification of cells, these are incubated with c-kit-PE cells and the c-kit + cells were classified by fluorescent activation (FACS) (FACstar, Becton Dickinson). The classified cells were again analyzed to determine its degree of purity with the cytometer, obtaining a degree of purity ≥ 95%. Figure 7B shows the result of the Slug expression, analyzed by RT-PCR, in the purified c-kit + cells from bone marrow and from spleen of control mice, S1 / S1 and W / W v. PCR products were transferred to a nylon membrane and analyzed by hybridization with an internal oligonucleotide probe, labeled in the end, specific for the Slug gene (upper panel). It was used  β-actin to verify integrity and cDNA load (bottom panel). Figure 7C is a scheme illustrative of the SCF / signaling path function model c-kit in development. The signaling route SCF / c-kit affects the development of three populations of cells: melanoblasts, hematopoietic stem cells and cells germinals The data shown indicates that Slug mediates the functions of the c-kit receptor and its ligand in melanoblasts and in hematopoietic cells. Inside the cells germinals, the SCF / c-kit function is mediated both by Slug as per PI3-kinase.

Detailed description of the invention

The invention relates, in general, to the use of the Slug gene as a genetic marker of functions mediated by SCF and as a therapeutic agent of the Slug gene, the complementary DNA (cDNA) to the RNA encoding the transcription or expression product of said gene Slug (hereinafter, cDNA of said Slug gene), the expression and translation product of the Slug gene (hereinafter, Slug protein) and drugs or substances that activate the expression of the Slug gene, in the mobilization of hematopoietic stem cells for transplantation or gene therapy, in the ex vivo expansion of hematopoietic stem cells, and / or in the treatment of male sterility problems.

The Slug gene is a gene present in vertebrates which encodes a transcription factor of the type "fingers of zinc "involved in transitions epithelial-mesenchymal (Nieto et al., Science 264: 835-849 (1994)). Now it has been discovered, surprisingly, that the Slug gene is responsible for the functions of the c-kit receptor and its SCF ligand and, consequently, said Slug gene, as well as the cDNA of said gene Slug, the Slug protein and drugs or substances that activate the Slug gene expression, can be used in them applications that SCF with the caveat that lack the effects SCF side effects derived from mast cell activation.

In order to know the molecular mechanisms that provide biological specificity to the signaling path SFC / c-kit in the formation and migration of various cells from the bone marrow, the relationship has been investigated between the SCF / c-kit signaling path and the Snail protein family, being discovered. surprisingly, that SCF / c-kit signaling path induces specifically to the expression of the Slug gene, both in cells natural c-kit + as in created cells artificially The analysis of a null directed mutation that removing all coding sequences from Slug revealed that the Slug-mutant mice, as well as mice defective c-kit and SCF, have a complex phenotype which includes pigmentation, gonadal and hematopoietic

In the sense used in this description the term "Slug-mutant mice" refers to mice with a phenotype other than the original (wild type, Slug + / +) due to a mutation in the Slug gene, and includes both mice heterozygous mutants (Slug +/-) as to mutant mice homozygous (Slug - / -).

Long-term transplant experiments showed that the effect in Slug-mutant mice, in which the c-kit + cells of mice Slug - / - have an SCF / c-kit signaling path functional, they presented migratory defects similar to those of c-kit + cells in S1 / S1 and W / W v mice, and It is intrinsic to the cell. Since mutations in the SCF, in the c-kit receptor and in the Slug gene have a phenotype similar pleotropic that affects pigmentation, cells germinals, and at hematopoiesis, the hypothesis that There would be some relationship between them. In fact, two different data they demonstrated the relationship: first, the primary cells c-kit + purified from the mice of control express Slug; and secondly, the Slug is not expressed in the primary c-kit + cells derived from W / W v and S1 / S1 mice. These two results together identify to the Slug gene as the molecular target that provides specificity biological to the SCF / c-kit signaling path.

The invention provides a composition. pharmaceutical comprising the Slug gene, the cDNA of said Slug gene, Slug protein and / or one or more drugs or substances that activate the Slug gene expression, along with one or more excipients pharmaceutically acceptable. The Slug gene, the cDNA of said Slug gene and the Slug protein can be obtained by conventional techniques of genetic engineering [see Example 1]. Drugs or substances which activate the expression of the Slug gene can be obtained by techniques conventional ones comprising, for example, the preparation of DNA constructs comprising the Slug gene or the cDNA of said Slug gene and a reporter gene that when it comes in contact with the drug or substance to be tested allows to determine whether said drug or substance activates the expression of the Slug gene. By way of illustration, said reporter gene may be the gene that codes for a protein for which specific antibodies are available, or a protein with enzymatic activity, for example, alkaline phosphatase, β-glucoronidase, GFP, etc., detecting said activity by adding the substrate or developing system correspondent.

The excipients that may be present in the pharmaceutical composition of the invention will depend, among others things, of the form of administration of said composition Pharmaceutical A review of the different forms of administration  of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galénica, C. Faulí i Trillo, Luzán 5, S.A. of Editions, 1993

When the pharmaceutical composition of the invention contains the Slug gene or the cDNA of said Slug gene, the pharmaceutical composition will comprise vectors or systems that they help the process of transferring an exogenous gene to a cell, facilitating intracellular delivery and bioavailability of it so that it can work properly. By way illustratively, said vectors may be viral vectors, by for example, based on retroviruses, adenoviruses, etc., or non-viral such as DNA-liposome complexes, DNA-polymer, DNA-polymer-liposome, etc. [see "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)].

As is known, both bone marrow transplantation and gene therapy strategies use hematopoietic stem cells as target cells. The number of hematopoietic stem cells in peripheral blood is limited, so it is necessary to mobilize hematopoietic stem cells from the bone marrow to the peripheral blood for different purposes, such as bone marrow transplantation, gene therapy, ex vivo manipulation of hematopoietic stem cells , etc. It has now been found, surprisingly, that the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of the Slug gene can effect said mobilization of hematopoietic stem cells.

Therefore, in another aspect, the invention is refers to the use of the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of Slug gene in the preparation of a pharmaceutical composition for hematopoietic stem cell mobilization for transplantation or gene therapy. The mobilization of these stem cells Hematopoietic is performed in the transplant recipient or in the patient undergoing gene therapy.

The Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of the Slug gene can allow the ex vivo survival of hematopoietic stem cells, which favors their maintenance and manipulation.

Therefore, in another aspect, the invention provides a method for ex vivo expansion of hematopoietic stem cells comprising the use of the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate expression. of the Slug gene. More specifically, said method comprises the administration to a culture of hematopoietic stem cells of my effective amount of the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of the Slug gene, or of a composition containing said products.

On the other hand, as is known, the decrease of Leydig cells generates fertility problems, in particular, of male sterility. Now it has been observed, surprisingly, that the Slug gene favors cell migration and / or survival of Leydig, so the administration to a male patient affected by sterility, in need of treatment, of a pharmaceutical composition provided by this invention that it comprises the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of the Slug gene, together with one or more pharmaceutically acceptable excipients, it can solve certain problems of male sterility.

Therefore, in another aspect, the invention is relates to the use of the Slug gene, the cDNA of said Slug gene, the Slug protein, and / or drugs or substances that activate the expression of Slug gene, in the preparation of a pharmaceutical composition for treatment of male sterility problems in particular for the treatment of male sterility due to a Leydig cell decline.

The invention will be illustrated below by an essay illustrating the induction of Slug expression by the c-kit receptor activation by SCF.

Example 1 Induction of Slug expression by receptor activation c-kit by SCF I. Materials and Methods Cell culture

The cell lines used include cells LAMA-84 and Ba / F3. The cells were kept in a modified Dulbecco Eagle medium (DMEM) supplemented with 10% of fetal calf serum (FCS). When necessary, it was added as source of interleukin 3 (IL-3) a medium WEHI-3B conditioned. The Ba / F3 cells that express the wild type of c-kit were obtained as It is indicated below. The PECE-kit plasmid, which contains the complete coding sequence of the cDNA of c-mouse kit (donated by Dr. D. Martín-Zanca), was used to transfect the line pro-B cell IL-3-dependent. The co-transfection with a plasmid resistant to neomycin (MCI-neo) and the primary selection with the Neomycin analog G418 generated a stable cell line Ba / F3 which expresses c-kit (Ba / F3 + c-kit). Ba / F3 cell populations stained monoclonal antibody CD117 specific to c-kit.

Mice

The animals were caged in conditions not Sterile in a conventional animal facility. The rats heterozygous and homozygous for the mutation Slugh1 Delta generated by sequence suppression genomics of the entire coding region of the Slugh protein (Slug? 1 mutant mice) have been described previously (Jiang et al., 1998, Developmental Biology 198: 277-285). The W / W v and S1 / S1 mice and the breeding pairs were obtained from Jackson Laboratory (Bar Harbor, Maine). In experimental mice, it was injected intraperitoneally phenylhydrazine (PHZ; 60 mg / kg weight bodily; Sigma Chem) for two consecutive days (Broudy et al., 1996, Blood 88: 75-81). In each of the three days after the second injection of PHZ, 5 mice died from cervical dislocation, and the bones and spleens were removed (under sterility conditions) for further in-depth analysis. All procedures were approved by the committee Animal care institutional.

Phenotypic cell analysis

Cellular morphology was analyzed according to standard criteria Unicellular suspensions were prepared at from individual tissues, including bone marrow, spleen, thymus and peripheral blood by standard procedures (García-Hernández et al., 1997, PNAS). For the most stains were used approximately 1 x 10 6 cells. Cell phenotypes were immunized with the following antibodies: conjugated phycoerythrin (PE) -TER1 19 [Ly-76, an antibody monoclonal that recognizes an antigen expressed in erythroid cells from erythroblasts to erythrocytes]: PE-CD4, PE-Gr-1, PE-CD117; PE-CD19: PE-B220, conjugated fluorescein (FITC) -CD8, FITC-IgM, FITC-Macl (all from Pharmingen). Cells, suspended in a phosphate buffer saline solution (PBS), without Ca ++ or Mg ++ with a 1% fetal bovine serum supply (v / v), were labeled with each antibody (approximately 1 mug / 10 6 cells) for 30 minutes on ice. The Cellular fluorescence was analyzed with the FACScan cytometric flow (Becton Dickison). Cells incubated with isotopic controls properly marked (Pharmingen) were used to avoid the output of non-specific fluorescence signals. Before the analysis, mature red cells were reduced by rupture hypotonic (0.38% ammonium chloride for 15 minutes on ice). Background controls were treated identically, with the except that the primary antibodies were omitted. Initially, the cells were analyzed by size and by dispersion to identify living cells. In some experiments, cell viability was assessed by exclusion with propidium iodide (5 µg / ml, Sigma) (in cytometry of flow).

Cell purification

Mononuclear suspensions of the spleen are prepared by cutting the spleens into small fragments in 5 ml of PBS solution, without Ca ++ or Mg ++, containing 10% FBS (v / v) and passing the cell suspension through needles progressively smaller. The bone marrow cells are removed from the femurs with a syringe containing 2 ml of PBS with 10% FBS. Low density mononuclear cells of the medulla and spleen were isolated by centrifugation about Ficoll-Paque (P = 1,077 g / m1) at 800 g during 20 minutes at room temperature. For the classification of cells, these were incubated with c-kit-PE and cells c-kit + were classified by a fluorescence activated cell sorter (FACS) (FACstar; Becton Dickinson). The classified cells were re-analyzed with the cytometer to determine purity.

Reverse transcription-Chain reaction of the PCR polymerase (RT-PCR)

To analyze the expression of Slug and Snail in the cell lines and in c-kit + cells purified an RT was performed, according to the manufacturer's protocol in a reaction volume of 20 µl containing 50 ng of hexamers randomized, 3 µg of total RNA, and 200 transcriptase units Reverse Superscript II Rnasa H (GIBCO / BRL). The parameters of thermocycling for the PCR reactions and the sequences of the Specific initiators were the following: mSlug. 30 cycles at 94 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 2 minutes minutes, direct starter (direction): 5'-GCCTCCAAAAAGCCAAACTA-3 '(SEC. ID. N °: 1) and reverse initiator (antisense): 5'-CACAGTGATGGGGCTGTATG-3 '(SEC. ID. N °: 2); mSnail, 30 cycles at 95 ° C for 2 minutes, 60 ° C for 2 minutes, v 72 ° C for 2 minutes, direct starter (direction): 5'-CAGCTGGCCAGGCTCTCGGT-3 '(SEQ. ID. N °: 3) and reverse initiator (antisense): 5'-GCGAGGGCCTCCGGAGCA-3 '(SEQ. ID. N °: 4). The amplification of the β-actin mRNA It served as a control to assess the quality of each RNA sample. The sequences of the internal probes were the following: mSlug, 5'-GACACACATACAGTGATTATTTCC-3 '(SEC. ID. N °: 5) and mSnail, 5'-TGCAACCGTGTTTTGCTGACCGCTCCAAC-3 ' (SEC. ID. N °: 6).

Bone marrow transplant (BMT) and sampling

C57 BL / 6J female receptor mice (from 8-12 weeks of age) were irradiated with two doses divided into 600 cGy separated by two hours. This dose is enough to completely eliminate endogenous hematopoiesis. Be injected BM cells into the tail vein of the mice irradiated at 2-4 x 10 6 cells per mouse to a long-term reconstitution. All receivers are kept in micro-insulating cages with sterilized food and acidified sterile water. The animals were sacrificed and the hematopoietic tissues were collected for analysis FACS

Hematopoietic Colony Assays

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El crecimiento de las colonias derivadas de la unidad formadora de colonias de eitroide (CFU-E) se estimuló únicamente con EPO (2 U/ml). Los cultivos se incubaron a 37°C en una incubadora humidificada que contenía 5% de CO^{2} en el aire y se observaron los resultados a 3 días (para colonias derivadas de CFU-E) o a los 7 días (para colonias derivadas de BFU-E) después de la iniciación del cultivo. La frecuencia de BFU-E y de CFU-E se determinó en los cultivos por triplicado.Bone marrow cells (0.25-1.0 x 10 5 cells / plate) and spleen cells (10 4 -10 5 cells per plate) isolated from normal mice and Slug-mutants were seeded on semi-solid FBS (Stem Cell Technology) free culture plates. Colony growth was stimulated with the following combinations of recombinant growth factors: rat stem cell factor (100 ng / ml; SIGMA), mouse IL-3 (10 ng / ml, SIGMA), and human erythropoietin ( hEPO) (2 U / mL; ROCHE) for the growth of the erythroid branching unit

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The growth of colonies derived from the eitroid colony forming unit (CFU-E) was stimulated only with EPO (2 U / ml). The cultures were incubated at 37 ° C in a humidified incubator containing 5% CO2 in the air and the results were observed at 3 days (for colonies derived from CFU-E) or at 7 days (for colonies derived of BFU-E) after the initiation of the culture. The frequency of BFU-E and CFU-E was determined in triplicate cultures.

Isolation of primary mast cells derived from the bone marrow bone, immunoprecipitation and western-blotting

Bone marrow cells were collected by irrigation of the cavity of the femur marrow, and mast cells were derived by selective growth in a medium containing IL-3 for 6 weeks (Opti-Mem I, GIBCOBRL, 10% fetal bovine serum. 0.5 ng / ml of Recombinant murine IL-3, R&D Systems Inc.). He medium was renewed every day and the cells were transferred to new plates to remove adherent cells, including macrophages and megakaryocytes The immunoprecipitation and western trials blotting were performed using 1 x 10 7 mast cell extracts per band In short, the cells were deprived of food during 12 hours in Opti-Mem I media without IL-3 and containing only 0.5% serum, before stimulate them with 100 ng / ml of murine SCF (R&D Systems Inc.) for 10 minutes at 37 ° C, as indicated. Kit was detected using a goat anti-serum purified by affinity towards the C-terminal end of the receptor murine kit, M-14 (Santa Cruz). Antibody Monoclonal 4G10 (UBI) was used to detect phosphotyrosine.

Histological analysis

Tissue samples were fixed during night in 10% formalin, and then processed, it embedded in paraffin and 6 µm sections were stained with hematoxylin and eosin, were histologically examined and they photographed All sections were taken from portions homogeneous and viable of the cut tissues. The mast cells are They stained with Giemsa. The number of mast cells was determined by square millimeter

TUNEL trial

End-tagging of the terminal dent with dUTP-biotin mediated by deoxynucleotidyltransferase (TUNEL) [from English "Terminal Deoxynucleotidyltransferase-mediated dUTP-biotin" Nick End Labeling] was performed using the in situ dead cell detection kit (Boehringer Manhein) , essentially following the manufacturer's instructions although with minor modifications depending on the type of preparation. Briefly, subsequently sections were fixed for 15 minutes in 4% paraformaldehyde, washed twice with PBS, and incubated in a 2: 1 mixture of ethanol and acetic acid for 5 minutes at -20 ° C. After two washes in PBS, the sections were digested in proteinase K (10 µg / ml in 10 mM Tris HC1, pH 8.0 and 1 mM EDTA), washed twice with PBS and counter-colored with green methyl.

II. Results Induction of Slug expression by receptor activation c-kit by SCF

The ability of the c-kit receptor to stimulate the expression of the Snail family members was mainly tested on naturally expressed c-kit + cells, using the LAMA 84 cell line (Figure 1A). As shown in Figure 1B, Slug expression rapidly increased in LAMA 84 cells treated with SCF. However, Snail's expression level was not modified in the presence of SCF. To some extent, these previous data indicate the ability of LAMA 84 cells treated with SCF to specifically activate Slug gene expression. Ba / F3 cells lacking endogenous c-kit (Palacios and Steinmetz, 1985, Ceil 41: 727) were manipulated to express a wild-type and full-length c-kit receptor (Ba / F3 + c-kit) (Figure 1 C). C-kit-transfected cells specifically expressed Slug with SCF stimulation (Figure ID). However, the Snail gene was expressed at similar levels in Ba / F3 + c-kit cells not stimulated by SCF and in Ba / F3 + c-kit cells stimulated by SCF. These experiments demonstrate that c-kit activation specifically induces Slug expression, thus indicating a clear relationship between c-kit / SCF activation and Slug expression. Because mutations in two different genes, the c-kit receptor and its ligand (SCF), have the same complex phenotype that affects pigmentation, germ cells and hematopoiesis, mice lacking the gene were thoroughly analyzed Slug to determine if the functions of the c-kit / SCF route in vivo were mediated by Slug.

Pigmentation, gonadal and hematopoietic defects in Slug-mutant mice

The most obvious phenotype of the SI and W mutants in vivo is the presence of severe dwarfism, which is observed shortly after birth. This characteristic is also observed in mice that carry a null mutation of the Slug gene (homozygous mutant mice Slugh? 1) that appeared considerably smaller than their bait partners (Jiang et al., 1998, Developmental Biology 198277 -285). As in the defective c-kit and SCF mice, the growth retardation of the homozygous mutant mice Slugh? 1 happened in the first three weeks of life. Therefore, it was then studied whether the Slug gene, such as the c-kit receptor and its ligand (SCF), are also important in dermal, gonadal and hematopoietic development.

1. Pigmentation deficiencies

Melanoblasts originate from the neural crest pluripotent and migrate along some characteristic roads. Both for their survival and for their migration they depend on numerous signaling systems (Ling et al., 2000, Development 127: 5379-5389). Heterozygous mutant mice (W / + or S1 / +) have a characteristic white spot on the forehead and additional areas of depigmentation in the ventral, tail and legs. Homozygous mutant mice (W / W v or S1 / S1) are much more affected and completely lack pigmentation of the skin and hair, whose melanocytes are derived from the neural crest.

Heterozygous mice for Slug do not they presented pigmentation alterations. However, the mice homozygous mutants for Slug had a diluted coat with areas additional depigmentation on tails and legs, and the characteristic white spot of the forehead (Figure 2A). These Dermal defects in Slug - / - mice consisted of several degrees of depigmentation.

However, the retina and the inner layer of the iris, whose melanocytes came from the optic hollow and are independent of the SCF / c-kit signaling path,  are systematically pigmented in Slug - / - mice. These Dermal defects observed in Slug - / - mice are similar to the dermal phenotype observed in the W / + and SU + mice and suggest a role of the Slug gene in the development of melanocytes coming from the neural crest.

2. Gonadal deficiencies

Slug-deficient females were Fertile and the ovaries appeared normal. Most males Slug - / - were also fertile. Although they seemed to copulate with normal, as indicated by the formation of vaginal plugs, more 15% failed to induce pregnancy in their partners. Those Slug - / - mice capable of procreating produced small litters (3-6 pups unlike a normal litter of 10-12 offspring). The size and weight of the testicles in the mutants - / - was reduced by approximately 40% compared with the wild-type bait mates. The sections histological testicles of 6-week-old mice Slug-deficient revealed that atrophy testicular came from a global reduction in tube size seminal, characteristic that can also be observed in some heterozygous mice for Slug (Figure 2B). However, the sperm were visualized in the lumina, according to the fact that fertility was not seriously compromised in These animals. Histological analysis also revealed a number reduced Leydig cells in the interstitial space of the Slug-deficient mice (Figure 2B). For him on the contrary, the interstitial space of the testicles of mice W / W v and S1 / S1 is disproportionately increased and full of Leydig cells. Therefore, the Slug gene has a function in the germ cell development of males but their loss it is insufficient to completely compromise the production of sperm cells

3. Hematopoietic deficiencies

Mice with SCF null mutation and c-kit have severe hematopoietic deficiencies. SCF acts in hematopoietic progenitor cells, where it has observed an increase in survival rather than recruitment within the cell cycle Therefore, the function was analyzed of Slug in normal hematopoiesis.

3.1 Macrocytic anemia in Slug mice - / -

Anemia is the most prominent hematopoietic phenotypic anomaly observed in S1 and W mutants in vivo and is responsible for growth retardation during the first weeks of life, a characteristic shared with Slug-deficient mice. Therefore, blood parameters in the Slug - / - mutant mice were then examined. The hematological parameters examined, in particular, hemoglobin (HGB), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), define a macrocytic anemia (Table I), an aspect of S1 and W mice, and of human phenotypes of piebaldism ("piebald"), which are due to loss of function mutations that occur naturally in the c-kit receptor or its SCF ligand, respectively.

TABLE I Hematological parameters of the peripheral blood of Slug + / +, +/- and - / - mice

 \ hskip-20mm Genotype - / - +/- + / + RBC (x 10 6 / \ mul) 8.3 ± 1.1 9.15 ± 1.0 10.2 ± 0.6 HGB (g / dl) 11.9 ± 1.6 14.8 ± 1.4 15.2 ± 0.9 HCT (%) 37 ± 3.7 48.3 ± 4.1 48.3 ± 3.4 MCV (fl) 55.5 ± 4.1 48.2 ± 3.1 49.3 ± 2.9 MCH (pg) 19.4 ± 1.4 18.3 ± 1.2 18.5 ± 1.1 Mchc (g / dl) 34.9 38.9 ± 3.5 36.7 ± 3.3 35.6 ± 3.2 RDW (%) 15.2 ± 1.3 12.2 ± 0.9 12.4 ± 1.1 Plaq (x103 / \ mul) 422 ± 24.5 437 ± 30.8 445 ± pm 34.4 MPV (fl) 5.2 ± 0.3 5.5 ± 0.3 5.4 ± 0.4 Wbc (x103 / \ mul) 8.4 ± 1.0 9.07 ± 1.2 11.5 ± 1.4 Neu (% N) 64.6 ± 3.9 65.9 ± 4.3 66.2 ± 4.7 Lym (% L) 35.4 ± 2.2 33.6 ± 2.9 33.8 ± 2.8 Monkey (% M) N.D. N.D. N.D. Eos (%AND) N.D. 0.5 N.D. Base (% B) N.D. N.D. N.D. Mean value ± SEM (standard deviation of the sample, for n = 10); RBC: erythrocytes; HGB: hemoglobin; HCT: hematocrit; MCV: average erythrocyte volume; MCH: hemoglobin middle corpuscular; MCHC: mean corpuscular hemoglobin concentration; RDW: amplitude of erythrocyte distribution; Plaq: platelets; MPV: average platelet volume; WBC: leukocytes; Neu: neutrophils; Lym: lymphocytes; Monkey: monocytes; Eos: eosinophils; Baso: basophils; N.D .: not detected.

Next, we analyzed the ability to expansion of erythropoiesis in mice Slug-mutants under hematopoietic stress. The vast expansion of erythropoiesis that takes place in the spleen of mice in response to hemolytic anemia or other situations of hematopoietic stress (during pregnancy) is due to BFU-E migration from the medulla to the spleen. Therefore, first, the effects on the erythropoiesis in the splenic red pulp of mice Slug-mutants during pregnancy. The gestation of Mice is characterized by a transient to medium splenomegaly pregnancy due to a large increase in the number of erythroblasts. This anemia associated with pregnancy is the main reason for the great change in size and cellular content of maternal spleen (Table II). On the contrary, the spleens of the mice Slug-mutants with a 12-day gestation are more smaller than those of control mice (Table II).

TABLE II Weights (grams) of the spleens of Slug + / +, +/- mice and - / - pregnant

Genotype + / + +/- - / - Do not pregnant 0.0726 ± 0.0033 0.0697 ± 0.0048 0.0706 ± 0.0029 Pregnant 0.1379 ± pm 0.0075 0.0864 ± 0.0039 0.0771 ± 0.0029

The histological examination of the spleens showed that the increase in splenic red pulp was much less obvious both in Slug +/- mice and in mice - / - (Figure 3A). The Flow cytometry results of expression analysis of the erythroid marker (TER-119) in cells of the bone marrow and spleen of normal mice and mice Slug-pregnant mutants are shown in the Table III.

         \ newpage
      

TABLE III Frequency (in percentage) of cells TER-119 + in the bone marrow and in the spleen of mice Slug + / +, +/- and - / -, and in mice recovered from induced anemia by gestation

Marrow that is Spleen + / + 7.7 ± 1.5 1.2 ± 0.9 + / + during the gestation 19.1 ± 2.1 18.3 ± 2.9 +/- 7.5 ± 1.7 1.1 ± 0.8 +/- during pregnancy 12 \ pm 2.2 6.0 ± 1.5 - / - 7.9 ± 1.1 1.3 ± 0.7 -/- during the gestation 10 ± 2.1 4.9 ± 1.6

Cell frequency TER-119 + was increased in both the bone marrow and in the spleen during recovery from anemia caused by pregnancy in control mice. On the contrary, the increase in TER-119 + cells resulted affected in Slug-mutant mice both in the Marrow as in the spleen. These results show the poor recovery from anemia due to pregnancy in mice Slug-mutants, indicating a defect in the generation and / or migration of mouse progenitor erythroid cells Slug-mutants. Therefore, then proceeded to quantify the number of BFU-E, which is the  most primitive erythroid progenitor cell, and of CFU-E, which are the most progenitor cells differentiated by colony formation assays hematopoietic in both bone marrow and spleen of control mice as in Slug-mutant mice under conditions physiological (without erythroid stress). The number of BFU-E and CFU-E in mice Heterozygous for Slug was similar to that of control mice. However, the number of BFU-E in bone marrow and the number of CFU-E in the spleen had been reduced by homozygous mice for Slug compared to mice of control. All these results show a baseline defect. Erythroid at BFU-E level in Slug - / - mice. Do not However, basal erythroid development appears normal in the Slug - / - mice, although hematopoietic stress (pregnancy) evidenced little recovery from anemia.

Then the number was quantified of BFU-E and CFU-E in mice Slug-mutants in which it had been induced previously a hemolytic anemia with phenylhydrazine (PHZ). The PHZ injection causes severe destruction of red blood cells followed by an expansion of erythropoiesis. Therefore, mice of the same age were injected with PHZ and its effect was systematically observed around day 3 in mice at PHZ was supplied resulting in a rapid reduction in the hematocrit and an increase in the number of reticulocytes (data not shown). In Slug +/- mice with induced hemolytic anemia by the PHZ, the number of CFU-E was reduced by BM in comparison with control mice and the expansion of the number of BFU-E and CFU-E in the spleen resulted affected (Table IV). Induction of hemolytic anemia with PHZ in Slug - / - mice resulted in an increase in marrow erythropoiesis, but the expected increase in Splenic erythropoiesis was not completely blocked (Table IV). These results show that the demand response Acute erythropoietic results in the expansion of erythropoiesis primarily at the level of BFU-E in Slug-mutant mice. A similar phenotype is observed in W / W v mice. Although spinal erythropoiesis increases around day 3 in the W / W v mice that are given provides PHZ, the expected increase in erythropoiesis Splenic does not occur on day 3. Cytometry results flow of marker expression analysis of c-kit (CD 117) in bone marrow cells and of control spleen, of Slug-mutant mice, mutants S1 and W mutants after induction of hemolytic anemia with PHZ shows that the increase in c-kit cells in the spleen of Slug-mutant mice, S1 / S1 and W / W v It was blocked compared to control mice (Figure 3B).  These results show that the cells c-kit + Slug deficient behave as S1- and W-c-kit + cells and the Erythroid development defect is similar in mice Slug-mutants, W / W v and S1 / S1.

TABLE IV Expansion of BFU-E numbers and CFU-E in the bone marrow (BM) and in the spleen of mice treated with phenylhydrazine (PHZ)

 \ hskip-10mm No. CFU-E (x10 4)  \ hskip-5mm No. BFU-E (x10 <3>) BM Spleen BM Spleen (+ / +) 4.3 ± 0.6 6.6 ± 1.5 4.2 ± 0.5 1.3 ± 0.4 PHZ - (+ / +) 16.2 ± 1.1 256 ± 29 4.4 ± 0.4 25 ± 2.4 (+/-) 3.9 ± 1.3 4.1 ± pm 0.8 4.2 ± 0.3 1.6 ± 0.2 PHZ - (+/-) 4.1 ± 0.9 2. 3 ± 3 4.9 ± 0.6 11 \ pm 0.8 (- / -) 4.0 ± 1.3 2.4 ± 0.5 1.6 ± 0.1 1.5 ± 0.2 PHZ - (- / -) 4.1 ± 1.4 2.0 ± 0.4 4.6 ± 0.3 1.7 ± 9.4 The numbers of BFU-E and CFU-E in the bone marrow and in the spleen of mice treated with PHZ and sacrificed on day 3 they were quantified by Hematopoietic colony formation assays. The values indi- every represent the mean ± SEM of 5 mice in each group.

3.2 T cells in mice Slug-mutants

In mice that lack functional expression Slug, peripheral blood T cell numbers are normal, although the analysis of the thymus composition of mice 4 weeks of age shows a reduction in cell production and differentiation towards CD4 + CD8 + cells that was similar to the mutant mice W and S1 (Figure 4). This specific lock T cell differentiation was also observed in +/- mice. The thymus of Slug - / - mice was small and studied in sections histological Morphological differences were detected between thymus of animals - / - and + / + of the same bait, the appearance being histological similar to the scams of mutant mice S1 and W (Figure 4). In thymus sections of deficient Slug mice, they were observed many cells at the cortical level that seemed to correspond to apoptopic bodies that are not frequently seen in sections of Thymus from wild-type mice (Figure 4). In accordance with this interpretation, a significant increase in TUNNEL-positive cells in thymus sections of Slug-deficient mice. The rise of the apoptosis in Slug-deficient animals was correlated with thymus atrophy. These results are consistent with the idea that SCF stimulates the growth of CD4 - CDS - thymocytes of the primitive mouse but not of the CD4 + CD8 + cells or individual CD4 + cells or CD8 + (J. Immunol 152: 4783, 1994; Cell Immunol. 157: 118, 1994).

3.3 The development of B cells, myeloid cells and mast cells appear normal in mice Slug-mutants

Although the interaction between c-kit and SCF is not necessary for the development of B cells and myeloids in vivo , extensive expression analysis was performed by flow cytometry of markers of cell surface differentiation in spleen and marrow cells Bone of 5-week-old wild-type mice, S1 and W mutants, and Slug-mutant mice. No reduction in myeloid and B cell lines was observed in Slug-mutant mice (Figures 5A-B). Therefore, unlike the important function of the Slug gene, such as the c-kit / SCF interaction, in the generation of erythroid and T-cell lines, the Slug gene does not appear to be necessary for normal B-cell and myeloid development. in adult mice.

As is known, the signaling route SCF / c-kit is necessary for the development of mast cells Therefore, the mast cells of mice were examined Slug-mutants 4 and 8 weeks old in sections histology of different tissues. Could not detect any morphological difference between animal mast cells - / - and + / + of the same litter (Figure 5C). In addition, the number of mast cells in the ear, an organ known to be rich in mast cells was equivalent in animals - / - and + / +. Therefore, the development and mast cell differentiation does not appear to have been affected by the absence of a functional Slug gene.

The defect in Slug-mutant mice is intrinsic to the stem cell

\hbox{c-kit/SCF-R}

era también quinasa activo y autofosforilaba restos de tirosina tras estimulación con SCF (Figura 6A).Since the signaling of the receptor depends on the interaction with the ligand, it is not surprising that the mutant forms of the c-kit receptor and its ligand produce almost identical developmental defects. However, transplant experiments reveal an important difference between two mutations: the hematopoietic stem cells of mice. If they function normally in wild-type receptors, while the same does not occur in those of the W mutants. Consequently, as the absence of the Slug gene affects the development of three stem cell populations: melanoblasts, hematopoietic stem cells and germ cells (such as in both W and SI mutations), it was first analyzed whether the Slug-mutant mice had a normal SCF / c-kit signaling pathway. To ensure that a normal transmembrane tyrosine kinase receptor encoded by c-kit for the SCF (c-kit / SCF-R) was available, the primary bone marrow mast cells of mice of the same age were examined + / +, +/- and - / -. The c-kit / SCF-R pair of mice - / -, +/- and control mice was the same size and expressed at comparable levels (Figure 6A). The pair

 \ hbox {c-kit / SCF-R} 

it was also active kinase and autophosphorylated tyrosine residues after stimulation with SCF (Figure 6A).

To define if the nature of the defect was extrinsic or intrinsic to the stem cell, the capacity was analyzed of hematopoietic stem cells of mice Slug-mutants to reconstitute a hematopoiesis in irradiated guests. Bone Marrow Cell Graft of a normal donor cures the hematopoietic phenotype observed in Slug mice - / -. On the other hand, wild-type receptors lethally irradiated and reconstituted with stem cells Hematopoietic Slug - / - mice had macrocytic anemia and the composition of the hematopoietic system was similar to that of the Slug - / - mice (Figure 6B). These mice had a development normal B-cell and myeloid, a block in the differentiation of T cells towards CD4 + CD8 + cells, and when treated with PHZ c-kit + cells could not migrate to the spleen These results indicate that the defect in mice Slug-mutants is intrinsic to the stem cell.

C-kit + primary cells do not express Slug in the W or S1 mutant mice

The Slug gene favors fundamental functions to promote the development, survival and proliferation of hematopoietic stem cells, those derived from the crest neural, and germ cells, function well illustrated by the reduction of erythroid precursors and associated macrocytic anemia, gonadal defects and hypopigmentation manifested in mice Slug-deficient. The discoveries that the c-kit receptor activation specifically induces  the expression of Slug mice and mice Slug-deficient have a phenotype similar to that of the mutant mice S1 and W, led the inventors to check if Slug gene expression levels are regulated as a consequence of SCF / c-kit activation in control cells against the primary c-kit + cells of S1 and W. Therefore, hemolytic anemia was induced with phenylhydrazine (PHZ) in control mice and in mutant mice S1 and W. On day 3, c-kit cells of the bone marrow and spleen were purified by classifying them into control mice and S1 mutants and W (Figure 7A). Then it was checked if the expression of the Slug gene was also present in c-kit + cells of control mice. The Slug gene expression test by RT-PCR revealed that the Slug gene was present in the primary c-kit + cells derived from the bone marrow and spleen of control mice (Figure 7B). Β-actin expression was used to assess the integrity and burden of each reaction RT-PCR (Figure 7B, lower section). The expression of the Slug gene was higher inside the migratory cells observed in the spleen than in c-kit + cells that were left in the bone marrow. On the contrary, using the same empirical conditions, the expression of the Slug gene in primary c-kit + cells purified from the bone marrow of W and S1 mutant mice. I only know observed expression of the Slug gene in the cells primary c-kit + from the spleen (cells migratory) of mutants W. These results, together with the discovery that receiver activation c-kit specifically induces gene expression Slug and that Slug-deficient mice have a phenotype similar to that of the S1 and W mutant mice, indicate that the Slug gene is the molecular target that provides specificity biological to the SCF / c-kit signaling path.

III. Discussion Defects in the development of the signaling path SCF / c-kit mediated by Slug

Since 1990, the SCF / c-kit in vivo migration pathway and developmental destinations have been well known, due to the existence of mutant mice in which both the receptor coding genes and those of their respective ligand are defective (Nature 335, 88, 1988; Cell 55: 185, 1988; Cell 63: 225, 1990; Cell 63: 203, 1990; Cell 63: 167, 1990; Cell 63: 75, 1990; Cell 63: 213, 1990). However, the mechanism that provides biological specificity to the SCF / c-kit signaling pathway in the formation and migration of c-kit + cells is much less known. A key aspect is the identification of c-kit signaling targets that reinforces the migratory behavior of c-kit + cells. In this regard, the biological events controlled by the c-kit signaling pathway are similar to those that occur in epithelial-mesenchymal transitions (EMT) during mammalian development and are controlled by transition factors of type "fingers zinc "of the Snail family (Nieto et al., 1994, Science 264: 835-849; Cano et al., 2000, Nature Cell Biology 2: 76-83). These proteins, which share an evolutionary conservation role in both vertebrates and invertebrates, are involved in the generation and migration of both mesoderm and neural crest cells in several vertebrate species. It has now been investigated whether the biological functions governed by the SCF / c-kit signaling pathway are mediated by the Snail family of proteins. The results obtained show that the activation of the c-kit receptor by its SCF ligand specifically induces the expression of Slug, a specific member of the Snail family, indicating a clear relationship between SCF / c-kit activation and gene expression Slug.

Because mice with mutations in the c-kit receptor and its ligand (SCF) have the same complex phenotype that affects pigmentation, germ cells and hematopoiesis, mice without the Slug gene were analyzed in order to observe in vivo whether the Slug gene mediated some functions of the c-kit / SCF route. Mice have been generated that experience mutations with loss of function at the Slug site. The expression pattern of the Slug gene suggested that this gene had a role in the development of the nervous system. Consequently, the analysis of the mutant mice focused on this system. Currently, the analysis of mutant mice focuses on those aspects of development that depend on the SCF / c-kit path. The results obtained show the presence of defects in dermatological, gonadal and hematopoietic development in Slug-mutant mice.

Only Slug - / - mice showed alterations in pigmentation, which indicates that the absence of Slug only affects the migration and / or survival of cells pigmented stem derived from the neural crest, so that it produce failures of melanoblasts in positions far from the neural crest, that is, on the forehead and extremities. This Slug function is consistent with the fact that the Slug gene of mouse is not expressed in premigratory cells of the neural crest but it does in migratory cells of the neural crest. These alterations observed in the pigmentation of Slug - / - mice are similar to the alterations described in mutant mice W and S1, explaining why some areas in mutant mice heterozygous W and S1 and in individuals with phenotype of piebaldism they are completely depigmented while others remain normal. These data also indicate that signaling intracellular mediated by c-kit must exceed a critical threshold in order for the Slug to become active and the Melanoblasts migrate and survive. Heterozygous mice S1 and W do not seem to reach this threshold. In fact, melanoblasts that migrate to the forehead and other affected areas may be in the low end of SCF gradient values (Development 109, 911-923, 1990).

In addition to this melanocyte deficiency, the homozygous mutant mice for Slug evidenced defects in the testicles. These defects involve both spermatogonia and Leydig cells. The spermatogonia defect is well known in homozygous mice W and S1 which are sterile and are specifically controlled by activation with IP Kit-mediated 3-kinase (Blume-Jensen et al., 2000, Nature Genetics, 24: 157-162; Kissel et al., 2000, EMBO J. 19: 1312-1326). However, interstitial space in homozygous mice W and S 1 and in the testicles of Kit Y719F / Kit Y719F has increased disproportionately and have been filled with Leydig cells. A possible explanation of these observations is that there are balancing mechanisms within of the Leydig line, such as FSH and factor-1 of insulin-like growth, which try to compensate for deficiencies in the compartments of primitive germ cells in mutant mice W, S1 and Kit Y719F / Kit Y719F, stimulating the proliferation or survival of Leydig cells. By on the contrary, in Slug mice - / - the main problem may reside in the compartment of Leydig cells, which derived of the neural crests and are c-kit +. This impairment of the development of Leydig cells could, as side effect, affect the maturation of cells germinals Therefore, the signaling route SCF / c-kit would have a double function in the testicle: in the development of germ cells controlled by the activation by kit-mediated PI 3-kinase and in the development of Leydig cells controlled by the Slug gene (Figure 7C). Hematopoietic development analysis in mice Slug-mutants evidenced a phenotype similar to that of the defective mice W and S1. Homozygous mutant mice S1, W and Slug had macrocytic anemia. These mutations impaired the development capacity of the cells of the progenitor of the erythroid and T cell lines and have a normal development in B cells and myeloids. The defect in the development of hematopoietic cells in mice Slug-mutants was intrinsic to the cell. The Slug-mutant mice have equal phenotypes, regardless of whether hematopoietic cells were isolated directly from the mutant mice or if they recovered from the transplant recipients Therefore, the phenotype of mice Slug-mutants are not due to inadequacies in the microenvironment (as in S1 mutant mice) but it is due to intrinsic defects of the hematopoietic cells of the parent (as in the W mutant mice).

Other lines that express kit, such as mast cells and most melanoblasts do not show phenotypes obvious in Slug-mutant mice, which suggests that the cellular context is of great importance for the Signal interpretation SCF / c-kit. In this type of cells, the Slug function is either not required or can be compensated for through its synergistic effect with other family members Snail Another question is why the loss of Slug function of heterozygotes produces phenotypic abnormalities. This is coming to indicate that a loss of mutation function in an allele does not can be compensated with the rest of the wild-type alleles of the same gene, defining the Slug as a semi-dominant gene.

S1, W or Slug mutations affect the development of three populations of stem cells: melanoblasts, Hematopoietic stem cells and germ cells. Therefore, Slug it is present in the c-kit + migratory cells and is not present in c-kit + cells in the bone marrow of homozygous S1 and W, which highlights the role of the Slug gene in the acquisition of migration capacity of the c-kit + cells. These results are consistent with the model in which stem cells that contain the c-kit receptor would express the Slug gene, causing cell survival regardless of the external signal required (SCF) and allowing cells to migrate out of its normal environment. If this is not achieved in a given period of time, they could experience apoptosis already that have been deprived of external signals to retain the expression of the Slug gene. This would prevent the entry of migratory cells into territories inadequate to their specification status. These dates indicate that the signals that regulate the fate of the cell (or the cell death) play an important role in maintenance of cell specification and differentiation patterns.

These results identify the Slug gene as a transcription factor that controls migration and survival of the c-kit + cells. In this sense, it know that p53 deficiency rescues androfertility of mice  W, but does not affect the survival of melanocytes and cells Ematopoietic Therefore, germ cell apoptosis male in the absence of kit is dependent on p53 (Dev. Biol. 1999, 215: 78-90). The results now obtained show that Slug is the survival factor in the lines of  melanocytes and hematopoietic cells. Slug protein normally acts as a repressor (Dev. Biol. 2000, 221: 195-205). Therefore, Slug could regulate genes whose expression needs to be excluded from the cells c-kit + to migrate.

Slug is a candidate gene for piebaldism and hereditary anemias in humans

\hbox{c-kit.}

Disorders in the development of melanocytes are characterized by a heterogeneous distribution of pigmentation, called "white mottled", typified by piebaldism and Waardenburg syndrome. At present it is clear that these disorders in the development of the cell pigment represent a subgroup of the neurocristopathies, which involve defects of several cell lines of the neural crest that include melanocytes. The results now obtained involve Slug as the cause of the piebaldism trait. The alteration in the Slug gene may be responsible for the phenotype of piebaldimso in some similar. Therefore, in some patients the trait of piebaldism can be found to result from deletions in this gene, rather than from mutations in the receptor gene

 \ hbox {c-kit.} 

Another characteristic of mice Slug-mutants is anemia. Human anemias congenital, such as Diamond-Blackfan anemia (DBA), which are characterized by a decrease in parents of marrow erythroids, they resemble in some respects the anemia of Slug-mutant mice. So, the alterations in the Slug gene may be responsible for anemia in some patients However, it has previously been observed that humans with pathological mutations of the KIT gene do not have anemia (Spritz, 1992, Blood 79: 2497). A direct test is now viable of this hypothesis.

SCF / c-Kit / Slug in transformation

\hbox{c-kit}

podría conferir propiedades invasivas a las células del tumor. En este contexto, Slug puede representar también un evento molecular relevante en la transformación de células. Descubrimientos recientes muestran que Slug también se expresa en células leucémicas t(17;19) y en células de rhabdomiosarcoma que expresan la traslocación PAX3-FKHR. Por tanto, Slug puede ser un componente de invasión en la biología del cáncer.The c-kit receptor is involved in both leukemia and solid tumors. Mutations that result in the constitutive activation of c-kit have been described in acute myeloid leukemias, in small lung cell cancers, in gynecological tumors, in breast carcinomas and in colon tumors derived from the interstitial cells of Cajal (one type cell that is SCF dependent). However, the oncological potential supposedly conferred to alterations in c-kit activity in malignancy is uncertain. The results obtained show that Slug confers migration and survival properties to c-kit + cells. Therefore, the constitutive activation of

 \ hbox {c-kit} 

It could confer invasive properties to tumor cells. In this context, Slug can also represent a relevant molecular event in cell transformation. Recent findings show that Slug is also expressed in t-leukemic cells (17; 19) and in rhabdomyosarcoma cells that express PAX3-FKHR translocation. Therefore, Slug can be an invasion component in cancer biology.

Potential uses of Slug

The mobilization of hematopoietic stem cells is important in clinical transplants, gene therapy, and in the ex vivo expansion of hematopoietic stem cells, as well as in male sterility. However, these and other SCF applications have been limited by mast cell activating properties (Broudy, 1997, Blood 90: 1345-1364). The results provided by this invention identify Slug as the molecule that mediates the function of the SCF / c-kit signaling pathway, suggesting that Slug could have the same clinical applications as SCF, with the advantage that Slug would not activate mast cells .

<110> SUPERIOR INVESTIGATION COUNCIL SCIENTISTS

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<120> EMPLOYMENT OF GEN SLUG AS A MARKER GENETIC OF FUNCTIONS MEDIATED BY SCF (STEM CELL FACTOR) AND APPLICATIONS

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\hskip-.1em\dddseqskip

cagctggcca ggctctcggt

\hfill

20

 \ hskip-.1em \ dddseqskip 

cagctggcca ggctctcggt

 \ hfill 

twenty

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 4

           \ vskip0.400000 \ baselineskip
        

<211> 18

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial sequence

           \ vskip0.400000 \ baselineskip
        

<223> Reverse initiator oligonucleotide

           \ vskip0.400000 \ baselineskip
        

<400> 4

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

gcgagggcct ccggagca

\hfill

18

 \ hskip-.1em \ dddseqskip 

gcgagggcct ccggagca

 \ hfill 

18

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 5

           \ vskip0.400000 \ baselineskip
        

<211> 24

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial sequence

           \ vskip0.400000 \ baselineskip
        

<223> Internal probe

           \ vskip0.400000 \ baselineskip
        

<400> 5

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

gacacacata cagtgattat ttcc

\hfill

24

 \ hskip-.1em \ dddseqskip 

gacacacata cagtgattat ttcc

 \ hfill 

24

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 6

           \ vskip0.400000 \ baselineskip
        

<211> 29

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial sequence

           \ vskip0.400000 \ baselineskip
        

<223> Internal probe

           \ vskip0.400000 \ baselineskip
        

<400> 6

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

\hskip-.1em\dddseqskip

tgcaaccgtg ttttgctgac cgctccaac

\hfill

29

 \ hskip-.1em \ dddseqskip 

tgcaaccgtg ttttgctgac cgctccaac

 \ hfill 

29

Claims (5)

1. A pharmaceutical composition comprising a product selected from the group formed by the Slug gene, the DNA complementary to the RNA coding for the product of transcription or expression of said Slug gene (cDNA of said gene Slug), the Slug protein, one or more drugs or substances that activate Slug gene expression, and mixtures thereof, along with one or more pharmaceutically acceptable excipients. 2. Pharmaceutical composition according to claim 1, comprising the Slug gene or the cDNA of said gene Slug in a viral or non-viral vector. 3. Use of a product selected from the group formed by the Slug gene, the complementary DNA to the RNA that encodes for the transcription or expression product of said Slug gene (CDNA of said Slug gene), the Slug protein, one or more drugs or substances that activate the expression of the Slug gene, and their mixtures, in the development of a pharmaceutical composition for mobilization of hematopoietic stem cells for transplantation or therapy gene. 4. A method for the ex vivo expansion of hematopoietic stem cells comprising the use of a product selected from the group formed by the Slug gene, the DNA complementary to the RNA encoding the transcription or expression product of said Slug gene (cDNA) of said Slug gene), the Slug protein, one or more drugs or substances that activate the expression of the Slug gene, and mixtures thereof. 5. Method according to claim 4, which comprises administration to a stem cell culture hematopoietic of an effective amount of a selected product of the group formed by the Slug gene, the cDNA of said Slug gene, the Slug protein, one or more drugs or substances that activate the expression of the Slug gene, and mixtures thereof, or of a composition that It contains such products.