WO2001062273A1

WO2001062273A1 - The use of gdnf family-related compounds for manufacturing products for treating testicular tumors

Info

Publication number: WO2001062273A1
Application number: PCT/FI2001/000173
Authority: WO
Inventors: Hannu Sariola; Xiaojuan Meng; Mervi Hyvönen; Maria Lindahl; Mart Saarma
Original assignee: Hannu Sariola; Xiaojuan Meng; Hyvoenen Mervi; Maria Lindahl; Mart Saarma
Priority date: 2000-02-22
Filing date: 2001-02-21
Publication date: 2001-08-30
Also published as: AU2001240723A1; FI20000403A0

Abstract

The present invention is related to glial cell line-derived neurotrophic factor (GDNF) family-related compounds, particularly GDNF and nucleic acids encoding functionally active, inactive or inactivatable GDNF-like compounds and their use for treating and diagnosing human seminoma. The invention also discloses methods for screening of substances capable of modulating the interaction between GDNF family-related compounds and their signal transmitting receptor (Ret) and/or their co-receptors, the GDNF family receptor α:s (GFRα1-4) and useful model animals for testing the efficacy of said compounds.

Description

THE USE OF GDNF FAMILY-RELATED COMPOUNDS FOR MANUFACTURING PRODUCTS FOR TREATING TESTICULAR TUMORS

The Field of the Invention

The present invention is related to glial cell line-derived neurotrophic factor (GDNF) family-related compounds, such as glial cell line-derived neurotrophic factor (GDNF), other GDNF-like factors, compounds acting like GDNF on its signal mediating receptor and/or co-receptors and products which have the capacity of modulating the signaling between the GDNF-like factors and its receptor. Also disclosed are the use of said compounds for studying the pathogenesis of human seminoma, treating and/or diagnosing seminoma as well as methods for screening to identify compounds, which are capable of modulating, stimulating or inhibiting, the signaling between GDNF-like factors and receptor tyrosine kinase Ret.

The Background of the Invention

Neurotrophic factors control development and maturation of neurons. Different neuronal subpopulations require specific trophic factors from their target sites. Glial cell line-derived neurotrophic factor (GDNF) and related factors, here so called GDNF-like factors or compounds, neurturin (NTN), artemin (ART) and persephin (PSP) form a subgroup in transforming growth factor β (TGF-β) superfamily. Responses to these GDNF family ligands are mediated by a receptor complex composed of the transmembrane receptor tyrosine kinase Ret and one or more of the GDNF-family α-receptors (GFRαl-GFRα4) (reviewed by Airaksinen, et al. Mol. Cell. Neurosci. , 13:313-325, 1999).

Glial cell line-derived neurotrophic factor (GDNF) family-related compounds, including the GDNF factor, other GDNF-like compounds, such as artemin (ART), neurturin (NTN) and persephin (PSP) as well as other derivatives thereof acting like said GDNF-like compounds on their signal mediating receptor and/or the co-receptors and their use for regulating and studying spermatogenesis, for inhibiting the differentiation of sperm cells, for developing male contraceptives or as a male contraceptive as well as their use for manufacturing male contraceptive compositions have been described in the International patent application (WO 00/10594). The GDNF-family related compounds have been described in the following patent publications. The International patent application WO 97/18240 discloses isolated receptors which bind glial cell line-derived neurotrophic factor (GDNF) as well as identification and isolation methods for these receptors. WO 97/33911 discloses persephin (PSP) and related growth factors. WO 97/33912 discloses a GDNF receptor (GFRαl), its variants and their use. WO 98/36072 discloses neurturin (NTN) receptor NTNR (GFRα2). Artemin (ART) is disclosed in WO 00/18799.

The International patent application W0 99/62332 describes the production of transgenic mice lacking a functional GFRα2-receptor whereas the International Patent Application W0 00/10594 describes transgenic animals, especially mice, which overexpress GDNF in testis. Ret receptor tyrosine kinase, GFRαl (the GDNF receptor) and GFRα2 (the NTN receptor) are expressed by testicular germ cells, whereas GDNF and neurturin (NTN) are expressed by Sertoli cells.

Cancer cells and tumors are known to arise from normal cells following mutations of cellular genes such as proto-oncogenes and tumor suppressor genes. Cancer has generally been connected to old age, but the fact that an increasing incidence of cancer is observed in younger generations and that tumor cells behave like undifferentiated cells has focused cancer studies to developmental biology. The main motivation for studying the molecular basis of cancer is to develop new therapies and methods for early diagnosis or including methods of finding a predisposition for developing cancer. Such prediction systems would provide possibilities for starting prophylactic treatments at a very early stage.

At present, tumors, especially testicular tumors are treated using radiation and cytostatic drugs, which as known are connected with severe side effects, e.g. male infertility. Accordingly, there is an increasing need of novel, alternative, more effective drugs with less side effects in order to provide efficient tools for combating cancer, including new animal models for testing the effects of potential drugs and treatment modalities. Tumors occur in many tissues and they also differ in properties and invasiveness. The present invention is focused on testicular tumors, especially seminomas.

Testicular germ cell tumors (TGCTs) are the most common solid tumors in young men and their frequency is increasing. The testicular tumors are grouped in two entities, seminomas and non-seminomatous TGCTs. Seminomas accounting for approximately half of all TGCTs, are further subdivided in two distinct subtypes, classic seminoma and spermatocytic seminoma (Ulbright, T. M., et al. , Armed Forces Institute of Pathology, Washington, D.C. pp 1-100, 1999). As said above the present invention is specifically related to seminoma.

The pathogenesis of seminomas has remained obscure. Therefore, an animal model for this tumor type would be highly beneficial, not only to approach the pathogenesis of seminomas, but also to develop new treatment modalities for testicular tumors and products useful for treating and diagnosing disorders in human seminomas. However, as said above spontaneous seminomas are extremely rare in animals (Mitsumori, K. and El well M. R. , Environ. Health Perspect. , 77: 11-21, 1988). This means that studying the regulation of seminoma and the effect of potential drugs for treating seminoma has been difficult because of lack of suitable animal models.

When studying testes of old GDNF overexpressing mice (WO 00/10594) it was surprisingly observed that said mice developed testicular tumors. Based on this observation a research project was initiated in order to develop new methods for treating and diagnosing testicular tumors, especially seminomas and for screening compounds capable of modulating the interactions between the GDNF family -related compounds and Ret and their use for manufacturing products, including animal models, cell-lines, vectors, constructs useful for studying and regulating pathogenesis in human seminoma and for developing new treatment modalities based on the results obtained.

The objective of the present invention is to use the GDNF family-related compounds or modulators thereof as active ingredients for manufacturing compositions useful for treating or diagnosing testicular tumors, particularly seminomas to block or inactivate GDNF family-related compounds and the activation of their receptors and thereby the formation of tumors. The invention is particularly targeted to the use of the GDNF signaling pathway and methods and means capable of modulating the interactions of said signaling pathway for treating seminoma.

Another objective of the present invention is to use the GDNF family-related compounds to diagnose the presence, predisposition or risk to develop seminoma and to follow up the rate of healing of seminoma.

The Summary of the Invention

In the present invention it is disclosed for the first time that old glial cell line-derived neurotrophic factor (GDNF) overexpressing mice develop testicular tumors. Further studies with said transgenic mice indicated that targeted overexpression of GDNF, a ligand for the receptor tyrosine kinase Ret, a proto-oncogene, in undifferentiated spermatogonia promotes malignant testicular germ line tumors, which are invasive and contain aneuploid cells. The data showed that a modulated, preferably deregulated stimulation of a known proto-oncogene by its ligand can be an initiative event in carcinogenesis. By their histological, molecular and histochemical characteristics the GDNF-induced tumors mimic classic seminomas in men, representing the first animal model for this tumor type. The similarity of the testicular tumors in GDNF overexpressing mice with human seminomas indicated that the GDNF overexpressing transgenic mice can be used as a new animal model for studying human seminomas. Furthermore, the data suggested that activity of the GDNF signaling pathway is involved in the pathogenesis of human seminomas and this signaling pathway may be a new target for the therapy of testicular tumors.

Accordingly, the present invention is related to the use of GDNF family-related compounds, derivatives or mixtures thereof as well as nucleic acids encoding a functionally active, inactive or inactivatable GDNF family-related compound, GDNF-like compounds, particularly GDNF, which compounds act like said GDNF-like compounds on the Ret receptor tyrosine kinase or co-receptors thereof. The compounds listed above are useful for screening for substances or modulators, i.e. active ingredients, which are capable of modulating the interaction or signaling between GDNF-like factors and Ret. Such modulators or active ingredients are useful for treating and/or diagnosing seminoma.

The GDNF family-related compounds of the present invention include a functionally active, inactive or inactivatable GDNF family-related compound GDNF, persephin (PSP), neurturin (NTN), artemin (ART) or compounds acting like the GDNF family-related compounds on the receptor transmitting signals of Ret or co-receptors, but above all the invention is related to GDNF-like compounds, particularly GDNF and modulator compounds or active ingredients capable of modulating the interaction between GDNF-like compounds and Ret and the co-receptors, GDNF family-receptor α:s (GFRαs), which activate the Ret receptor tyrosine kinase or transmits the signal of the GDNF family-related compounds. Thus, the present invention is related to the use of the interactions occuring in the GDNF-signaling pathway as a response to an outside stimulus and the use of said interaction for providing new therapies for seminoma.

The invention is also related to the use of nucleic acid sequences which encode GDNF-like factors, constructs or vectors carrying said nucleic acid sequences, which are characterized by encoding a functionally active, inactive or inactivatable GDNF family-related compound. Said sequences and vectors can either be used to manufacture transgenic animals, e.g. rodents, particularly mice, useful as model animals, which when overexpressing GDNF family-related compounds are useful for testing the safety and efficacy of selected substances as drugs against seminoma or if they have aberrant sequences which inhibit expression or encode functionally inactive or inactivatable GDNF-compounds they can be used in gene therapy.

The invention is also related to a method for treating seminoma. The method comprises the administration to a subject suffering from a disorder in seminoma, especially a disorder caused by an aberrant expression of GDNF-like compounds, a therapeutically effective amount of a substance, i.e. the active ingredient capable of modulating the interaction between GDNF family-related compounds and receptor tyrosine kinase Ret. Preferably, the treatment of seminoma should be targeted to the signaling pathway involving the GDNF family-related compound. The targeting to the signaling pathway is provided by substances capable of modulating, stimulating or inhibiting GDNF-like factors and their interaction with Ret. The substances modulating the GDNF-like compounds, the modulators or active ingredients act by modulating the binding of GDNF to the receptor, by modulating the formation of receptor complex or by modulating the phosphorylation of Ret.

The modulator substances or active ingredients comprise substances which can modulate the interaction between GDNF-like compounds and Ret by binding to said GDNF family-related compounds. The modulator substances can also be nucleic acid sequences encoding substances, which can interact with the expression, e.g. disturb or down-regulate the expression of GDNF-like compounds. The modulator substance or active ingredient is advantageously a nucleic acid sequence, which modulates the expression of a GDNF family -related compound and thereby, when introduced into a cell modulates the interaction of the GDNF family -related compound and Ret. The nucleic acid sequence can advantageously be an antisense nucleic acid fragment of the nucleic acid sequence encoding a functionally active GDNF family-related compound. In other words, it disturbs the production of the GDNF-like compound on mRNA level. Alternatively, the nucleic acid sequence can be a sequence, which can hybridize with a genomic nucleic acid sequence encoding functional GDNF-like products and by said hybridization disturb the expression of functionally active GDNF-like compounds.

Alternatively, the substance acting as a modulator or active ingredient can be a substance binding to the GDNF family-related compounds and which when introduced into a cell modulates the interaction of the GDNF family-related compound. Such binding substances can be found among small molecules, which by binding to the GDNF-like compound, the signal mediating receptor or the co-receptors, modulate the complex formation and consequently the interaction or signaling between the GDNF-like compound and Ret, i.e. the GDNF-signaling pathway. The binding substance can also be an antibody, including polyclonal or monoclonal antibodies, which acts as the small molecule described above.

In order to facilitate the treatment of seminomas, the compounds modulating the GDNF family-related compounds or the inactivated or inactivatable constructs of said GDNF-like compounds, the modulating drugs or active ingredients, are administered to the subject suffering from seminoma in combination with at least one pharmaceutically acceptable carrier and/or additive, which should be compatible with the substance capable of modulating the GDNF family-related compound and the route of administration. In connection with surgical intervention the modulating drugs or active ingredients can be administered locally in a controlled or sustained-release matrix. Nucleic acid delivery systems, such as liposomes can advantageously be placed in such matrixes. The modulating drugs or active ingredients can also be administered as topical, oral, parenteral or injectable compositions. When injectable compositions are administered it is important that the modulator or active ingredient is soluble.

Consequently, the substance capable of modulating GDNF-like compounds, i.e. the active ingredient, can be administered by cell therapy or gene therapy. In such case the cells have been modified to produce and secrete modified GDNF family-related compounds or derivatives or mixtures having the capacity of modulating GDNF-like compounds.

The present invention is also related to a method for diagnosing the presence, predisposition or risk of developing seminoma as well as the progress of healing. The method comprises determination of the amount of GDNF family related compound, particularly GDNF, secreted by the seminoma. The sample is preferably, a blood or serum sample or a tissue sample obtained by biopsy. The determination of the GDNF-like compounds is carried out by per se known immunochemical or biochemical methods. Especially, alkaline phosphatase seems to be an indicator of predisposition of seminoma. Determination of changes in phosphorylation of Ret can accordingly be applied in said diagnostic determinations or assays.

The invention is also related to a method for screening a library of known or unknown chemical substances for identifying compounds useful for treating seminoma. A library of substances may be obtained by combinatorial chemistry, genomics, proteomics, phage-display, etc. Said substances are screened for their capacity of modulating, i.e. stimulating respective inhibiting the complex-formation, i.e the interaction and signaling between GDNF-like compounds and Ret. The safety and value of the substances capable of modulating the interaction can be further evaluated by using for example the GDNF-like factor overexpressing mice, particularly GDNF overexpressing mice of the present invention.

In the present invention compositions for treating seminoma are discussed. The modulating substances having the capacity of modulating the interaction between GDNF-like compounds and pharmaceutically acceptable additives or carriers conventionally applied in sustained-release, topical, oral, parenteral or injectable administration forms are as discussed above. An injectable dosage form should in addition to the active ingredient preferably comprise pharmaceutically acceptable additive such as a buffered solution having a pH and electrolytes in a physiologically acceptable range.

The compositions used in gene therapy or in implants intended to be inserted e.g. during a surgical intervention should advantageously comprise in addition to the active ingredient, a suitable delivery system, e.g. liposomes and other compatible pharmaceutically acceptable additives. Advantageously, the whole system can be inserted into a pharmaceutically acceptable controlled or sustained release matrix. This approach would be especially advantageous for local application of nucleic acid containing delivery systems.

The composition may in addition to the active ingredient or modulating substance be combined with at least one second substance effective against seminoma, e.g. other cytostatic drugs or other medically active ingredients e.g. antibiotics.

In short, the characteristics of the present invention are as defined in the claims. The present invention describes a use of GDNF family-related compounds, a method, whereby it is possible to diagnose the presence, predisposition and rate of healing of the human seminoma and secondly it provides methods related for treating testicular tumors by blocking/inactivating GDNF family-related signaling pathway and/or formation of tumors. It is characterized in that GDNF family-related compounds, including GDNF, artemin, neurturin and/or artemin, but especially GDNF, or co-receptors thereof are useful for studying the regulation of pathogenesis in human seminoma.

A Short Description of the Drawings

Figure 1. Development of seminomatous tumors in old transgenic mice targeted to overexpress GDNF in testes. Scale bar 100 μm.

Figure la. Seminiferous tubules in a normal testis. The spermatogonia are situated at the peripheral rim of the tubules. Scale bar 100 μm.

Figure lb. A testis from a 4- week-old transgenic mouse. Note the clusters of spermatogonia within seminiferous tubules. Scale bar 100 μm.

Figure lc. Small groups of spermatogonia-type cells (arrow) invading the interstitium in a transgenic testis from a 7-month-old mouse. These cells were only observed by the microscopic analysis. Scale bar 100 μm.

Figure Id. Solid sheet of invasive spermatogonia-type cells in a 1-year-old transgenic mouse developing the testicular tumor. Scale bar 100 μm.

Figure le. A normal 4-week-old wild type testis. Spermatogonia at the periphery rim are labeled by TRA98 antibody. Scale bar 100 μm.

Figure If. A 4-week-old transgenic testis with clusters of spermatogonia within seminiferous tubules (star). The testicular germ line cells are labeled with TRA98 antibody. Scale bar 100 μm.

Figure lg. A 7-month-old testis with microinvasive spermatogonia in the interstitium (arrow). The dotted lines mark the shape of a seminiferous tubule. The testicular germ line cells are labeled with TRA98 antibody. Scale bar 100 μm.

Figure lh. The testicular germ line cells in a testicular tumors labeled with TRA98 antibody in a 1-year-old mouse. Scale bar 100 μm.

Figure li. A normal 4-week-old wild type testis. The EE2 antibody is highly specific to spermatogonia in the periphery of seminiferous tubule. Scale bar 100 μm.

Figure lj. A 4-week-old transgenic testis with clusters of spermatogonia within seminiferous tubules (star). Scale bar 100 μm.

Figure Ik. A 7-month-old testis with microinvasive spermatogonia in the interstitium (arrow). The dotted lines mark the shape of a seminiferous tubule. The EE2 antibody is highly specific to spermatogonia. Scale bar 100 μm.

Figure 11. The germ line markers are also expressed by the testicular tumors. Scale bar 100 μm. Figure lm. In situ hybridization for the GDNF transgene in the testicular tumors. The grains in the dark field image depict the signals of in situ hybridization. Scale bar 100 μm.

Figure In. In situ hybridization for the Ret in the testicular tumors. The grains in the dark field image depict the signals of in situ hybridization. Scale bar 100 μm.

Figure lo. In situ hybridization for the GFRal in the testicular tumors. Scale bar 100 μm.

Figure 2. Phosphorylation of Ret and its downstream targets in wild type testis and GDNF-induced testicular tumors.

Figure 2a. Immunoprecipitated Ret blotted with Ret antibody (Ret).

Figure 2b. Immunoprecipitated Ret blotted with phosphotyrosine antibody (pTyr).

Figure 2c. Immunoprecipitated Ret blotted with ERK1/2.

Figure 2d. Immunoprecipitated Ret blotted with phosphoERKl/2 (pERKl/2).

Figure 2e. Immunoprecipitated Ret blotted with AKT.

Figure 2f . Immunoprecipitated Ret blotted with phosphoAKT (p AKT) .

Figure 3a. Karyotype of mouse spleen cells which serve as diploid control cells.

Figure 3b. A mmor-free testis of a 9-month-old transgenic mouse shows a diploid karyotype.

Figure 3c. A testicular tumor of an old GDNF overexpressing transgenic mouse exhibits aneuploidy. The peaks marked with an arrow are from cells with a hypoploid DNA content, the peak marked with a star indicates the diploid cells, and the peak marked with a plus-sign represents a triploid cell population.

Figure 4a. The wild type testis before 1 week of age expresses L-Fng (insert), but the expression is no longer detectable at 4 weeks of age. The grains in the dark field image depict the signals of in situ hybridization. Scale bar 100 μm. Figure 4b. Spermatogonial clusters in a 4-week-old transgenic mouse continuously expressing L-Fng indicated by grains in in situ hybridization. Scale bar 100 μm.

Figure 4c. High L-Fng expression in a GDNF-induced testicular tumor indicated by grains in in situ hybridization. Scale bar 100 μm.

Figure 4d. A 4-week-old wild type testis shows the alkaline phosphatase reactivity only in the basement membranes, while germ cells are unlabeled. Scale bar 50 μm.

Figure 4e. A subset of cells in the spermatogonial clusters of the transgenic mouse testes positive for alkaline phosphatase. Scale bar 50 μm.

Figure 4f. The testis tumors exhibit high alkaline phosphatase reactivity. Scale bar 50 μm.

The Detailed Description of the Invention

Definitions

In the present invention the terms used have the meaning they generally have in the fields of cell biology, neurology, biochemistry, pharmacology, recombinant DNA technology, including transgenic animal production, but some terms are used with a somewhat deviating or broader meaning than in the normal context. Accordingly, in order to avoid uncertainty caused by terms with unclear meaning some of the terms used in this specification and in the claims are defined in more detail below.

The term "testicular tumors" means testicular germ cell tumors (TGCTs) which are common solid tumors in men. The tumors are grouped in two entities, seminomas and non-seminomatous TGCTs. A few testicular malignancies are comprised of mixed seminomatous and non-seminomatous areas (Ulbright, T. M. , et al. , Armed Forces Institute of Pathology, Washington, D.C. pp 1-100, 1999).

The term "seminoma'' means a form of testicular tumors (testicular germ cell tumors, TGCTs). Seminomas accounting for approximately half of all TGCTs, are further subdivided in two distinct subtypes, classic seminoma, and spermatocytic seminoma. The present invention is focused on classic seminoma composed of fairly uniform medium-sized cells with clear cytoplasm and well-defined cell borders. Human classic seminoma is supposed to originate from carcinoma in situ (CIS) cells. These cells arise early in development and resemble gonocytes, the immature germ cells, and proliferate but are unable to differentiate. Both carcinoma in situ (CIS) cells and classic seminomas express placental alkaline phosphatase.

The term "carcinoma in situ (CIS) cells" means malignant cells that are not yet invasive and have not yet left the site of their origin. CIS cells arise early in the development and resemble gonocytes, the immature germ cells, and proliferate, but are unable to differentiate. They present the first stage of cancer formation.

The term "glial cell line-derived neurotrophic factor (GDNF) family-related compounds" i.e. GDNF-family-related compounds are also called GDNF-like compounds or factors and comprise glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP) and/or artemin (ART) and derivatives thereof having the same effect as GDNF-like compounds on their signal mediating receptor(s) or co-receptor(s) . Said receptor(s) or co-receptor(s) comprise(s) Ret receptor tyrosine kinase and GDNF family-receptor α:s (GFRα:s), respectively.

The term "glial cell line-derived neurotrophic factor (GDNF)" means the glial cell line-derived neurotrophic factor (GDNF) , which is a member of the transforming growth factor-β (TGF-β) family (Lin, L. F. , et al. , Science, 260: 1130-1132, 1993). GDNF maintains the dopaminergic, noradrenergic, cholinergic and motor neurons in the nervous system and also protects peripheral, parasympathetic, ciliary and sensory nerve cells (Lin, L.F. , Neural Notes, 2:3-7, 1996). GDNF is expressed also in other tissues, such as embryonic kidney and testis (Suvanto, P. , et al. , Eur. J. Neurosci. 8:816-822, 1996). The properties and structure, amino acid sequence and their functions are disclosed in the review article (Airaksinen, M. S. , et al. , Mol. Cell. Neurosci. , 13:313-325, 1999).

In the present invention the term "GDNF-like factors" or "GDNF-like compounds" include in addition to GDNF itself growth factors which have a similar structure or act like GDNF on its receptor or co-receptors. Thus, the term "GDNF-like factor" or "GDNF-like compound" above all relates to functionally active, inactive or conditionally inactivatable GDNF, persephin, artemin and neurturin. All four growth factors of the GDNF family are structurally similar and Ret receptor tyrosine kinase acts as the common signal transmitting receptor of GDNF, artemin, persephin and neurturin.

The term "derivatives" means compounds which act as GDNF-like factors on the receptor transmitting signals of GDNF or co-receptors thereof. "Derivatives" include functionally active, inactive or conditionally inactivatable polypeptides "substantially homologous" at amino acid level having a significant similarity or identity of at least 80%, preferably 85%, most preferably more than 90% with human forms of GDNF, neurturin, artemin and persephin.

The term "GDNF-like factors and derivatives thereof" comprises polypeptides having the structure, properties and functions characteristic of GDNF-like molecules. Thus, the term "GDNF-like molecules and derivatives thereof" includes GDNF-like molecules, wherein one or more amino acid residues are substituted by another amino acid residue. Also truncated, complexed or chemically substituted, forms of said GDNF-like molecules are included in the term. Chemically substituted forms include for example, alkylated, es- terified, etherified or amidized forms with a low substitution degree, especially using small molecules, such as methyl or ethyl, as substituents, as long as the substimtion does not disturb the properties and functions of the GDNF-like molecules. The truncated, complexed and/or substituted variants of said polypeptides are producible by synthetic or semisynthetic, including enzymatic and recombinant DNA techniques. The only other prerequisite is that the derivatives still are substantially homologous with and have the properties and/or express the functions characteristic of GDNF-like factors. Additionally, such structurally similar GDNF-like factors, which are inactivated or conditionally mactivatable are include in the present invention. They are so called modulators and provide the active ingredients of the present invention.

The GDNF-like factors or compounds can exist in different isoforms. The term "isoform" refers to the different forms of the same protein, which originate from different sources, e.g. different mammalian species, e.g. human and murine sources in this case. In the present invention the term, thus, includes fragments, complexes and their derivatives originating from different sources. Isoforms of GDNF-like factors can be generated by the cleavage of the proprotein. Different reactions, including different enzymatic and non-enzymatic reactions, proteolytic and non-proteolytic, are capable of creating truncated, derivatized, complexed forms of the molecules. Preferably, all GDNF-like compounds and their derivatives should be recognizable using binding substances capable of recognizing and specifically binding to natural mammalian, including human and murine GDNF-like factors or at least one specific portion of said molecules.

The term "modulator" means the active ingredient, a substance which is capable of modulating, i.e. stimulating or inhibiting the complex formation, i.e. the interaction or signaling between the GDNF-like compounds and Ret. Modulators can be both nucleic acids, small molecules and/ or antibodies.

The term "active ingredient" means compounds which act as modulators. Such compounds are found among substances binding to GDNF-like compounds. Such binding substances are found among antibodies and fragments thereof but also among a multitude of small molecules, including substances known to have an effect on GDNF-signaling.

The terms "markers" and "probes" in the present invention include substances such as antibodies for a spermatogonial marker EE2 and a germ line marker TRA98, which can be labeled using for example affinity labeling, including for example biotin-streptavidine detection. Ret can be detected using a polyclonal antibody and immunoprecipitation-western blotting with phosphotyrosine antibodies. Downstream signaling molecules of Ret, AKT and ERK1/2, were probed with polyclonal antibodies in western blotting. GDNF, Ret, GFRαl , L-Fng, WT1 and 3β-HSD which were used as antisense and sense probes in in situ hybridization were preferably labeled with ³⁵S-uridine. The lunatic fringe and Notch molecules are also used as markers for different purposes.

The markers and probes as well as the GDNF-like compounds, modulators thereof including binding substances, nucleic acids, including DNA or RNA sequences can be labeled using direct or indirect labeling systems, which are well known in the art.

In the disclosure of the present invention radioactive labels, including isotope 35s-_uridine, has been used but it is evident that radioactive labels can be replaced by other more convenient labels mentioned above for routine screening and diagnosis.

The term "binding substances" includes for example antibodies or ligands specifically recognizing or binding to GDNF-like factors or at least a specific portion of said molecule, receptors of GDNF-like factors such as GFRαs or other binding proteins or peptides, comprising e.g. specific portions of said GFRαs, but above all they mean antibodies capable of specifically recognizing one or more GDNF-like factors alone or in any combination. The antibodies include both polyclonal and/or monoclonal antibodies as well as fragments or derivatives thereof. Preferably, such binding substances recognize and bind to specific epitopes or active sites of the GDNF-like factors. Said binding substances can be produced using specific domains of GDNF family related compounds, their isomers as well as their fragments, derivatives and complexes with the prerequisite that they are capable of functioning in respective signaling pathway.

The GDNF family-related compounds not only act as "ligands", they also act as "antigens" and are as such or as compositions capable of eliciting an antibody response specific to respective GDNF family-related compound. Said antibodies are producible by conventional techniques for producing polyclonal antibodies as well as monoclonal antibodies. The methods for preparing monoclonal antibodies include hybridoma techniques. Fragments of antibodies or other binding proteins like specific binding peptides can be developed by phage display techniques and produced by recombinant DNA techniques. All methods are well known by those skilled in the art and described in laboratory handbooks.

The binding substances are useful for manufacturing "test kits" i.e. a packaged combination of reagents needed for diagnosing testicular tumors and pathogenesis of seminomas. The function of the GDNF-like compounds and their derivatives can be followed (recorded) based on their signaling, which can be assayed as described in the Examples.

In the present invention the term "nucleic acid sequence" means any isolated and purified nucleic acid sequence encoding functionally active, inactive or inactivatable mammalian GDNF-like factors or nucleic acid sequences with substantial similarity encoding GDNF-like factors with substantially the same properties.

The nucleic acids sequences of the present invention belong to active GDNF family-related compounds of the present invention and they can be used as such or introduced into suitable transformation or expression vectors, which in turn can be introduced into suitable host organism to provide prokaryotic or eukaryotic organisms as well as transgenic animals capable of expressing altered levels of GDNF-like factors e.g. knockout or overexpressing mice.

Modulators, which can include both stimulators and/or inhibitors of gene activity, such as antisense nucleic acid sequences and antigenes, oligonucleotides and ribozymes are important forms of compounds used in gene -based drugs or active ingredients. These compounds are composed of strands of DNA, RNA or their modified forms. They inhibit the function of the target gene either at the level of gene transcription, translation or RNA splicing. Therefore, gene expression at too high level or in incorrect form can be altered with these technologies.

The "nucleic acid sequences" of the present invention are not in their natural state but are isolated and purified from their natural environment as transiently expressed mRNAs. Thereafter the mRNAs are purified and multiplied in vitro in order to provide by technical means new copies, which are capable of encoding said functionally active, inactive or inactivatable mammalian GDNF-like factors or derivatives thereof. In the present context the term "cDNA" means a complementary DNA sequence obtainable by reversed transcription of mRNA transcribed from the genomic DNA sequence. The term "genomic sequence" means the corresponding sequence present in the nuclei of the mammalian cells and comprising introns as well as exons.

The term "nucleic acid sequence encoding GDNF-like molecules" means nucleic acid sequences encoding functionally active, inactive or inactivatable GDNF, artemin, neurturin and/or persephin as well as substantially homologous nucleic acid sequences. Said sequences or their complementary sequences or nucleic acid sequences containing said sequences or parts thereof, e.g. fragments truncated at the 3 '-terminal or 5 '-terminal end, as well as such sequences containing point mutations, are especially useful as probes, primers and for preparing DNA constructions useful for stimulating or inhibiting the level of expression in the tumorous testicular tissue.

It is, however, evident for those skilled in the art that other nucleic acid sequences capable of encoding functionally active, inactive or inactivatable GDNF-like factors and useful for their productions can be prepared. The nucleic acid sequences encoding functionally active, inactive or inactivatable GDNF-like factors should be capable of hybridizing under stringent condition (Sambrook, J. , et al. , Molecular Cloning: A Laboratory Manual. , Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989). The nucleic acid sequences of the present invention should have a substantial similarity with the sequences encoding a functionally active, inactive or inactivatable GDNF, neurturin, artemin and/or persephin. "Substantial similarity" in this context means that the nucleotide sequences fulfill the prerequisites defined above and have a significant similarity, i.e. a sequence identity of at least 60% , preferably 70% , most preferably more than 80% with said sequences.

The term "nucleic acid sequences encoding functionally active, inactive or inactivatable human GDNF-like factors" include their truncated or complexed forms as well as point mutations of said nucleic acid sequences as long as they are capable of encoding functionally active, inactive or inactivatable amino acid sequences having the essential structural features as well as the properties and/or functions of said GDNF-like factors. The nucleic acids are useful as such or inserted in transformation or expression vectors or hosts comprising said nucleic acids or vectors. Said nucleic acids are capable of encoding a functionally active, inactive or inactivatable GDNF-like factors, which are recognizable by binding substances specifically recognizing said nucleic acids encoding said factors. The GDNF family-related compounds include in addition to the polypeptides also nucleic acid sequences encoding functionally active, inactive or inactivatable GDNF family related compounds and derivatives thereof. They include primers, probes, antibodies, receptors as well as binding peptides or ligands. Said substances are useful especially for diagnosing and treating testicular tumors as well as for studying pathogenesis of human seminomas.

The term "diagnosing" means judging, predicting, assessing or evaluating from the recorded results if a person is susceptible to or suffers from seminoma. The diagnoses also enable to evaluate the severity of the condition, therapy required as well as the efficacy of treatment modalities or medical treatment. Especially, early identification of the disease and precancerous lesions in order to start prophylactic and/or other treatments before the onset of the actual disease is a desirable feature, enabled by the present invention. The results are recordable with means for performing immunoassays using GDNF-like molecules and/or their binding substances as well as parts thereof or means for performing amplification and hybridization methods using sequence specific probes or primers, which can be selected from the parts of the nucleic acid sequences encoding the functionally active, inactive or inactivatable domains of mammalian, especially human GDNF-like factors.

The term "immunoassay" refers to a immunochemical method or procedure capable of detecting and/or measuring at least one substance, either a GDNF-like factor or an antibody recognizing said factors using per se known means for performing an immunoassay, which means including a substance capable of specifically recognizing the substance to be determined, i.e. either at least one binding substance or a GDNF-like molecule or fragments thereof, for the desired application, respectively.

Well known examples of immunoassays are radioimmunoassays (RIA), radio immunomet- ric assays (IRMA), fluoroimmunometric assays (IFMA) enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), fluoroimmunoassays (FLA), luminescence immunoassays, immunoagglutination assays, turbidimetric immunoassays, nephelometric immunoassays, etc. All methods are well known by those skilled in the art and described in laboratory handbooks.

Any immunochemical test methods can in principle be used for diagnosing testicular tumors as well as for longitudinal or latitudinal screening of the progress of disease and effect of medical treatment. However, visual agglutination, flow-through and immunochro- matographic methods are best suited for rapid assays or tests.

The "quantification" of GDNF-like factors is preferably determined or screened b per se known immunoassays using known amounts of GDNF family-related compounds or their respective binding substances as standards.

The term "therapeutic treatment" includes methods for treating persons with administration of the compositions comprising functionally active, inactive or inactivatable GDNF-like polypeptides, GDNF-like factor binding substances and/or nucleic acid sequences encoding functionally active, inactive or inactivatable GDNF-like compounds, gene therapy or preventing the genes causing the disease from expressing the gene products causing the diseases. The amounts of GDNF family-related compounds administered are such that a desired effect is obtained in the treated tissue.

The term "routes of administration" includes locally, topically, orally, parenterally or intravenously.

They should be provided as pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.

The term "compositions" means the active ingredients of the present invention including the nucleic acid molecules encoding functionally active, inactive or inactivatable GDNF-like factors, GDNF-like factor modulators, e.g. inhibitors or activators and/or antibodies thereof in combination with at least one pharmaceutically acceptable carrier, which is compatible with the active ingredient and the route of administration. The nucleic acid molecules of the present invention can be used in gene therapy by introducing the molecules to suitable vectors or other er se known delivery systems. The pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for use.

The nucleic acid molecules, proteins, protein homologues, modulators, i.e. stimulators or inhibitors, and antibodies, i.e. the active ingredients described can be used in drug-screening assay, diagnostic assays, methods of treatment, pharmacogenomics, and monitoring of effects during clinical trials.

The nucleic acids of the present invention can be used to provide mammalian vectors which in turn can be introduced into mammalian cells. Suitable expression systems for pro- and eukaryotic cells are described in Sambrook, J. , et al. , Molecular Cloning: A Laboratory Manual. , Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. The host cells can be used for expressing the GDNF-like factors of the present invention.

The "host cells" of the present invention can also be used in production of non-human transgenic animals useful for testing the safety and value of modulator compounds, i.e. the active ingredients, which are capable of ameliorating detrimental systems and disorders related to human seminoma by modification of the interaction between GDNF-like compounds and Ret. Especially useful are knockout mice expressing altered levels of GDNF-like factors or mice overexpressing GDNF-like factors for studying the role of GDNF-like factor mediated signaling in seminoma. Said GDNF-like factors can be used either as separate entities or in any combinations or complexes thereof.

As used herein, "a GDNF-like factor responsive cell" includes a cell which has a biological activity that can be modulated (e.g. , stimulated or inhibited) by at least one of the GDNF-like factors listed above. Depending on the type of cell, the response elicited by the GDNF-like factors is different. For example, GDNF-like factors in testis regulate spermatogenesis. Abnormal or aberrant activity of proteins involved in specific signaling pathways seems to lead to a variety of disorders in the testicular system, e.g. , abnormal growth or development of tumors as well as abnormal differentiation or function of the cells, ultimately leading to seminoma-like tumors.

Thus, abnormal or aberrant activity of GDNF family -related compounds, or abnormal or aberrant nucleic acid expression of the nucleic acid encoding functionally active GDNF-like compounds in testis may cause disorders or testicular tumors being or resembling seminoma.

In addition, GDNF-like factors, including GDNF, neurturin, artemin and persephin as such or in interaction with, receptors and GFRα-co-receptors also promote development of a variety of cell types. For example, GDNF-like factor/receptor/co-receptor interactions regulate spermatogenesis of germ line. Abnormal or aberrant activity in the signaling pathways in the testicular organs can lead to disorders including tumors associated with cellular development of cells of these organs. The present invention is aimed at providing cures for such diseases using the GDNF signaling pathway and means methods for modulating its signaling as a target.

General Description of the Invention

The present invention is based on a method, whereby it is possible to study pathogenesis of human seminoma by old, glial cell line-derived neurotrophic factor (GDNF)-like factor, particularly GDNF, overexpressing mice with disturbed regulation of differentiation of germ cells and secondly it provides a system for studying seminoma and thereby developing drugs or active ingredients and therapies useful for treating seminoma. The present invention discloses results which demonstrate with transgenic mice that targeted overexpression of particularly GDNF, a ligand for the Ret proto-oncogene, in undifferentiated spermatogonia promotes malignant testicular germ line tumors, which are invasive and contain aneuploid cells. Thus, the data show that modulation, e.g. a deregulated stimulation, of a proto-oncogene by its ligand can be an initiative event in carcinogenesis. By their histological, molecular and histochemical characteristics the GDNF-induced tumors mimic classic seminomas in men, representing the first animal model for this tumor type. The results are obtained using GDNF, but it does not exclude that other GDNF-like compounds or factors can be used in a similar way.

GDNF-like factor

Glial cell line-derived neurotrophic factor (GDNF), a ligand for Ret receptor tyrosine kinase, is a distant member of the transforming growth factor β (TGF-β) superfamily. It maintains several sets of neuronal cells, is required for enteric innervation, regulates ureteric branching in the embryonic kidney, and controls cell fate decision of undifferentiated spermatogonia in the testis (WO 00/10594). The signal transducing receptor complex for GDNF is composed of Ret and GDNF family receptor α (GFRαl) (reviewed by Saarma, M. and Sariola, H. , Microsc. Res. Tech. 46:292-302, 1999).

Mice overexpressing GDNF show accumulation of undifferentiated spermatogonia and they are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. In older mice overexpressing GDNF nonmetastatic tumors are regularly formed. GDNF contributes to paracrine regulation of spermatogonial self- renewal and differentiation (Meng, X. , et al. , Science 287: 1489-1493, 2000).

GDNF is expressed in the testis by Sertoli cells, the paracrine regulators of spermatogenesis, whereas the GDNF receptors are being expressed by the undifferentiated spermatogonia (Meng, X. , et al. , Science 287: 1489-1493, 2000). In order to test the role of GDNF signaling in spermatogenesis the full-length human GDNF was targeted to the testis by the translation elongation factor lα promoter, which directs the transgene expression specifically to spermatogonia (Meng, X. , et al. , Science 287: 1489-1493, 2000). The GDNF overexpressing mice do not produce sperm and all male mice are infertile. In short, spermatogenesis is disturbed and undifferentiated spermatogonia accumulate within the seminiferous tubules. The spermatogonial clusters occlude the seminiferous tubules and are dissolved subsequently by apoptosis. The transgenic mice develop testicular atrophy, but the spermatogonia remain in the periphery of seminiferous tubules. The older transgenic mice develop testicular tumors. It is now shown that said tumors mimic classic seminomas in man.

Seminoma development in mice

The consequence of the continuous overexpression of GDNF by spermatogonia in several mouse strains were studied by histological analysis. Seminomatous tumors developed in old transgenic mice, which were targeted to overexpress GDNF in testes were followed for a few months. In a normal testis the spermatogonia are situated at the peripheral rim of the seminiferous tubules (Figure la), whereas clusters of spermatogonia within tubules can be observed in a transgenic mouse (Figure lb). Spermatogonia-type cells invading the interstitium in a transgenic testis were observed first by the microscopic analysis only in testes from a 7-month-old transgenic mouse (Figure lc). The transgenic mice frequently (90%) developed macroscopic testicular tumors starting at 1 year of age. In contrast, neither testicular tumors nor microinvasive germ line cells were observed in the same wild type mouse strains at any age. In most transgenic mice the testicular tumors were bilateral (56%), and all tumors were histologically uniform. They were composed of round cells with only scant cytoplasm and they invaded the interstitial tissue of the testes leaving the seminiferous tubules mostly untouched. In the largest tumors, tumor cells also colonized seminiferous tubules (Figure Id). No distant metastases were found, indicating a low or nil metastatic potential of the tumor cells or a too short follow-up time.

Demonstration of tumor origin

Spermatogonial markers, including EE2, and germ line markers, including TRA98 were used to confirm the germ line origin of the microinvasive and tumor cells (Figure le - Figure 11). The testicular germ line cells were detected by TRA98 antibody (Figure le - Figure Ih), whereas the EE2 antibody is highly specific to spermatogonia (Figure li - Figure 11). The germ line markers were also expressed by the testicular tumors (Figure lh and Figure 11). Clusters of spermatogonia were detected within seminiferous tubules of an approximately one month old testis (Figure If and Figure lj) whereas microinvasive spermatogonia in the interstitium of 7 month old testis were detected (Figure lg and Figure Ik). Leydig or Sertoli cell contribution to tumor development were excluded by specific markers, including 3β-HSD and WTl cRNA probes. Like the intratubular spermatogonial clusters in young transgenic mice (Meng, X., et al. , Science 287: 1489-1493, 2000), the tumor cells expressed the GDNF transgene (Figure lm), Ret (Figure In) and GFRal (Figure lo) detected by in situ hybridization, as well as elevated levels of hyperphosphorylated Ret protein detected by immunoprecipitation-western blotting (Figure 2a - Figure 2b) .

Demonstration of activation of Ret Downstream signaling molecules of Ret, such as AKT (protein kinase B) and mitogen-activated protein kinases (MAPK) ERK1/2, are phosphorylated upon Ret activation (reviewed by Airaksinen, M. S. , et al. , Mol. Cell Neurosci. , 13:313-325, 1999). Consequently activation can be measured as phosphorylation of Ret and its downstream targets and used for studies of wild type testis and GDNF-induced testicular tumors. Immunoprecipitated Ret was first blotted with Ret antibodies and thereafter with phosphotyrosine antibodies (pTyr). ERK1/2, phosphoERKl/2 (pERKl/2), AKT and phosphoAKT (pAKT) were detected by western blotting (Figure 2a - Figure 2f). Only about two-fold increase in the ERK1/2 phosphorylation was found in the experiments performed, but the phosphorylation of AKT increased about six-fold in the tumors as compared to the wild type testis (Figure 2d and Figure 2f). Since the Ret-mediated transforming effect is known to be critically dependent on the activation of the AKT pathway in several cancer types, its high activity in the GDNF-induced seminomas indicates that AKT is involved in the oncogenesis of these tumors as well.

Karyotype and DNA contents of the GDNF overexpressing testes and testicular tumors were further studied using flow cytometric analysis (Figure 3a - Figure 3c). Karyotyping was done either from frozen tissues or paraffin embedded material. Mouse spleen cells served as diploid controls (Figure 3a). When the DNA-ploidy was analyzed in young transgenic testes without tumors, no aneuploidy was found. A tumor- free testis of a transgenic mouse at 9 months of age showed a diploid karyotype (Figure 3b). However, mitoses with a tripolar organization indicating a triploid DNA content were occasionally observed in the spermatogonia of atrophic testes. Such cells might represent carcinoma in situ (CIS) cells, but they were too few to be detected by flow cytometry. The testis tumors of old transgenic mice exhibited aneuploidy (Figure 3 c). The mitotic index of the tumor cells was constantly high (approximately 10 mitotic figures / 40 x magnification) and the mitotic figures were often atypical. A distinct triploid peak was always observed, while a considerable number of tumor cells appeared to be hypodiploid. No peak at the tetraploid or higher level was found. The differentiation state of tumor cells was characterized with in situ markers for prepubertal spermatogonia and embryonic gonocytes. More details are presented in the Example 8.

Tumor phenotype and alkaline phosphatase

The tumor phenotype was further characterized by alkaline phosphatase reaction on frozen sections. Placental alkaline phosphatase is expressed by embryonic germ line cells, the gonocytes and it is normally downregulated postnatally (Figure 4d). Placental alkaline phosphatase is applicaple for demonstrating presence of tumor cells. It was for example demonstrated that in young wild type testis only the basement membranes showed alkaline phosphatase reactivity, while germ cells were unlabeled (Figure 4d) whereas tumor cells showed a strong alkaline phosphatase reactivity (Figure 4f). In addition, it was also seen in a few cells in the spermatogonial clusters of young transgenic mice (Figure 4e) indicating that susceptibility to develop seminoma could be detected at much earlier stage.

Comparing properties of GDNF-tumors and human seminoma

The GDNF-induced tumors resemble classic human seminomas in several important aspects. Firstly, the tumors consisted of a gonocytic cell type without the thread-like chromatin and giant cells that are typical for spermatocytic seminomas. Secondly, no peaks at the tetraploid or higher level were found in the flow cytometric analysis. Instead, a triploid peak was present which is a characteristic karyotype in classic seminomas. Thirdly, all GDNF-induced tumors were alkaline phosphatase-positive like in classic seminomas. In spermatocytic seminomas such cells are only sporadically observed. Fourthly, the development of the GDNF-induced tumors resembled that of classic seminomas. In mice immature spermatogonia-like germ cells, which seem to be comparable to CIS cells in man, were present before macroscopic tumors appeared. Human CIS cells are also alkaline phosphatase positive and are unable to undergo differentiation.

In the present invention the first microinvasive spermatogonia appeared already at 7 months of age, indicating that the pathogenesis of these tumors can be traced back to a young age. A possible difference between the mouse and human tumors is the absence of large lymphocyte infiltrates in the GDNF-induced tumors while these are present in most but not all classic seminomas (Looijenga, L. H. J. and Oosterhuis, J. W. , Rev. Reproduct. , 4:90-100, 1999).

The morphology, histochemical and molecular characteristics, and aneuploidy of the GDNF-induced tumors are similar to human classic seminomas. Thus, these transgenic mice represent the first animal model for this tumor type that is the most common testicular tumor in men. Consequently, it seems highly probable that the GDNF-expressing mice can be used as a model for developing drugs or active ingredients for treating human seminomas. The high frequency of tumors in this mouse model provides further evidence that a deregulated stimulation of a normal receptor tyrosine kinase by its normal ligand has a carcinogenic potential.

The present invention can be divided in two separate lines. One line is related to the use of GDNF-like compounds and especially to that which aberrant or detrimental expression of said compounds may cause in testis. This line of the present invention, ultimately, provides transgenic animals which overexpress GDNF-like compounds and can be used as test animal models when studying seminoma. For the preparation of transgenic animals, vectors, plasmids, cells or cell-lines can be provided with instructions for use. Said line of the present invention also provides GDNF-like factors, which can be used for screening compounds for their capacity of modulating the interaction between GDNF-like compounds and Ret including the co-receptors thereof. Said GDNF-like compounds produced by recombinant DNA techniques can be provided as test kits for screening libraries of chemical substances, but recombinant GDNF-like compounds can also be used to protect testicular tissue against detrimental effects of radiation.

The other line of the present invention is related to means and methods for overcoming the detrimental effects of overexpression of GDNF-like compounds. This line is related to different types of modulating substances or active ingredients, including small molecules, antibodies and nucleic acids, which by different means and methods are capable of modulating, preferably inhibiting the interactions between GDNF-like molecules.

Accordingly, one aspect of the invention pertains to providing functionally active GDNF-like compounds and nucleic acids encoding functionally active GDNF-like compounds to combat the effects of the activities of GDNF-like compounds. Nucleic acids encoding functionally active GDNF-like compounds are provided as vectors, e.g. recombinant expression vectors, containing the nucleic acid molecules of the invention and as host cells into which such nucleic acid sequences, vectors or constructs have been introduced. In one embodiment, a host cell is used to produce GDNF-like factors by cultivating the host cell in a suitable medium. If desired, the GDNF-like factor can then be isolated from the host cell or the surrounding medium and used for screening compounds capable of modulating the interactions between GDNF-like compounds and Ret.

Alternatively, the host can be a transgenic animal, which can be used as a test animal model for further testing of the value and safety of molecules which can modulate GDNF-like factors. Yet another aspect of the invention pertains to vectors, cells, cell-lines or transgenic non-human animals in which a gene encoding a functionally inactive or conditionally inactivatable GDNF-like factor has been introduced. Such test animals could be useful for studying pathogenesis of seminoma.

In one embodiment, the genome of the cell, cell-line or non-human animal has been altered by introduction of a nucleic acid encoding an inactive or an inactivatable GDNF-like factor as a transgene. In another embodiment, an endogenous gene or nucleic acid encoding a functionally inactive or conditionally inactivatable GDNF-like compound is characterized by having an altered, e.g. , functionally disrupted gene obtained by homologous recombination. It can be provided to the subject suffering from seminoma as an active ingredient by cell or gene therapy.

Still another aspect of the invention pertains to an isolated GDNF-like factor, e.g. a biologically active portion, thereof. In a preferred embodiment, the isolated GDNF-like factor or portion thereof can bind to its respective GFRα-receptor or parts therof and thereby modulate the interaction between the GDNF-like compound and Ret, but it can also be used for determining the presence or absence of GDNF-like factors.

Another aspect of the invention pertains to methods for modulating, i.e. stimulating or inhibiting, a GDNF-like factor mediated cell activity, e.g., the function, proliferation or differentiation of the cell. Such methods include contacting the cell with a modulating substance, which stimulates or inhibits a GDNF-like factor activity or the expression of a functionally active, inactive or inactivatable nucleic acid encoding a GDNF-like factor in such a way that a cell associated activity is altered relative to the cell associated activity of said cell in the absence of the modulating substance.

In one preferred embodiment, the cell is capable of responding to a GDNF-like compound or neurotrophic factor through a signaling pathway involving a GDNF-like factor. The modulating substance, which modulates a GDNF-like factor can be an antisense or defective nucleic acid or an antagonist, i.e. a substance which inhibits the activity of GDNF-like factors or the GDNF-factor expressing nucleic acids or modulates the interaction between GDNF-like factors and Ret.

Examples of modulating substances, which modulate, i.e. stimulate or inhibit, GDNF-like factor activity or GDNF-like factor expressing nucleic acids include small molecules, antibodies or nucleic acids encoding inactive or inactivatable GDNF-like factors that have been introduced into a cell. Examples of substances, which modulate or preferably inhibit GDNF-like activity or expression include small molecules, antisense nucleic acids encoding GDNF-like factors, which disturb the expression of GDNF-like compounds on mRNA level. Also nucleic acid molecules with aberrant expression which are capable of hybridizing to the correct nucleic acid and thereby disturbing its function, are modulating substances as well as antibodies and small molecules that specifically bind to GDNF-like factors.

In a preferred embodiment, the modified or altered nucleic acid, vectors, constructs, cells or cell-lines are present as such or in a controlled release matrix which can be placed within the subject, whereby the modulator or active ingredient can be administered to the subject even locally during a surgical intervention.

The present invention particularly pertains to methods for treating subjects suffering from seminoma or testicular tumors mediated by abnormal GDNF-like factor activity and/or expression. For example, the invention pertains to methods for treating a subject having a disorder characterized by aberrant GDNF-like factor activity or nucleic acid expression. Such conditions can easily be diagnosed by determining respective GDNF-like compound. These methods include administering to the subject one or more GDNF-like factor modulating, stimulating or inhibiting, small molecules, antibodies, etc. , such that treatment of the subject occurs.

In another embodiment, the invention pertains to methods for treating a subject suffering from testicular tumors, especially seminoma, comprising administering to the subject a substance modulating the interactions between GDNF-like factor and Ret, i.e. a modulator, stimulator or inhibitor, such that treatment occurs.

In other embodiments, the invention pertains to methods for treating a subject suffering from cancer comprising administering to the subject a GDNF-family related compound as a substance protecting spermatogonia against radiation or drug injuries or alternatively a modulating compound activating the GDNF-like compound mediated signaling pathway, so that protection occurs.

Another aspect of the invention pertains to methods for diagnosing by detecting the presence or abscence of a GDNF-like factor, or fragments thereof, in a biological sample. In a preferred embodiment, the methods involve contacting a biological sample, e.g. , any tumor (biopsy) sample, with a compound capable of recognizing, e.g. specifically binding or detecting a GDNF-like factor protein or nucleic acid encoding a functionally active, inactive or inactivatable mRNA such that the presence of GDNF-like factor is detected in the biological sample. The compound can be, for example, a labeled nucleic acid probe capable of hybridizing to a specifically recognizable fragment of a nucleic acid encoding a functionally active, inactive or inactivatable GDNF-like factor or a mRNA. Alternatively, the compound can be a labeled antibody capable of binding to GDNF-like factor protein. The invention further provides methods for diagnosis of a subject with, for example, a testicular system disorder caused by aberrant expression of a GDNF-like factor, e.g. , based on detection of GDNF-like factor or mRNA. In one embodiment, the method involves contacting a cell, tissue, or fluid sample (e.g., a tumor sample) from the subject with an substance capable of detecting, e.g. by specifically binding, a GDNF-like factor or mRNA, determining the amount of GDNF-like factor or mRNA expressed in the sample to a control sample and forming a diagnosis based on the amount of GDNF-like factor or mRNA expressed in the sample as compared to the control sample. The sample should preferably be a tumor cell sample. Kits for detecting a GDNF-like factor, or fragments thereof, in a biological sample are also within the scope of the invention.

Still another aspect of the invention pertains to methods, e.g. , screening assays, for identifying a modulating compound, the active ingredient for treating a disorder characterized by aberrant expression, particularly overexpression of GDNF-like compounds, particularly GDNF or aberrant protein activity in a testicular cell.

These methods typically include assaying the ability of a compound to modulate, i.e. stimulate or inhibit the expression of the GDNF-like factor encoding gene or the activity of the GDNF-like factor thereby identifying a compound for treating a disorder characterized by nucleic acid encoding aberrant GDNF-like factor or protein activity. In a preferred embodiment, the method involves contacting a biological sample obtained from a subject suffering from a pathogenic disorder, e.g. seminoma with a compound e.g. a co-receptor or ligand, and thereby determining the amount of GDNF-like factor expressed and/or measuring the activity of the GDNF-like factor in the biological sample, comparing the amount of GDNF-like factor expressed in the biological sample and/or a recordable GDNF-like factor biological activity in the cell to that of a control sample. An alteration in the amount of GDNF-like factor expression or GDNF-like factor activity in the cell exposed to the modulator compound or active ingredient in comparison to the control is indicative of a stimulation or inhibition of GDNF-like factor expression and/or GDNF-like factor activity .

The present invention also pertains to methods for treating testicular tumors particularly seminomas to block or inactivate GDNF family-related compounds and thereby the formation of tumors by administering to a subject suffering from seminoma a therapeutically effective amount of a modulator substance or active ingredient using cell implantation or gene therapy. In gene therapy nucleotide containing compounds, such as nucleic acid, e.g. DNA, RNA, including other macromolecules, including proteins, polypeptides, antibodies and parts therof, as well as combinations of said nucleotides and/or polypeptides are used to produce, for example, a protein or a polypeptide, which has a desired effect on the disease to be treated. Gene transfer may result in stable or transient, aberrant or correct expression of the transferred gene by the cells. Gene therapy can be practiced either in vivo by direct gene transfer to the target cells in the body or ex vivo by gene transfer to cell cultures to be transplanted into the body. In both cases ability to transfer DNA in active form into cells is essential for success. After gene transfer, gene expression is regulated by the machinery of the cell and the regulatory elements of the transgene.

Depending on the gene sequence, gene expression may be limited to the cell population of interest or it may be induced with exogeneously administered compounds such as small molecular weight drugs. Successful gene transfer or introduction of exogeneous DNA into target cells is a prerequisite in gene therapy as well as in many other applications of gene technology. It is also essential to transfer DNA in intact form into the target cells in the body either after injection or by application on mucosal surface.

The present invention further pertains to administering to a subject suffering of seminoma gene-based drugs. Inhibitors of gene activity, such as antisense and antigene oligonucleotides and ribozymes are important forms of gene-based drugs. These compounds are composed of strands of DNA, RNA or their modified forms. They inhibit the function of the target gene either at the level of gene transcription, translation or splicing. Therefore, aberrant gene expression at too low or too high level or in incorrect form can be altered with these technologies. Other forms of gene-based drugs include gene correction and gene modifier oligonucleotides. Gene correction oligonucleotides can for example be composed of chimeric oligonucleotide structures with modified RNA or DNA. These compounds are able to provide gene correction in the target cells at low frequency. Importantly, the gene correction is permanent and thus gene correction oligonucleotides can be used to treat genetic diseases. Gene modifier oligonucleotides are able to turn on or off gene expression e.g. at the level of gene promoter.

In all aforementioned forms of gene therapy, DNA, RNA or their modified forms as such or as plasmids, vectors, etc. , must be able to permeate into the cytoplasm or nucleus of the cells. Permeation is not optimal due to the hydrophilicity and the large molecular weights of these nucleotide containing compounds. They are also prone to degradation in body fluids and they bind to proteins in the cytoplasm of cells.

In addition to its medical applications gene transfer is an important part of modern research of cell biology, molecular biology and many other sub-disciplines of biology. Gene transfer is used frequently in laboratories in order to study the functions of particular gene sequences. Likewise transfer of antisense oligonucleotides is utilized to block the function of a certain gene and thereby elucidate its role in the cell biology. Importantly gene transfer is used in order to genetically engineer cells that express a certain gene in a stable fashion or under the control of a drug inducible gene promoter. Efficient gene transfer reagents are needed for the gene transfer protocols also in research laboratories.

Potential medical indications of the aforementioned technologies include treatment of seminoma based on disorders in gene expression. Gene therapy and administration of DNA, RNA or their modified forms may be practiced using different delivery or administration routes, including intravenous, oral, nasal, pulmonary, intramuscular, ocular, topical, subconjunctival, intravitreal, subretinal, dermal, topical, transdermal, electrically assisted or local application to different sites in the body e.g. during surgical inventions of semioma or as an injection or a solid controlled release device or matrix, as microparticles or as implants.

In vitro transfer of nucleotide containing compounds is performable using cell cultures, the cells of which can be obtained from different sources and can be cultivated by per se known methods. The transfection efficiencies of nucleotide containing compounds can be evaluated by several per se known methods. In vivo gene transfer of nucleotide containing compounds can be tested in animal models using plasmid complexes. The transfer is recorded as marker gene expression in germ cells or seminoma.

When gene transfer is used for treating diseases having a genetic component, including cancer or other inherited conditions, the active ingredient can be provided as liposomes, which are used as a part of a therapeutic formulation in combination with other physiologically and/or pharmaceutically acceptable additives. The characteristics of the components in the delivery system depend on the route of administration. The delivery system formulation may contain, in addition to the nucleotide containing compound and the liposome-forming compound, also other components, including lip ids, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.

The preferred formulation used in the present invention may be nucleotide containing compounds combined with pharmaceutically acceptable additives and with the cationic lipids or polymers. These are generally provided as thin films, lamellar layers or liposomes. Administration of the nucleotide containing compounds can be carried out in a variety of conventional ways, such as by cutaneous, subcutaneous, intramuscular, or intravenous injection. When administering formulations intravenously, cutaneously or subcutaneously they should be in form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. In addition to the liquid formulations of the complexes the complexes can be dried using e.g. freeze drying, spray drying and other known methods to provide the complexes in powder form. These complexes can be reformulated into solid and semi-solid materials, such as controlled release polymers (leachable, bioerodible, biodegradable, channel forming). These preparations can be manufactured as matrices, reservoir devices, microspheres, or semi-solid pastes. Such technologies are well known in the state of art and they could provide controlled release of the nucleic acid containing compounds and would provide administration of active ingredients over prolonged periods of time. Such devices could be placed in several tissues and body cavities to treat or prevent various diseases.

The amounts of nucleotide containing compound in the formulation of the present invention and the duration of treatment will depend upon the nature and severity of the condition being treated, on the nature of prior treatments which the patient has undergone, and on the responses of the patient. Ultimately, the attending physician will decide the amounts of nucleotide containing compound with which to treat each individual patient and the duration of treatment. Initially, the attending physician will administer low doses of the formulation and observe the patient's response. Larger doses of the formulation are administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.

The invention also pertains to methods for identifying a compound which interacts with (e.g. , specifically binds to) a GDNF-like factor. These methods can include the steps of contacting the GDNF-like factor, a fragment thereof, or a cell expressing GDNF-like factor, with the detector compound or marker or probe under conditions, which allow binding of the detector compound to the GDNF-like factor to form a complex and detecting the formation of a complex of the GDNF-like factor and the detector compound in which the ability of the compound to bind to the GDNF-like factor is indicated by the presence of the compound in the complex.

The invention further pertains to methods for identifying a modulating compound, which stimulates or inhibits the interaction of the GDNF-like factor with a target molecule, this can be e.g. , persephin, artemin or neurturin interacting with the tyrosine kinase receptor Ret. In these methods, the GDNF-like factor is contacted, in the presence of the modulating compound, with the target molecule under conditions which allow binding of the target molecule to the GDNF-like factor to form a complex. An alteration, e.g. , an increase or decrease, in complex formation between the GDNF-like factor and the target molecule as compared to the amount of the complex formed in the absence of the compound is indicative of the ability of the compound to stimulate or inhibit the interaction of the GDNF-like factor with a modulating compound molecule.

The present invention also enables the production of a transgenic non-human animal capable of overexpressing GDNF-like factors and non-human transgenic animals lacking a functional GDNF-like factor and which is useful as a test animal model for studying GDNF-like factor-mediated signaling in germ cell line tumors, particularly in seminoma. Said animals are obtainable from a genetically modified compatible cell-lines, containing a genomic gene coding for a functional GDNF-like receptor, which genomic gene is functionally inactivated or conditionally inactivatable.

The present invention also provides a method for obtaining a cell-line useful in the production of the transgenic non-human animal characterized by having a genomic gene encoding a functionally active GDNF-like factors inactivated by introducing into stem cells a nucleic acid sequence, which is capable of integrating to said genomic GDNF-like factor encoding gene and by said integration functionally inactivating said genomic gene or making it conditionally inactivatable.

The cell-line lacking a functionally active, inactive or mactivatable GDNF-like factor encoding nucleic acid is obtainable by established gene targetmg methods described e.g. in the International patent applications WO 99/62332 and/or WO 00/10564. A nucleic acid sequence capable of being integrated into the genomic gene coding for a functionally inactivated or conditionally mactivatable GDNF-like factor is prepared and inserted into a targeting vector or construct with a suitable selectable nucleic acid sequence acting as a marker. Said targeting construction is capable of functionally inactivating the nucleic acid encoding functionally active GDNF-like factors. By the integration, it functionally inactivates the genomic gene or makes it conditionally inactivatable by per se known methods.

The cell lines of the present invention are useful not only for producing transgenic non-human animals but also for studying in vitro GDNF-like factor-mediated signaling in seminal cells, especially in testicular carcinoma cells, such as seminoma.

The present invention describes a method, whereby it is possible to study pathogenesis of human seminoma by old GDNF-overexpressing mice with disturbed differentiation of germ cells and secondly it provides a system for studying and treating testicular tumors. The invention is further illustrated in the following examples, which disclose the material and methods used in the experiments used to demonstrate the involvement of GDNF in the pathogenesis of human seminomas and the possible involvement of the pathway for the therapy of testicular tumors as well as the results obtained. Said experimental part should not be construed to limit the scope of the invention. Those skilled in the art are able to use different applications based on the results obtained in the following experimental part.

Methods

DNA and Karyotype Analysis. Flow cytometric analysis of the DNA contents of testicular tumor cells was performed by FACScan flow cytometry (Becton Dickinson) after ethidium bromide labeling of the nuclei (CellFIT Cell-Cycle Analysis version 2.01.2). Mouse spleen cells served as diploid cell controls. Karyotyping was done either from frozen tissues or 60 μm sections of paraffin embedded material.

Histology and Immunohistochemistry. For histology and immunohistochemistry, testes and other tissues were freshly dissected and frozen in liquid nitrogen or fixed in either Bouin or 4% paraformaldehyde for 2-24 hours depending on the size of the sample. The paraffin embedded samples were sectioned at 5 μm and stained by hematoxylin/eosin. Paraffin-embedded sections were used for immunohistochemistry with EE2 and TRA98 antibodies (Koshimizu, U. , et al. , Mol. Reprod. Devel. , 40:221-227, 1995; Tanaka, H., et al. , Int. J. Androl. , 20:361-366, 1997). After deparaffinization, sections were incubated with the EE2 and TRA98 antibodies diluted 1:500 and 1: 1000, respectively, followed by standard biotin-streptavidin-peroxidase labeling (Vector Laboratories Inc. , CA). The enzymatic method for alkaline phosphatase activity on frozen sections was as described by Looijenga, L. H. J. and Oosterhuis, J. W. , Rev. Reproduct. , 4:90-100, 1999.

In Situ Hybridization. cRNA in situ hybridization was performed as described previously (Wilkinson, D. and Green, P. , In: Postimplantation Mammalian Embryos. A Practical Approach (eds. A. Copp and D. Cockroft). Pp. 155-171. Oxford University Press, London, UK, 1990). 35s-_Uridine-labeled antisense and sense cRNAs to GDNF, Ret, and GFR l were as described previously (Meng, X. , et al. , Science 287: 1489-1493, 2000). WTl probe was from Dr. Jordan A. Kreidberg (Departments of Medicine, Harvard Medical School, Boston, USA). The L-Fng probe and 3&-HSD probe were as described previously (Harada, H. , et al. , J. Cell Biol. , 147: 105-120, 1999; Koga, M. , et al. , Biol. Reprod. , 58:261-265, 1998). The hybridization temperature was 52°C and slides were exposed at +4°C for 2 weeks. The slides were photographed with an Olympus Provis microscope equipped with a CCD camera (Photometries Ltd) . The dark field images were processed in PhotoShop 4.0 program. Sense controls did not show labeling above background (not shown) .

Immunoprecipitation and western blot of Ret phosphorylation assay. Homogenisation of samples, either freshly-dissected or frozen, was performed in lysis buffer (50 mM HEPES, 1 % Triton X-100, 50 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 % Aprotinin, 1 mM PMSF, 0.5 mM sodium vanadate). After 30 min incubation in ice the homogenate was centrifuged at 13,000g for 2 minutes, and the supernatant was analysed by either immunoprecipitation-western blot (IP) or western blotting. The ly sates were immunoprecipitated with rabbit polyclonal antibody to human Ret cross-reacting with mouse Ret (Viglietto, G. , et al. , Int. J. Oncol. , 16:689-694, 2000; Ret antibody is a gift from Dr. M. Santoro, University of Naples, Italy), separated on a 7.5% SDS-PAGE gel, and blotted on Hybond ECL membrane (Amersham). Ret protein was blotted by the same Ret antibody and phosphorylation of Ret was detected by monoclonal phosphotyrosine antibody (Transduction Laboratories). Signals were detected using an ECL plus kit (Amersham, RPN 2132).

MAPK and AKT phosphorylation assays. Total protein lysates mentioned above were separated on a 12% SDS-PAGE gel, blotted, and probed with polyclonal antibodies to phosphorylated forms of either ERK1/2 (Promega) or AKT (New England Biolabs). The blots were then reprobed with the antibodies, which recognize both the phosphorylated and unphosphorylated forms ERKl/2 (Promega) or AKT (New England Biolabs).

Example 1

GDNF-transgene carrying mouse strains

A GDNF-transgene carrying transgenic mouse strain, the GDNF t-mouse strain (the transgene transferring mouse strain), is described in the International patent application WO 00/10594. Said mouse is a model for infertility caused by disturbances in the differentiation of sperm cells. It is characterized in that GDNF, compounds acting like said GDNF, another Ret receptor, another GDNF-receptor activating compound or a compound, which activates the Ret receptor signal transmitting tubules in spermatogonia, are suitable as a male contraceptive.

It was shown that the GDNF t-mouse model is suitable as an animal model for studying the pathogenesis of human seminoma, especially testicular tumors, by demonstrating that testicular tumors developed regularly in old GDNF overexpressing mice. While their testes showed atrophy below a year of age and the transgenic spermatogonia remained dormant for several months. After a year of age, these mice formed non-metastatic testicular tumors. Out of 12 mice older than a year of age, ten had bilateral and two unilateral tumors. Said transgenic mice looked healthy, but often showed swelling of the scrotum. When the mice were sacrified, the macroscopic and microscopic features of all organs were normal except in the enlarged testes, the longest diameter of which was about 5 cm. In transsections, the testicular tumors were macroscopically composed of white amorphous tissue.

Microscopic survey revealed some atrophic seminiferous tubules with a rim of spermatogonia and Sertoli cells. However, the tumors consisted predominantly of homogeneous fields of round unorganized, middle-sized cells with a large dark-staining nucleus and scarce cytoplasm. Although the morphology of individual tumor cells was similar to the cluster cells in the young GDNF overexpressing mice, the general histoarchitecture of the tumors resembled human seminomas. In contrast to the highly invasive human seminomas and metastatic malignancies, the tunica albuginea that surrounds the testis was intact in the transgenic mice. Tumor cell invasion in the surroundin 'ge tissues or distant metastases were not found.

Example 2

The overexpression of GDNF in transgenic mice causes testicular tumors

The long-term consequence of the continuous overexpression of GDNF by spermatogonia is studied using FVB and NMRI mouse strains. For histological analysis testes and other tissues are freshly dissected and frozen in liquid nitrogen or fixed in either Bouin or 4% paraformaldehyde for 2-24 hours depending on the size of the sample. The paraffin embedded samples are sectioned at 5 μm and stained by hematoxylin/eosin.

The overexpression of GDNF by spermatogonia was followed until 15 months of age (37 mice older than 5 months from FVB and NMRI mice strains). The atrophic testes of 19 mice between 6 and 11 months of age were analyzed. In a normal testis the spermatogonia were situated at the peripheral rim of the seminiferous tubules (Figure la), whereas clusters of spermatogonia within tubules could be observed in a transgenic mouse at 4 weeks of age (Figure lb). One 7-month-old mouse testis showed small groups of spermatogonia-type cells invading the interstitium (Figure lc). These cells were only observed by the microscopic analysis. All other testes showed typical atrophy at these ages. Thereafter, the transgenic mice frequently developed macroscopic testicular tumors starting at 1 year of age (16 out of 18 mice). In contrast, neither testicular tumors nor microinvasive germ line cells were observed in the same wild type mouse strains at any age (n > 200). In most transgenic mice the testicular tumors were bilateral (56%), and all tumors were histologically uniform. They were composed of round cells with only scant cytoplasm and they invaded the interstitial tissue of the testes leaving the seminiferous tubules mostly untouched. In the largest tumors, tumor cells also colonized seminiferous tubules (Figure Id). Solid sheet of invasive spermatogonia-type cells in a transgenic mouse developed the testicular tumor at the age of one year. No distant metastases were found, indicating a low or nil metastatic potential of the tumor cells or a too short follow-up time.

Example 3

Characterization of tumor cells in old GDNF overexpressing mice

To further characterize the tumor cells in the old GDNF overexpressing mice, they are analyzed with markers of spermatogonia and with GDNF probes and receptors. The mRNA expression of the GDNF transgene and its receptors (Ret and GFRαl) is analyzed by in situ hybridization; GDNF, Ret and GFRal transcripts are highly expressed by the tumor cells. Thus, the tumor cells mimic the cells in the testicular clusters of young GDNF overexpressing mice by their receptor and transgene characteristics. No Sertoli cell markers, such as GATA-1 and WT-1, are seen in tumor cells by immunohistochemistry or in situ hybridization. The absence of 3β-HSD, a Ley dig cell marker, excludes the Ley dig cell contribution to tumor.

Thus, the similarity of the testicular tumors in GDNF overexpressing mice with human seminomas indicates that the GDNF overexpressing transgenic mice can be used as a new mouse model for human seminomas. Furthermore, the data suggest that activity of the GDNF signaling pathway is involved in the pathogenesis of human seminomas and this signaling pathway may be a new target for the therapy of testicular tumors.

Example 4

Immunohistochemistry with EE2 and TRA98 antibodies

A spermatogonial marker EE2 and a germ line marker TRA98 (Koshimizu, U. , et al., Mol. Reprod. Devel. , 40:221-227, 1995; Tanaka, H. , et al. , Int. J. Androl. , 20:361-366, 1997) are used to confirm the germ line origin of the microinvasive and tumor cells. Testes and other tissues are freshly dissected and frozen in liquid nitrogen or fixed in either Bouin or 4% paraformaldehyde for 2-24 hours depending on the size of the sample. The paraffin embedded samples are sectioned at 5 μm and stained by hematoxylin/eosin. After deparaffinization, sections are incubated with the EE2 and TRA98 antibodies diluted to 1:500 and 1: 1000, respectively, followed by standard biotin-streptavidin-peroxidase labeling (Vector Laboratories Inc. CA).

The testicular germ line cells were detected by TRA98 antibody (Figure le - Figure lh). The EE2 antibody is highly specific to spermatogonia (Figure li - Figure 11). The germ line markers were also expressed by the testicular tumors (Figure lh and Figure 11). Clusters of spermatogonia were detected within seminiferous tubules in a 4-week-old transgenic testis (Figure If and Figure lj). In a 7-month-old testis microinvasive spermatogonia in the interstitium were detected (Figure lg and Figure Ik). A possible Leydig or Sertoli cell contribution to the tumors was excluded by 3R-HSD and WTl cRNA probes, respectively, that did not label the tumor cells (data not shown). Like the intratubular spermatogonial clusters in young transgenic mice (Meng, X. , et al. , Science 287: 1489-1493, 2000), the tumor cells expressed the GDNF transgene (Figure lm), Ret (Figure In) and GFRal (Figure lo).

Example 5

Immunoprecipitation and western blot of Ret phosphorylation assay

Phosphorylation of Ret and its downstream targets were studied in wild type testis and GDNF-induced testicular tumors. Homogenisation of samples, either freshly-dissected or frozen, was performed in lysis buffer (50 mM HEPES, 1 % Triton X-100, 50 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 % Aprotinin, 1 M PMSF, 0.5 mM sodium vanadate), after 30 min incubation in ice the homogenate was centrifuged at 13,000g for 2 minutes, and the supernatant was analysed by either immunoprecipitation-western blot (IP) or western blotting. The lysates were immunoprecipitated with rabbit polyclonal antibody to human Ret cross-reacting with mouse Ret (Viglietto, G. , et al. , Int. J. Oncol. , 16:689-694, 2000; Ret antibody is a gift from Dr. M. Santoro, University of Naples, Italy), separated on a 7.5% SDS-PAGE gel, and blotted on Hybond ECL membrane (Amersham). Ret protein was blotted by the same Ret antibody and phosphorylation of Ret was detected by monoclonal phosphotyrosine antibody (Transduction Laboratories). Signals were detected using an ECL plus kit (Amersham, RPN 2132).

Tumor cells expressed elevated levels of hyperphosphorylated Ret protein as detected by immunoprecipitation-western blotting (Figure 2a - Figure 2b) .

Example 6

MAPK and AKT phosphorylation assays Downstream signaling molecules of Ret, such as AKT (protein kinase B) and mitogen-activated protein kinases (MAPK) ERK1/2, are phosphorylated upon Ret activation (reviewed by Airaksinen, M. S. , et al. , Mol. Cell Neurosci. , 13:313-325, 1999; Trupp, M., et al. , J. Biol. Chem. , 274:20885-20894, 1999). Phosphorylation of Ret and its downstream targets were studied in wild type testis and GDNF-induced testicular tumors. Homogenisation of samples, either freshly-dissected or frozen, was performed in lysis buffer (50 mM HEPES, 1 % triton X-100, 50 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 1 % Aprotinin, 1 mM PMSF, 0.5 mM sodium vanadate), after 30 min incubation in ice the homogenate was centrifuged at 13,000g for 2 minutes, and the supernatant was analysed either by immunoprecipitation-western blot (IP) or western blotting.

Total protein lysates were separated on a 12% SDS-PAGE gel, blotted and probed with polyclonal antibodies to phosphorylated forms of either ERK1/2 (Promega) or AKT (New England Biolabs). The blots were then reprobed with the antibodies, which recognize both the phosphorylated (phosphoERKl/2 (pERKl/2), AKT and phosphoAKT (pAKT)) and unphosphorylated forms of ERK1/2 (Promega) or AKT (New England Biolabs) and detected by western blotting (Figure 2a - 2f) .

Only two-fold increase in the ERK1/2 phosphorylation was found, but the phosphorylation of AKT increased about six-fold in the tumors as compared to the wild type testis (Figure 2d and Figure 2f). Since the Ret-mediated transforming effect in both MEN2A and MEN2B cancer syndromes is critically dependent on the activation of the AKT pathway (Murakami, H., et al. , Biophys. Res. Commun. , 262:68-75, 1999; Segouffin-Cariou, C. and Billaud, M. , J. Biol. Chem., 275:3568-3576, 2000; De Vita, G. , et al. , Cancer Res., 60:3727-3731, 2000), its high activity in the GDNF-induced seminomas indicates that AKT is involved in the oncogenesis of these tumors as well.

Example 7

DNA contents and karyotype analysis of testicular tumor cells

Flow cytometric analysis of the DNA contents of testicular tumor cells is performed by FACScan flow cytometry (Becton Dickinson) after ethidium bromide labeling of the nuclei (CellFIT Cell-Cycle Analysis version 2.01.2). Karyotyping is done either from frozen tissues or 60 μm sections of paraffin embedded material.

Karyotype and DNA contents of the GDNF overexpressing testes and testicular tumors were studied using mouse spleen cells as diploid cell controls (Figure 3a). When the DNA-ploidy was analyzed in young transgenic testes without tumors, no aneuploidy was found (n= 10 mice between 3 to 10 months of age). A tumor-free testis of a transgenic mouse at 9 months of age showed a diploid karyotype (Figure 3b). Three tumors However, mitoses with a tripolar organization indicating a triploid DNA content were occasionally observed in the spermatogonia of atrophic testes. Such cells might represent carcinoma in situ (CIS) cells, but they were too few to be detected by flow cytometry. Three tumors were analyzed. A testis tumor of an old transgenic mouse exhibited aneuploidy (Figure 3 c). The mitotic index of the tumor cells was constantly high (approximately 10 mitotic figures / 40 x magnification) and the mitotic figures were often atypical. A distinct triploid peak was always observed, while a considerable number of tumor cells appeared to be hypodiploid. No peak at the tetraploid or higher level was found.

Example 8

The characterization immature phenotype of GDNF-induced seminomas

Further characterization of the tumor phenotype is done with molecules involved in Notch signaling. Notch has been implicated in the pathogenesis of leukemia, cervical and colon carcinoma, and Alagille syndrome (Lee, J. S. , et al., FEBS Lett. , 455:276-280, 1999). Lunatic fringe (L-Fng) is a modulator or active ingredient of Notch signaling. It inhibits Notch activation via the ligand Serrate, but enhances Notch activation via Delta (Irvine, K. D. , Curr. Opin. Genet. Devel. , 9:434-441, 1999). cRNA in situ hybridization is performed as described by Wilkinson, D. , et al. , In: Postimplantation Mammalian Embryos. A Practical Approach (eds. A. Copp and D. Cockroft). Pp. 155-171. Oxford University Press, London, UK, 1990. 35s-_uriciine-labeled antisense and sense GDNF, Ret, and GFRal cRNAs are as described by Meng, X., et al. , (Science 287: 1489-1493, 2000). WTl probe is from Dr. Jordan A. Kreidberg (Departments of Medicine, Harvard Medical School, Boston, USA). The L-Fng probe and 3&-HSD probe are as described (Harada, H., et al., J. Cell Biol., 147:105-120, 1999; Koga, M., et al., Biol. Reprod., 58:261-265, 1998). The hybridization temperature is 52°C and slides are exposed at +4°C for 2 weeks. The slides are photographed with an Olympus Provis microscope equipped with a CCD camera (Photometries Ltd). The dark field images were done using PhotoShop 4.0 software.

L-Fng was expressed in wild type testes only within a narrow time window during prepuberty, and it became undetectable at the onset of spermatogenesis (Figure 4a). In contrast, the intratubular spermatogonial clusters in young transgenic mice and the tumors invariably expressed L-Fng (Figure 4b - Figure 4c) . Sense controls did not show labeling above background (not shown) .

Example 9

Alkaline phosphatase reactivity

The tumor phenotype is further characterized by alkaline phosphatase reaction (Looijenga, L. H. J. and Oosterhuis, J. W. , Rev. Reproduct. , 4:90-100, 1999). Placental alkaline phosphatase is expressed by the embryonic germ line cells, the gonocytes (Looijenga, L. H. J. and Oosterhuis, J. W. , Rev. Reproduct., 4:90-100, 1999), and it is normally downregulated postnatally (Figure 4d). Testes and other tissues are freshly dissected and frozen in liquid nitrogen.

In a 4-week old wild type testis only the basement membranes showed alkaline phosphatase reactivity, while germ cells were unlabelled (Figure 4d). The tumor cells showed a strong alkaline phosphatase reactivity (Figure 4f) and, in addition, it was also seen in a few cells in the spermatogonial clusters of young transgenic mice (Figure 4e).

The reappearance of alkaline phosphatase by the transgenic spermatogonia and the continuous expression of L-Fng that is normally downregulated at puberty indicate that they initially maintained prepubertal features that gradually regressed to or were replaced by an embryonic gonocytic phenotype.

Example 10

Use of GDNF overexpressing mice for testing cytostatic drugs

A GDNF t-mouse strain (the transgene carrying mouse strain) overexpressing GDNF can be used for testing of cytostatic drugs or active ingredients for treatment of seminoma and to study the pathogenesis of seminomas.

Mice overexpressing GDNF are injected with substances inhibiting the formation of clusters of spermatogonia. At present a known specific inhibitor (Novartis ST1571) is used for treating lymphomas. The aim of the current experiment is to find similar substances for treating seminomas and which can be tested in cell culture and thereafter in GDNF overexpressing mice.

Example 11

The inhibition of Ret signaling and treatment of seminomas The downstream signaling pathways of Ret receptor tyrosine kinase are activated in the GDNF-induced seminomas (Figure 2) and drugs or active ingredients inhibiting these pathways are supposed to lead to tumor regression and therefore such molecules might be useful in treatment of human seminomas. The seminomas are treated with drugs or active ingredients that inhibit Ret signaling. The mechanisms by which Ret signaling is inhibited include the inhibition GDNF binding to receptor, inhibition of receptor complex formation and the inhibition of phosphorylation of Ret.

The drugs or active ingredients inhibiting Ret signaling include molecules that inhibit the ligand binding to receptor, inhibit dimerization of Ret receptor molecules or activity of downstream signaling cascade of Ret, inhibitors of phosphorylation of Ret (for example tyrphostins), or antibodies to Ret or GFRαl that are tagged with cytostatic drugs such as radioactive agents, pertussis toxin or cholera toxin, or other cytostatic agents. Radionuclides, which emit gamma-radiation are useful in diagnostics and treatment of seminoma. It is advantageous if the half-lifes of said radionuclides are from one hour to 40 days. As examples of gamma-emitting radionuclides gallium-67, indium-Ill, technetium-99m, iodine- 123, iodine-131, berium-169, rhenium- 186, rhenium- 188 and boron can be mentioned. Generally the metabolism of the compound according to the invention has to be taken into account.

The goal would be a small molecule that penetrates all the tissues and is only affective against seminomas. It would be administered locally, topically, orally, parenterally or intravenously and directed to testis using a conventional carrier. For example, an antibody specifically recognizing a tumor and tagged with a radioactive agent is targeted to the tumor causing high local increase in radiation and leading to destruction of tumor.

The effect of cytostatic agents on the tumor can be monitored by histology, cell proliferation and apoptosis assays, western blotting of phospho-Akt or similar downstream molecules in Ret signaling.

Example 12

The protection of spermatogonia with GDNF during cytostatic treatment

Different cancer types are commonly treated with radiation and cytostatic drugs. The side effect of present cancer therapies is that they cause frequently male infertility. Because GDNF maintains the pool of spermatogonia, local injections of GDNF in the epididymis or GDNF-like molecules that would activate Ret can protect spermatogonia during the drug or radiation therapies of cancer. This treatment would be appropriate for short time use in order to protect spermatogonia and germ line cells.

A Detailed Description of the Drawings

Figure 1. Development of seminomatous tumors in old transgenic mice targeted to overexpress GDNF in testes. Figure la: Seminiferous tubules in a normal testis. The spermatogonia are situated at the peripheral rim of the tubules. Figure lb: A testis from a transgenic mouse at 4 weeks of age. Note the clusters of spermatogonia within seminiferous tubules. Figure lc: Small groups of spermatogonia-type cells (arrow) invading the interstitium in a transgenic testis at 7 months or age. These cells were only observed by the microscopic analysis. Figure Id: Solid sheet of invasive spermatogonia-type cells in a transgenic mouse developing the testicular tumor at the age of one year. Figure le - Figure lh: TRA98 antibody labels the testicular germ line cells. Figure li - Figure 11: The EE2 antibody is highly specific to spermatogonia. Figure le and Figure li: Wild type testis. Figure If and Figure lj: A 4-week-old transgenic testis with clusters of spermatogonia within seminiferous tubules (star). Figure lg and Figure Ik: A 7-month-old testis with microinvasive spermatogonia in the interstitium (arrow). Figure lh and Figure 11: The germ line markers are also expressed by the testicular tumors. In situ hybridization for the GDNF transgene (Figure lm), Ret (Figure In) and GFRal (Figure lo) in the testicular tumors. In m to o the grains depict the signals of in situ hybridization and the dotted lines in Figure lg and Figure Ik mark the shape of a seminiferous tubule. Scale bar 100 μm.

Figure 2. Phosphorylation of Ret and its downstream targets in wild type testis and GDNF-induced testicular tumors. Immunoprecipitated Ret was first blotted with Ret antibodies (Figure 2a) and thereafter with phosphotyrosine antibodies (pTyr) (Figure 2b). ERK1/2 (Figure 2c), phosphoERKl/2 (pERKl/2) (Figure 2d), AKT (Figure 2e) and phosphoAKT (pAKT) (Figure 2f) were detected by Western blotting.

Figure 3. Karyotype in the GDNF overexpressing testes and testicular tumors. Figure 3a: Mouse spleen as a diploid control. Figure 3b: A tumor-free testis of a transgenic mouse at 9 months of age shows a diploid karyotype. Figure 3c: A testis tumor of an old transgenic mouse exhibits aneuploidy. The peaks marked with an arrow are from cells with a hypoploid DNA content, the peak marked with a star indicates the diploid cells, and the peak marked with a plus-sign represents a triploid cell population. Figure 4. The GDNF overexpressing mice accumulate undifferentiated spermatogonia that gradually regress to an embryonic gonotypic phenotype. Figure 4a - Figure 4c: In situ hybridization for L-Fng. Figure 4a: The wild type testis before 1 week of age expresses L-Fng (insert in Figure la), but the expression is no longer detectable at 4 weeks of age. Figure 4b: In contrast, the spermatogonial clusters in a 4 weeks old transgenic mice continuously express L-Fng. Figure 4c: High L-Fng expression by a GDNF-induced testicular tumor. Figure 4d - Figure 4f: Alkaline phosphatase reactivity. Figure 4d: In a 4- week old wild type testis, only the basement membranes show alkaline phosphatase reactivity, but germ cells are unlabeled. Figure 4e: A subset of cells in the spermatogonial clusters of the transgenic mouse testes becomes positive for alkaline phosphatase. Figure 4f: The testis tumors exhibit high alkaline phosphatase reactivity. Scale bar in Figure 4a - Figure 4d 100 μm, in Figure 4e - Figure 4f 50 μm. Note: in a to c grains depict in situ hybridization in dark field images.

Claims

1. Use of glial cell line-derived neurotrophic factor (GDNF) family-related compounds, derivatives or mixtures thereof as well as nucleic acids encoding said compounds, characterized in, that the GDNF family-related compounds act as GDNF-like compounds on their signal mediating receptor tyrosine kinase (Ret) or co-receptor(s) thereof for manufacturing products useful for treating or diagnosing seminoma as well as screening for compounds having a capacity of modulating the activity of said GDNF-like compounds.

2. The use of the GDNF family-related compounds according to claim 1, characterized in, that the GDNF family-related compound is the GDNF-like factor, including GDNF, persephin, neurturin or artemin or mixtures or derivatives thereof.

3. The use of the GDNF family-related compounds according to claim 1, characterized in, that the GDNF family-related compound is GDNF or derivatives thereof.

4. The use according to claim 1, characterized in, that the co-receptors which activate the receptor tyrosine kinase Ret or transmit the signal of the GDNF family-related compounds are GDNF family-receptor α:s (GFRα).

5. The use according to claim 1, characterized in, that the product manufactured using GDNF family-related compounds is a nucleic acid sequence encoding a functionally active, inactive or conditionally inactivatable GDNF-like compound, a construct or a vector carrying said nucleic acid sequence.

6. The use according to claim 1, characterized in, that the product is a cell-line comprising the nucleic acid sequence, construct or vector according to claim 5.

7. The use according to claim 1, characterized in, that the product is a non-human transgenic animal carrying the nucleic acid, construct or vector according to claim 5 and capable of expressing various amount of GDNF-like compounds.

8. The use according to claim 1, characterized in, that the non-human transgenic animal is a mouse.

9. The use according to claim 1, characterized in, that the mouse is overexpressing the GDNF family-related compound.

10. A method for treating seminoma, characterized in, that it comprises the administration to a subject suffering from seminoma caused by an aberrant expression of GDNF-like compounds, a therapeutically effective amount of a modulator substance or active ingredient capable of modulating the activity of a glial cell line-derived neurotrophic factor (GDNF) family-related compound.

11. The method according to claim 10, characterized in, that the treatment of seminoma is targeted to the receptor tyrosine kinase Ret signaling pathway.

12. The method according to claim 10, characterized in, that the treatment targeted to the receptor tyrosine kinase Ret signaling pathway is provided by a modulator substance or active ingredient capable of modulating the complex formation or interaction between the GDNF-like compound and receptor tyrosine kinase Ret.

13. The method according to claim 10, characterized in, that the modulator substance or active ingredient capable of modulating GDNF-like compounds acts by stimulating or inhibiting the binding of GDNF-like compounds to receptor tyrosine kinase Ret, modulating the complex formation between GDNF-like compounds and receptor tyrosine kinase Ret or modulating phosphorylation of receptor tyrosine kinase Ret.

14. The method according to claim 10, characterized in, that the modulator substance or active ingredient capable of modulating the interaction between GDNF-like compounds and receptor tyrosine kinase Ret comprises a substance capable of specifically binding to a GDNF-like compound.

15. The method according to claim 14, characterized in that the binding substance is a small molecule, which by specifically binding to the GDNF-like compound is capable of modulating the complex formation or modulating the interaction between the GDNF-like compound and receptor tyrosine kinase Ret.

16. The method according to claim 14, characterized in that the binding substance is an antibody, which by specifically binding to the GDNF-like compound is capable of modulating the complex formation or modulating the interaction between the GDNF-like compound and receptor tyrosine kinase Ret.

17. The method according to claim 10, characterized in, that the modulator substance or active ingredient capable of modulating the interaction between GDNF-like compounds and receptor tyrosine kinase Ret comprises a nucleic acid sequence encoding functionally inactive or conditionally inactivatable GDNF-like substances, said nucleic acid sequences being capable of modulating or down-regulating the expression of GDNF-like compounds.

18. The method according to claim 17, characterized in, that the modulator substance or active ingredient capable of modulating or down-regulating the expression of GDNF-like compounds is an antisense mRNA of a nucleic acid sequence encoding a GDNF-like compound.

19. The method according to claim 10, characterized in, that the GDNF family-related compounds are administered to the subject in combination with at least one pharmaceutically acceptable carrier and/or additive.

20. The method according to claim 10, characterized in, that the pharmaceutically acceptable carrier and/or additive is compatible with the GDNF family-related compound and the route of administration.

21. The method according to claim 10, characterized in, that the modulator substance or active ingredient is administered locally, topically, orally, parenterally or by injection.

22. The method according to claim 10, characterized in, that the modulator substance or active ingredient is administered by cell implantation or gene therapy.

23. A method for diagnosing the presence, predisposition or risk of developing seminoma as well as the progress of healing, characterized in, that it comprises determination of the amount of GDNF-like compound, particularly GDNF expressed or secreted by the seminoma.

24. The method according to claim 23, characterized in, that the determination is carried out from a blood or tissue sample by per se known immunochemical or biochemical methods.

25. A method for screening a library of substances for identifying compounds potentially useful for treating seminoma, characterized in, that the substances are screened for their capacity of modulating, stimulating respective inhibiting or down-regulating, the complex formation, interaction or signaling between GDNF-like compounds and receptor tyrosine kinase Ret.

26. A composition for treating seminoma characterized in, that it comprises as an active ingredient a modulator substances having the capacity of modulating the complex formation or interaction between GDNF-like compounds and receptor tyrosine kinase Ret in combination with pharmaceutically acceptable additives or carriers.

27. The composition according to claim 26, characterized in, that the additives or carriers are compatible with the modulator and the route of administration.

28. The composition according to claim 27, characterized in, that the route of administration is local, topical, oral, parenteral or injectable.

29. The composition according to claim 26, characterized in, that the active ingredient or modulator substance is provided in a controlled release matrix for local administration during surgery.

30. The composition according to claim 26, characterized in that the active ingredient or modulator substance is provided in combination with at least one second substance effective against seminoma.