MXPA00001410A - Determining the transforming capability of agents - Google Patents

Abstract

The invention relates to activation of apoptin-induced apoptosis by different cell-transforming agents in normal and/or cancer-prone cells. Apoptin or also called VP3 is a viral protein derived from the Chicken anemia virus. Also the invention relates to preventive anti-tumor therapies of normal and/or cancer-prone cells. Treatment of normal and/or cancer-prone cells with tumor-inducing agents will activate apoptin-induced apoptosis, resulting in the elimination of potential tumor cells. Also the invention relates to diagnosis of cancer agents. Agents with tumor activity can be examined by expressing them in normal cells and analyzing their capability of enabling apoptin-induced apoptosis.

Description

DETERMINATION OF THE TRANSFORMATION CAPACITY OF THE AGENTS Field of the Invention The present invention relates to the field of diagnosis and treatment of cancer, as well as to the field of determination of the transforming capacity of tumorigenic agents or promoters of suspicious tumors (the two terms will be used interchangeably here).

Background of the Invention The common denominator of the present invention is that all of the above fields are fields in which apoptin or derivatives and / or fragments thereof (all here referred to later as apoptin and with apoptin-like activity) can be applied according to the invention. Apoptin is a protein originally found in the Chicken Anemia Virus (Noteborn et al., 1991) and was originally called VP3. The apoptotic activity of this protein was discovered by the Rβf. 32794 group of the present inventors (Noteborn et al., 1994). As stated above, the present invention makes use of the apoptosis inducing effect of apoptin. Apoptosis is an active and programmed physiological process to eliminate malignant and excessively damaged, superfluous cells (Earnshaw, 1995, Duke et al, 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus and fragmentation of the cytoplasm, condensation and segmentation of DNA into domain-sized fragments, in most cases followed by internucleosomal degradation. The apoptotic cells become fragmented in the apoptotic bodies enclosed in the membrane. Finally, neighboring cells and / or macrophages will rapidly perform phagocytosis of these dye cells (Wyllie et al., 1980, White, 1996). The cells grew under the tissue culture conditions and the tissue cells can be analyzed to verify the signals of apoptosis with agents that stain the chromosomal DNA, such as for example DAPI or propidium iodide, which stain normal DNA ( chromatic) in a strong and regular manner, but apoptotic chromatin in a weak and / or irregular manner (Noteborn et al., 1994, Telford et al., 1992). The apoptotic process can be initiated by a variety of regulatory stimuli (Willie, 1995, White 1996, Levine, 1997). Changes in the survival rate of cells play an important role in human pathogenesis, for example in the development of cancer, which is caused by the improved and / or reduced proliferation of cell death (Kerr et al. , 1994, Paulovich, 1997). A variety of chemotherapeutic agents and radiation have been shown to induce apoptosis in tumorigenic cells which, in many cases, is mediated by the p53 tumor suppressor protein (Thompson, 1995, Bellamy et al., 1995, Steller, 1995, McDonell et al., 1995). Many tumors, however, acquire a mutation in p53 during their development, often correlating with a poor response to cancer therapy. The transforming proteins of the DNA tumor viruses inactivate p53 by binding directly or indirectly to it (Teodoro, 1997). An example of such an agent is the large T antigen of the SV40 DNA tumor virus. In certain hemopoietic tumors, a high level of expression of Bcl-2-oncogene is associated with strong resistance to several chemotherapeutic agents that induce apoptosis (Hockenberry 1994, Sachs and Lotem, 1997). For such cancers, which are resistant to many cytotoxic agents, alternative antitumorigenic therapies based on the induction of apoptosis are being developed (Thompson, 1995 and Paulovich et al., 1997). Apoptin is a small protein derived from chicken anemia virus (CAV, Noteborn and De Boer, 1995, Noteborn et al., 1991, Noteborn et al., 1994), which can induce apoptosis in malignant cell lines. and transformed human, but not in untransformed diploid human cells. In vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, fibroblastic, epidermal, endothelial and smooth muscle cells. However, when normal cells are transformed for example by the transforming SV40 genes, they become susceptible to apoptosis by apoptin. (Danen-van Ooschot, 1997 and Noteborn, 1996). The long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells. In normal cells, apoptin was found to be located predominantly in the cytoplasm, whereas in transformed or malignant cells, it is located in the nucleus, suggesting that apoptin localization is related to its apoptosis-inducing activity. death (Danet-van Oorschot et al., 1997). In addition, it has been established that apoptin can induce apoptosis in the absence of functional p53 (Zhuang et al., 1995a), and can not be inhibited by Bcl-2, Bcr-abl (Zhuang et al., 1995) , the BAG-2 association protein Bcl-2 and cow pustular eruption protein, caspase inhibitor CrmA (Danen-Van Oorschot, 1997, Noteborn, 1996). Finally, it seems that cells that are only immortalized and therefore minimally transformed, can also be killed by apoptin. Therefore, apoptin is an extremely potent antitumour agent, also for tumors that are not or that are less susceptible to (chemo) therapeutic agents due to the lack of functional p-53, (over) -expression of Bcl- 2 or other inhibitory genes of apoptosis. The fact that apoptin does not induce apoptosis in normal human cells suggests that a toxic effect of the apoptin treatment in vivo will be very low. In addition, it seems that even minimally transformed, premalignant cells may be sensitive to the apoptin killing-inducing effect. Knowing that apoptin is very safe in normal cells, but that as soon as a cell becomes' transformed and / or immortalized (the terms can be used interchangeably here) the present inventors designed some uses based on this discovery. Accordingly, the invention provides a method for determining the transformation capacity of a possible transformation agent, which comprises providing a non-transformed cell with an apoptotic activity similar to inducible apoptin, exposing the cell to the transformation agent and determining the location of the apoptotic activity within the cell or the determination of the induction of apoptosis in the cell. It is to be understood that the above apoptotic activity also refers to the entity that has said activity. It is preferred to provide the cell with apoptotic activity by transducing the cell with a recombinant nucleic acid molecule encoding said activity. Apoptin-like activity is defined herein as any substance (preferably proteinaceous) that has a similar activity as VP3 or chickenpox virus apoptin. Specifically included in said definition are the allelic variants, derivatives and / or fragments of apoptin, wherein the derivatives are defined because they have amino acid replacements which do not lead to the loss of all apoptotic activity. It is to be understood that a similar activity means that the activity class is the same, although the quantity may differ. The methods according to the invention are especially suitable in applications whereby the possible transforming agent is a proteinaceous substance. This allows the proteinaceous substance to be coexpressed in the untransformed cell with the apoptotic activity. The exemplified proteinaceous substance is the SV40 large T antigen or a functional equivalent thereof. The invention also provides modifications of the Apoptin gene leading to changes on the apoptin protein which makes it possible for apoptin to be introduced into the nucleus in the transformed and untransformed tumor cells, leading to the induction of apoptosis. The apoptin protein is enlarged with a nuclear localization signal of SV40. Allelic variants, derivatives and / or apoptin fragments are specifically included in the definition of apoptin, wherein the derivatives are defined having amino acid replacements which do not lead to the loss of all apoptotic activity. This allows the apoptin protein to be expressed in non-transformed cells with apoptotic activity. Fragments of apoptin with apoptotic activity but which are not capable of being introduced into the nucleus of transformed or untransformed cells by their own sequences are capable of being introduced into the nucleus through modifications and induce apoptosis. The invention further provides a method for determining the predisposition of a cell to become a tumorigenic cell, providing the cell with an activity apoptotic similar to inducible apoptin and subjecting the cell to relatively mild tumorigenic activity and determining apoptosis in the cell and / or determining the location of apoptotic activity in the cell. In this case the suspect transforming agent as described herein above is already present in the cell as a mutation that leads to an oncogenic or tumorigenic activity. In this case, the fact that apoptin only induces apoptosis in cells that have already been transformed, leads to the possibility of verifying whether the cells have a mutation which leads to immortalization or transformation during mild exposure to a transforming activity, such as treatment with UV irradiation and X-rays. In this way the probability of a set of cells leading to cancer can be determined. This leads of course to applications in the field of diagnosing the probabilities that people who have a hereditary risk of contracting cancer have and to provide them with preventive treatment, which is another object of the present invention. This kind of diagnosis can also be applied to warn people of the likelihood that their children are predisposed to cancer. Accordingly, the invention also provides a method for determining the predisposition of a subject towards the hereditary types of cancer, which comprises subjecting a sample of a relevant subset of cells of such subject to a method as described herein. And the invention further provides a method for determining a mutation of genes having an oncogenic and / or transforming activity in a cell, which comprises subjecting the cell to a method according to the invention. As stated hereinabove, it is another object of the present invention to provide means for the prophylactic treatment of subsets of cells in a person, such a subset of cells being prone to cancer. These means include a nucleic acid encoding apoptin-like activity, preferably provided in the form of a carrier for the delivery of the genes. The vehicles for the supply of the genes may be of viral or other origin. Many vehicles have been described in the art and are known to the person skilled in the art. They include but are not limited to adenoviral vectors, preferably in the form of adenoviral particles; retroviral vectors, preferably as recombinant retroviruses; the same class of vectors but derived from other viruses; liposomes or other carrier molecules, etc. The invention also provides a set or diagnostic test set for carrying out a method according to the invention in determining the tumorigenic capacity of an agent, comprising an untransformed cell transduced with an apoptin encoding the nucleic acid or a derivative or functional fragment thereof and optionally any other material necessary to carry out the test and detect the result. The invention further provides a set or diagnostic test set for carrying out a method according to the invention for determining the cancer propensity of cells, comprising a nucleic acid encoding an apoptin or a derivative or functional fragment thereof. capable of transducing a eukaryotic cell and capable of being expressed in such a cell and optionally any other material necessary to carry out the test and detect the result. Preferably a means for subjecting a cell to a mild tumorigenic activity, such as UV irradiation and X-ray treatment is also provided. The invention also provides a method for studying the induction of apoptosis induced by apoptin, transforming agents such as chemicals, viruses, UV and X irradiation in a transgenic mouse model, which leads to inhibition of apoptosis. the formation of tumors. Transgenic apoptin-treated mice can be used to analyze the antitumor effect of Apoptin in the transgenic chimeras that carry the hereditary types of cancer and capable of expressing apoptin. In addition, the effect of apoptin expression in an in vivo model can be studied by means of the described transgenic apoptin-treated mice.

Detailed description of the invention It has previously been shown that viral protein apoptin induces apoptosis in cultured transformed cells of both human and rodent origin, but not in normal human cells. It has now been observed that apoptin fails to induce apoptosis in cultured murine (or rat) embryonic fibroblasts. (The cultures were derived from mouse (rat) embryos 16-18 days old). This shows that apoptin can also be expressed in the intact embryo without causing toxicity, at least in embryos of a stage not too early in embryonic development. It has now been able to produce transgenic apoptine-treated mice, which are available. Evidence is provided that the constitutive expression of apoptin in a transgenic mouse does not lead to lethal effects or other life-threatening / life-limiting effects. It has been observed that the cotransfection of normal human fibroblasts cultured with the apoptin gene and the SV40 transformation genes will activate the apoptotic process, which is accompanied by the translocation of the apoptin protein from the cytoplasm, where it is accumulates initially, until the core. The invention described provides the basis for additions of amino acids to the apoptin protein or its fragments making it possible for them to be introduced and / or accumulate in the cell nucleus, leading to the induction of apoptosis. The invention provides the basis for a diagnostic test for the detection of potential transformation genes. Normal diploid mammalian cells, such as human and / or rodent cells, are used for such a test. For this purpose, normal diploid cells are cotransfected with a plasmid containing the gene (s) to be studied and the apoptin that encodes the plasmid, or transfected with the gene (s) that are going to be studied and infected with an apoptin that expresses the viral vector. The induction of apoptosis induced by apoptin and / or the presence of apoptin in the nucleus shows that the examined gene retains or contains the tumorigenic / transformation potential. In addition, it has been discovered that human diploid cells isolated from individuals carrying a linear germline mutation in a tumor suppressor gene, and as a result are predisposed to develop a certain spectrum of tumors (also referred to herein as being prone to cancer), they are resistant to the apoptosis inducing effect of apoptin, precisely when the diploid cells of healthy individuals, however, become sensitive if the cell cultures are irradiated with ultraviolet light. This allowed to develop a diagnostic test to predict the propensity to cancer. In families with a hereditary predisposition to cancer due to a linear germline mutation in a tumor suppressor gene, it is often not possible, without extensive analysis of chromosomal DNA, to predict whether a family member is afflicted and carries the gene for the disease. The results of the invention show that this is carried out in a simple manner, making use of the apoptin gene. For this purpose, diploid skin fibroblasts or lymphocytes are isolated from the individuals to be tested, and the cultured cells are transfected with the apoptin gene, followed by irradiation with UV light (266 nm). If the transfected cells become apoptotic after irradiation with UV light but fail to introduce apoptosis without UV exposure, then this is an indication (strong) that the individual is prone to cancer. Not all types of cancer predisposition, which are due to a mutation in a tumor suppressor gene, have been tested with the UV / Apoptin assay yet. However, there is no reason to suppose that the same phenomenon will not be observed in other cells prone to cancer. A similar diagnostic test to predict the propensity to cancer can also be carried out using X-ray treatment instead of UV irradiation. The invention also allows to obtain more information on the molecular basis of the propensity to cancer and its relationship with certain responses to stress, such as Enhanced Reactivation (ER) (Abrahams et al., 1996). Enhanced Reactivation is one of the reactions of normal (human) cells to certain agents that damage DNA, and they seem to reflect the susceptibility of cells to oncogenic transformation. The invention will be explained in more detail in the next experimental part. This is for illustration purposes only and should not be construed as limiting the scope of protection.

Experimental Part Cells and cell culture conditions Rat embryo fibroblasts (REF) were prepared from 14-day-old rat embryos. The cells were liquefied from liquid nitrogen, cultured in DMEM supplemented with 10% fetal bovine serum, and transfected with the plasmid DNA in the passageway or step 2 of the cell. Mouse embryo fibroblasts (MEF) were prepared from p53 + / + mice or from unconscious mice p53 - / - (Tyier Jacks, 1994 and 1996, Tyier Jacks et al., 1994). The cells were grown on Corning boxes in an F15 medium supplemented with 10% fetal bovine serum. P19 cells are derived from a mouse embryonal carcinoma / teratocarcinoma (Burney et al., 1982). The cells were grown on gelatinized Petri dishes in DMEM supplemented with 8% fetal bovine serum. The BRK / xho cells are prepared from baby rat kidney cells, by transformation with the adenovirus type 5 of the El region (Schrier et al, 1983). The cells were cultured in DMEM supplemented with 10% fetal bovine serum. Fibroblasts VH10 and VH25 of the foreskin, diploid, human (Klein et al, 1990) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. The primary cultures of human epidermal keratinocytes (FSK-1) were initiated in a complete medium as described (Rhein ald and Green, 1975), with minor modifications according to M. Ponec et al., 1981, and cultured in a serum free medium of keratinocytes (KSFM) after this. For the experiments described here, the passage or step number 3 was used. F9605 cells are diploid fibroblasts which are pl6 - / -, derived from patients with the nevi dysplastic syndrome, which is postulated to be a precursor of a mola syndrome / multiple melanoma typical of family a (FAMMM) (Gruis et al., 1995). The cells were grown in DMEM with 10% fetal bovine serum. GM1492 cells are human diploid fibroblasts, which do not express p53 and are derived from patients with Bloom Syndrome, an autosomal recessive disorder with a high incidence of cancer (Van Laar et al., 1994). Cells were grown in DMEM containing 10% fetal bovine serum. LF2675T are skin fibroblasts, diploid, from patients with Fraumeni syndrome (LSF). This disease is characterized by a mutation of the germline in an allele of the p53 gene and in an initial attack of several types of cancer (Srivastava et al., 1990; Abrahams et al., 1996). The cells were grown in DMEM supplemented with 10% fetal bovine serum. 401 cells are diploid skin fibroblasts from individuals of the Lynch type 2 family with an increased incidence of several types of cancer. The cells were derived from an individual who died of breast cancer (Abrahams et al., 1996). The cells were grown in DMEM supplemented with 10% fetal bovine serum. All culture media were obtained from GIBCO / BRL and contained penicillin and streptomycin antibiotics.

Irradiation of cell cultures The conditioned medium was removed from the cultures and the cells were rinsed twice with PBS. After removal of the PBS, the cultures were irradiated with UV as previously described (Abrahams et al., 1984), or treated with X-rays (5 gray) using an Andrex 225 SMART device (Andrex St, Copenhagen) at 200 KV , 4 A with a 1 mm Al filter. The dose and the speed of the dose were verified with a PTW dosimeter. After UV treatment, the conditioned medium was returned, and the cultures were incubated at 37 ° C.

Plasmids The plasmid of the expression pCMV-VP3 contains the CAV DNA sequences encoding the apoptin protein exclusively (nt 427-868; Noteborn et al., 1991, Noteborn and De Boer, 1966), and the plasmid pCMV-des encodes the desmin, a structural protein of muscle cells (Menke et al., 1997), the pCMV-neo of the plasmid is used as the "empty" negative control for the plasmids encoding the gene products with a potential effect on apoptosis induced by apoptin. All genes were expressed under the regulation of the enhanced cytomegalovirus promoter (initial). The plasmid SV40 contains the clone of the initial region of defective origin (ori-) SV40 which includes the regions encoding both the small T antigen and the SV40 large T antigen regulated by its own promoter (Dinsart et al, 1984). Plasmid pR-S884 expresses a large T SV40 antigen complete and a truncated small T antigen under the transcriptional control of the long terminal repeat (LTR) of the Rous sarcoma virus (RSV; De Ronde et al., 1989; Smits et al., 1992). Plasmid PR-SVt contains the cDNA sequences encoding the SV40 small T gene that was fused to the RSV LTR (Philips and Bundell, 1988). The expression 21EcoA plasmid, which consists of the promoter / gene regions of the histocompatibility antigen of the murine H-2Kb (Mellor et al., 1982) and the pBr327 sequences, are a gift from Prof. Dr. Frank Grosveld, Erasmus University , Rotterdam, Holland. Plasmid 21EcoA contains a Notl within the first exon of the H-2Kb gene, which makes possible the integration of a foreign gene that becomes regulated by the H-2Kb promoter. The BamHI fragment containing the sequence encoding apoptin was isolated from pCMV-VP3 and cloned into the NotI site of 21EcoA using the Notl-BamHI linkers. The final plasmid containing the apoptin gene under the regulation of the H-2Kb promoter was called p21EcoA-VP3. Subsequently, the EcoRl site following the apoptin gene was deleted by the linearization of the plasmid p21EcoA-VP3 at this specific EcoRI site, and the treatment with the Klenow polymerase treatment. The plasmid is called p21EcoA-Vp3-Eco. The DNA fragment containing the H-2Kb expression unit with the apoptin gene is separated from the prokaryotic DNA sequences by means of EcoRI digestion and agarose gel electrophoresis. The plasmid DNA is purified by centrifugation in a column chromatography and gradient of CsCl in Sephacryl S500 (Pharmacia, Sweden).

Temporary Transfection Cells were transfected in single layer cultures with DOTAP transfection agent (Boehringer, Mannheim, FRG) essentially as described by Fischer et al., 1996, or transfected with the plasmid DNA by precipitation of calcium phosphate as it was described by Graham and Van der Eb (1973).

Indirect immunofluorescence.

All cells were grown on glass microscope slides. The slides were either uncoated (VH10, VH25), or coated with 3-amino-propyltriethoxysilane (TESPA; FSK-1). The cells were fixed with 80% acetone for 10 minutes at room temperature, and used for indirect immunofluorescence as described (Ben den Heuvel, 1990). To demonstrate the presence and / or cellular localization of apoptin in the transfected cells, the mouse monoclonal antibody (Mab) CVI-CAV-85.1 (85.1; Noteborn et al., 1991); for the human desmin the mouse Mab 33 (Monosan, Uden, Holland); for the SV40 T antigens the Pab 419, provided gently by A.-G Jochemsen, University of Leiden, The Netherlands, were used. The goat anti-mouse antibody labeled with fluoroscein isothiocyanate (Jackson Immunoresearch Laboratories Inc., West Grove PA, USA) was used as the second antibody. The nuclear DNA was stained with 2,4-diamino-2-phenylindole (DAPI).

The generation of the mouse treated with apoptin-transgenic.

For the generation of the mouse treated with transgenic apoptin, the fertilized oocytes of the FVB mouse strain and the mice of the murine strain were used as nurseries. The microinjections were carried out in the male pronuclei according to Brinster et al. (1981). By microinjection, 500 copies of the EcoRI DNA fragment, derived from the p21EcoA-Vp3-Eco plasmid, containing the required H-2Kb transcription unit and the complete apoptin gene, were injected.

Results and Description Apoptin induces apoptosis in transformed rodent cells but not in normal embryonic cells.

To examine whether apoptin fails to induce apoptosis in the cells of normal embryonic rodents, the cultures of the mouse embryonic cells and the embryonic cells of the rat were temporarily transfected with a plasmid encoding apoptin. As a negative control, the cells were transfected with a plasmid encoding desmin, which has no apoptotic activity. Cells expressing apoptin were selected by means of indirect immunofluorescence with Mab 85.1, and cells expressing desmin with mouse Mab 33. The induction of apoptosis in positive cells of apoptin or desmin was analyzed with the help of DAPI, which causes a regular staining in the intact nuclei, but a weak and / or irregular staining in the apoptotic nuclei. Five days after transfection, about 10-20% of the desmin positive cells were apoptotic, which is the most likely basal level due to the transfection event (data not shown, Menke et al., 1997, Danen- van Oorschot, 1997a). Two, 3, 4 or 5 days after transfection the percentage of apoptotic apoptine-positive cells did not significantly exceed the percentage of apoptotic cells observed in positive desmin cultures, indicating that apoptin does not induce apoptosis in embryonic cell cultures normal. Temporary transfection of rat or kidney rat embryonic mouse cells transformed with the plasmid encoding apoptin proved that apoptin is capable of inducing apoptosis in these cells. The results of apoptin expression in "normal" embryonic rodent cells against transformed rodent cells is shown in Figure 1. These data show that apoptin fails to induce apoptosis in embryonic and adult rodent cells. normal, but does not induce apoptosis in the virally transformed derivatives, at least under the conditions of cell culture.

Coexpression of large T antigen of SV40 and apoptin leads to apoptosis induced by apoptin in normal human diploid cells.

The effect of the expression of transforming genes on apoptosis induced by apoptin in normal human cells derived from healthy individuals has been examined. For this purpose, the human VH10 diploid fibroblasts and the diploid keratinocytes of FSK-1 were temporarily transfected with the pCMV-VP3 plasmid encoding apoptin and either the pSV40 plasmid encoding both a large T antigen and the small T antigen. , the pR-s884 encoding the large T antigen, the pR-SVt encoding the small T antigen, or the negative control pCMV-neo plasmid. By indirect immunofluorescence, the cells were analyzed to verify the apoptosis induced by apoptin. Cells of both normal VH10 and FSK-1 did not suffer from apoptosis when apoptin was transfected with the control plasmid. The results showed, as expected, that the expression of apoptin alone was not able to induce apoptosis in normal human diploid cells, confirming the data described by Danen-Van Oorschot (1997). However, normal diploid human fibroblasts and keratinocytes that express both apoptin and SV40 large T antigen, alone or together with the small T antigen, suffered apoptosis induced apoptosis (Figure 2). The rate of induction of apoptosis was considerably increased in the presence of the viral transformation genes. The coexpression of the small T antigen of SV40 with apoptin did not lead to the induction of apoptosis by apoptin. The transition of normal cells, from apoptin resistance to apoptin susceptibility, can probably be explained by the fact that the apoptin protein is translocated from a cytoplasmic location to a nuclear location. This transition becomes apparent approximately two days after the transfection of the SV40 plasmids (Figure 3). It can be concluded that an event takes place, in this example due to the expression of a transformation product from a tumorigenic virus-DNA, which leads to the translocation of apoptin from the cytoplasm to the nucleus, which is followed by the Induction of apoptosis.

The coexpression of apoptin and SV40 large T antigen leads to apoptosis induced by apoptin in normal rodent diploid cells.

Next, we examined the effect of co-expression of transforming genes and apoptin on the induction of apoptosis in the fibroblasts of normal mouse embryos (MEF) derived from p53 + / + mice or from p53 mice. - transgenic. Both types of temporarily transfected MEFs co-express the large T-gene of transforming SV40 with or without the small T antigen in combination with apoptin, they suffer from a very rapid apoptosis, while the MEF expressing the apoptin together with a control plasmid or with a plasmid encoding the non-transforming small T antigen, does not lead to an apoptosis induced by apoptin. The results are shown in Figure 4. Immunofluorescence analyzes also revealed that the coexpression of apoptin and SV40 large T antigen led to the translocation of apoptin. In the MEFs examined apoptin was located in the cytoplasm. During expression of SV40 large T antigen, apoptin is introduced into the nucleus, followed by induction of apoptosis. For comparison, the percentage of transformed mouse cells, positive in apoptin, are also given in Figure 5. These results indicate that apoptin did not induce apoptosis in p53 mouse fibroblasts both + / + and - / -, but not during the expression of a transforming protein. This information is important since it is known that "normal" p53 - / - cells are very susceptible to spontaneous transformation and progress easily to more highly transformed phenotypes. The loss of p53 alone, however, is not enough to create a transformed character. In addition, this discovery shows that apoptin can induce apoptosis during the expression of a transforming protein in other mammalian cells different from human cells.

Coexpression of SV40 large T antigen and apoptin led to apoptosis induced by apoptin in normal diploid cells derived from human individuals prone to cancer.

The effect of apoptin on the normal fibroblasts of F9605 and GN1492, which are derived from individuals showing an increased incidence of cancer due to a genetic defect, was also examined by means of temporary transfection and immunofluorescence. Apoptin is not capable of inducing apoptosis in the normal diploid cells of these cancer-prone individuals. However, during expression of SV40 large T antigen, apoptin induces apoptosis (Figure 6) after introduction to the nucleus (data not shown). These data confirm that diploid cells, from hereditary cancer-prone syndromes, are not susceptible to apoptin, whereas they become so when they express a transforming gene. Accordingly, the diploid cells of such hereditary syndromes are identical to the "normal" diploid human cells in this assay.

The effect on the induction of apoptosis of the covalent bond of a nuclear localization signal of the large SV40 antigen with respect to the apoptin protein.

Next, we examined whether the expression of a chimeric protein consisting of apoptin and the nuclear localization signal of the SV40 LT antigen (amino acids N-Proline-Proline-Lysine-Lysine-Lysine-Arginine-Lysine-Valine- C of the SV40 large T antigen covalently linked to the N-terminus of apoptin) leads to the induction of apoptosis in transformed and untransformed human cells. The chimeric protein is called NLS-apoptin. For this purpose, untransformed VH10 human fibroblast cells and transformed human osteosarcoma derived Saos-2 cells (Danen-van Oorschot et al., 1997) were transfected with a plasmid encoding the protein NLS-apoptin. chimerical In both cell types, the expression of NLS-apoptin led to the nuclear localization of apoptin and the induction of apoptosis. The expression of the Green Fluorescent Protein of the non-apoptotic protein (GFP; (Pines, 1995) covalently linked to the NLS introduced to the nucleus, did not lead to the induction of apoptosis.This data proves that a modified apoptin in the nuclear localization of a Independently of cell transformation, it will be capable of translocation in the nucleus, followed by the induction of apoptosis.A fusion product of the first 69 N-terminal amino acids of apoptin and the non-apoptotic GFP protein, does not lead to the induction of apoptosis, and which coincides with the fact that this chimeric protein is not introduced into the nucleus (Noteborn and Pietersen, 1998) Now the 8 amino acids of the NLS have been covalently linked to the N extremity of the apoptin fragment which consists of amino acids 1-69 (NLS-apoptin / 1-69), the transfection of both untransformed VH10 cells and human tumorigenic cells ( it is like the Saos-2 cells derived from human osteosarcoma) with a plasmid encoding NLS-apoptin / 1-69 led to the nuclear localization of NLS-apoptin / 1-69 followed by the induction of apoptosis. These data indicate that in addition to the C-terminal part of apoptin, also the N-terminal part (1-69 a.) Does so, when it is translocated in the nucleus. In these experiments, as expected, NLS-GFP translocated into the nucleus but did not lead to the induction of apoptosis.

Normal diploid cells from cancer-prone individuals suffered apoptosis induced by apoptin after UV radiation.

The effect of irradiation with UV light on the apoptosis induction by apoptin on diploid cells has been examined. Diploid fibroblasts derived from healthy people (VH25) or from individuals with a cancer-prone syndrome (LF2675T cells from a patient with Li Fraumeni Syndrome, and 401 cells from a patient with Lynch Type Syndrome) were temporarily transfected with a plasmid encoding apoptin. Some of the cells were irradiated with UV. As a negative control, the cells were transfected with a plasmid encoding the desmin protein. All 3 cell types, VH25, LF2675T and 401, did not reveal apoptosis induced by apoptin without irradiation with UV light. In combination with UV irradiation, however, LF2675T and 401 cells, but not VH25 cells, suffered apoptosis induced apoptin very rapidly. Although there is no explanation for this phenomenon, it seems that it correlates with other cellular property. The diploid cells of patients who are prone to cancer due to a germline mutation in a tumor suppressor gene, showed an unexpected reaction to UV irradiation. When normal diploid fibroblasts are treated with UV or another agent that damages DNA, they react with a large variety of temporal responses, including the activation of signal transduction pathways, the induction of expression of a variety of genes, the inhibition of cellular DNA replication and the activation of SOS-like phenomena such as Enhanced Reactivation (ER) and Enhanced Mutagenesis (EM). Abrahams et al. (1996) have found that normal diploid fibroblasts from patients with a predisposition to hereditary cancer due to a germline mutation in a tumor suppressor gene, show the same responses to UV irradiation as the cells of individuals normal, except for one response: Enhanced Reactivation. The response of ER in the cells of these patients is much greater than in the cells of normal individuals, hence these patient cells are called ERsuper (+). The molecular biological basis of the ER phenomenon is not yet clear. The detection of ER is a time-consuming approach, because it is based on the measurement of survival (improved) of the UV-irradiated virus in the cells damaged with UV (or damaged with X-rays), compared to the survival in undamaged cells. An assay based on apoptosis induced by apoptin during UV radiation is considerably simpler and faster (see below). The fact that apoptin becomes active in cancer-prone cells during UV radiation also makes it possible to study the ER process. There is evidence indicating that ER plays an important role in the process of cancer induction by agents that damage DNA.

Normal diploid cells of cancer-prone individuals suffer apoptosis induced by apoptin after X-ray treatment.

Next, the effect of X-ray treatment on the apoptosis induction by apoptin on human diploid cells was examined. Diploid fibroblasts derived from healthy individuals (VH10) or from people with a cancer-prone syndrome such as LF2675 and 401 cells, were transfected with a plasmid encoding apoptin. Before, transfection, part of the cells were treated with X-rays (dose, 5 grays). As a negative control, the cells were transfected with a plasmid encoding the desmin protein. As expected, all unirradiated cells analyzed from cell lines: VH10, LF2675 and 401, did not show apoptosis induced by apoptin. In combination with X-ray treatment, however, cell lines derived from cancer-prone individuals suffered from apoptosis, while those derived from healthy individuals did not. Five days after transfection, most of these cancer-prone cells positive for apoptin, treated with X-rays, became apoptotic. Cells treated with X-rays and expressing the non-apoptotic agent of desmin did not suffer apoptosis induced by apoptin. These results imply that X-ray treatment, which causes DNA damage such as the UV-C treatment described above, leads to the induction of apoptosis by apoptin in normal, non-transformed human cells.

Diagnostic assay for genes that induce cancer based on apoptosis induced by apoptin.

Danen-Van Oorschot et al. (1997a) has reported that the cellular localization of apoptin is different in transformed / tumorigenic human cells compared to the location in normal non-transformed cells. In addition, the accumulation of apoptin in the nucleus correlates with the induction of apoptosis, while the cytoplasmic localization correlates with the feasibility or availability of the cells and the normal proliferative capacity. Based on this report, we had the ability to develop a diagnostic assay for the identification of genes or agents for transformation and / or induction of cancer. A first type of assay consists of transfecting "normal" cells, for example from human or rodent origin, with a plasmid encoding apoptin, or infecting cells with viral vectors expressing apoptin, together with a plasmid encoding a cancer-inducing gene / of putative or putative transformation. Subsequently, the cells will be examined, (1) to verify the ability to undergo apoptosis by the apoptin gene and (2) for a shift in the location of the apoptin from the cytoplasm to the nucleus. The intracellular localization of apoptin can be determined, using an immunofluorescent assay with monoclonal antibodies specific for apoptin, such as CVI-CAV-85.1. Whether the percentage of apoptosis in normal cells co-expressing apoptin and the putative or putative cancer-inducing or putative transformation gene is significantly higher in apoptin-positive control cells expressing a control plasmid, it can be concluded that the analyzed gene can actually have a cancer / transforming induction activity. A second example of a diagnostic test is based on the treatment of normal diploid cells cultured with a putative or putative carcinogen. The agent can be added, for example, to the culture medium for several time intervals. Subsequently, the cells are transfected with a plasmid encoding apoptin or infected with a viral vector expressing apoptin. This approach can also be carried out by first transfecting / infecting normal cells, and then treating the cells with the agent to be tested. The subsequent steps of the assay are the same as those described for the first type of diagnostic assay.

Diagnostic assay for cancer propensity The data presented in this report allowed us to develop a trial to determine if an individual with an unknown genetic / cellular background is prone to cancer compared to normal healthy people. The normal diploid cells of an individual prone to cancer are insensitive to apoptosis induced by apoptin, but they do so after treatment with UV or X rays or another agent that damages the DNA. Below, an example of such a diagnostic test based on the effect of irradiation with UV light is described. This assay can also be carried out with other mutagenic / carcinogenic agents. Primary normal diploid cells are isolated from a biopsy of the skin of the individual to be tested and cultured in an appropriate medium. The cells are then irradiated with UV and subsequently transfected with a plasmid encoding apoptin, or infected with a viral vector expressing apoptin, or the cells are transfected / infected first and then irradiated. In parallel, the diploid cells of a normal healthy individual will be used as a control. Using an indirect immunofluorescence assay based on specific Mabs of apoptin, the cells are analyzed to verify the presence of apoptin in the nucleus and / or to verify the condition of apoptosis. If the percentage of cells suffering from apoptosis between cells treated with apoptin-positive UV is significantly higher than the percentage of apoptosis in cells treated with UV rays of a normal individual, this will be strong evidence that the individual from whom the cells are isolated, will be prone to cancer.

Use of apoptin proteins in pharmaceutical formulations for anticancer therapy.

Based on the results mentioned above, methods can also be developed to apply apoptin in anticancer therapy, not as a gene (DNA) but as a protein. Apoptin is a comparatively small protein, which makes it possible for it to enter the cell as a protein. (If fragments of the apoptin protein still have the desired apoptotic effect on cancer cells, the protein fragments will be used instead of the intact protein). Our objective is to develop effective pharmaceutical formulations that ensure the stability of the active component (= apoptin or a fragment thereof) and, if possible, the specificity for the tumorigenic cell that is to be targeted. Neoplasms that are expected to be treated with appropriate apoptin-containing formulations, both curative and preventive, include: hereditary forms of colorectal cancer (familial adenomatous polyposis) (APC) and colorectal cancer different from polyposis, hereditary (HNPCC) , cancer of the liver (or other organs that can be treated with perfusion techniques), leukemias and lymphomas (which are going to be treated by means of blood circulation), skin tumors and possibly tumors of the lungs (through the respiratory tract).

The construction and analysis of an expression plasmid for the generation of mice treated with transgenic apoptin.

The fact that apoptin has been found to fail to induce apoptosis in cultured murine embryonic fibroblasts leads to the fact that apoptin can also be expressed in intact embryo and adult mice without causing toxicity, at least in embryos of a not too early stage of embryonic development. We have chosen an expression system based on the murine H-2Kb transcription unit, which allows the constitutive expression of foreign genes during embryogenesis and in adult stages in various organs (Drezen et al., 1992; Morello et al. al., 1986). Thus, the expression plasmid p21EcoA-Vp3-Eco has been constructed, which expresses the apoptin under the regulation of the murine H-2Kb promoter. In addition, the expression vector contains the other elements of H-2Kb, which will allow the expression of the apoptin gene. Figure 8 shows a schematic representation of the expression vector of the transgenic apoptin p21EcoA-Vp3-Eco. By means of the temporary transfections of the Saos-2 cells transformed with the p21EcoA-Vp3-Eco plasmid we had the ability to prove that apoptin could actually be expressed in the context of the H-2Kb sequences. In addition, the expressed apoptin led to the induction of apoptosis to a degree too similar to that of apoptin expressed by means of the plasmid pCMV-VP3 (see Figure 9). These results imply that the expression vector p21EcoA-Vp3-Eco used expresses apoptin in such a way that the transformed cells will suffer apoptosis.

Generation of mice treated with transgenic apoptin.

In total 300 fertilized oocytes were microinjected with a DNA fragment comprising the transcription unit of H-2Kb and the apoptin gene and were transferred to 11 breeding mice. In total a progeny of 51 newborn mice has been collected. By Southern blot analysis (Southern, 1975) of mouse tail DNA digested with BamHI and Xbal using a 32P labeled DNA fragment consisting of the entire VP3 gene, it has been shown that the apoptin unit / H-2Kb was integrated into the genomic DNA of a total of 7 breeding mice (FO). All mice treated with transgenic apoptin were observed healthy. For unknown reasons, however, 1 mouse treated with apoptin, transgenic, died at an age of 5-6 weeks. The transgenic apoptin-treated mice were compared with male or female FVB. Progeny tail DNA was analyzed for the presence of the apoptin gene using a polymerase chain reaction (PCR) analysis using the primers Pl (5 '-CTCTCCAACAACATACT-CCACCCGG-3') and P2 (CTTATACGCCTTTTT -GCGGTTCGGG-3 '). Of all FO mice, 1 or more Fl mice treated with apoptin, transgenic (Figure 10). All the mice of the Fl generation of the transgenic apoptin-treated animals were shown to be available and therefore do not show any pathological defect, which should be correlated with the expression of apoptin. By means of Northern blot analysis (Noteborn et al., 1992) the expression of the apoptin gene, as expected, could be determined in several organs.

Description of the Figures Figure 1 shows the activity of apoptosis induced by apoptin in the fibroblasts of "normal" rodent embryos against the cells of the transformed rodent cell lines. The cells were transfected temporarily with pCMV-VP3. Subsequently, cells were fixed at various time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin positive cells that were abnormally stained with DAPI is given as a relative measure for the induction of apoptosis. Figure 2 shows the effect of large SV40 T antigen and / or small T antigen on apoptosis induced by apoptin in fibroblasts and keratinocytes of normal individuals. The VH10 and FSK-1 cells were temporarily transfected with plasmid pCMV-VP3 and pCMV-neo or pSV40 expressing the small T antigen and SV40 large T, pR-s884 expressing SV40 large T antigen, and the pR-SVt expressing the SV40 small t antigen. Subsequently, cells were fixed at various time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin positive cells that were abnormally stained with DAPI is given as a relative measure for apoptosis. Figure 3 shows the location of apoptin in normal human diploid cells expressing only apoptin or together with the large T antigen and the small SV40 T antigen. The same cells analyzed in Figure 2, considering the induction of apoptosis were also examined to verify the localization of apoptin in the nucleus or cytoplasm. The percentage of apoptin-positive cells that contain apoptin in the nucleus and that have not yet undergone apoptosis, are given as a relative measure of the location of apoptin in the nucleus. Figure 4 shows the effect of the small T antigen and / or SV40 large T antigen on apoptosis induced by apoptin in mouse fibroblasts, which are derived from the normal mouse p53 + / + or from an unconscious mouse p53 - / - transgenic The cells were transfected temporarily with the plasmid pCMV-VP3, which expresses apoptin and the control plasmid pCMV-neo or with PSV40 expressing SV40 large T antigen, pR-s884 expressing the large T antigen, and pR-SVt encoding the small T antigen. Subsequently, cells were fixed at various time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin-positive cells that are abnormally stained with DAPI is given as a relative measure for apoptosis. Figure 5 shows the location of apoptin in mouse p53 + / + and p53 - / - embryonic fibroblasts expressing only apoptin or together with small T antigen and / or SV40 large T antigen. The same cells that were analyzed in Figure 4 considering the induction of apoptosis, were also examined now to verify the location of apoptin in the nucleus or cytoplasm. The percentage of apoptin-positive cells that contain apoptin in the nucleus and that are still apoptotic are given as a relative measure of apoptin localization in the nucleus.

Figure 6 shows the effect of SV40 large T antigen on the apoptosis activity induced by apoptin in human diploid fibroblasts 9605 or G4905"normal" derived from cancer-prone individuals. The cells were temporarily transfected with pCMV-VP3 and pCMV-neo, or pSV40 expressing both the small T antigen and the SV40 large T antigen, pR-s884 expressing the large T antigen and pR-SVt expressing the small T antigen . Subsequently, cells were fixed at various time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin positive cells that were abnormally stained with DAPI is given as a relative measure of apoptosis. Figure 7 shows the effect of UV irradiation on apoptosis induced by apoptin in "normal" diploid fibroblasts derived from normal healthy individuals against cancer-prone patients. The cells were treated in a simulated manner or treated with UV light and subsequently transfected temporarily with pCMV-VP3 or with pCMV-des. Finally, the cells were fixed at several time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin positive cells that were abnormally stained with DAPI is given as a relative measure for apoptosis. Figure 8 shows a schematic representation of the transgenic apoptin expression vector. Figure 9 shows the activity of apoptosis induced by apoptin in the cells of Saos-2 The cells were temporarily transfected with p21EcoA-Vp3-Eco, pCMV-VP3 (both expressing apoptin) or with pCMV-des, which expresses the non-apoptotic desmin protein. Subsequently, the cells were fixed at various time intervals after transfection and analyzed by indirect immunofluorescence using Mab 85.1 specific for apoptin. The percentage of apoptin positive cells that were abnormally stained with DAPI is given as a relative measure for the induction of apoptosis. Figure 10 is a schematic representation of the lineage of the transgenic- (treated with apoptin) mice of VP3. The white boxes are nude mice. The yellow and green boxes represent the progeny (Fl) of several transgenic breeds treated with apoptin.

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It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (14)

1. A method for determining the transformation capacity of a possible transformation agent, characterized in that it comprises providing an untransformed cell with the apoptotic activity similar to inducible apoptin, exposing the cell to the transformation agent and determining the location of the apoptotic activity within of the cell or determine the induction of apoptosis in the cell.

2. The method according to claim 1, characterized in that the apoptotic activity is provided by transducing the cell with a recombinant nucleic acid molecule encoding the activity.

3. The method according to claim 1 or 2, characterized in that the possible transforming agent is a proteinaceous substance.

4. The method according to claim 3, characterized in that the proteinaceous substance is co-expressed in the non-transformed cell with the apoptotic activity.

5. A method to determine the predisposition of a cell to become a tumorigenic cell, providing the cell with an apoptotic activity similar to apoptin and subjecting the cell to a relatively mild tumorigenic activity and determining apoptosis in the cell and / or determining the location of the apoptotic activity in the cell.

6. The method according to claim 5, characterized in that the soft tumorigenic activity is UV irradiation.

7. A method for determining the predisposition of a subject to the hereditary types of cancer, characterized in that it comprises subjecting a sample of a relevant subset of the cells of such subject to a method according to claims 5 or 6.

8. A method for determining a mutation of the gene having an oncogenic and / or transforming activity in a cell, characterized in that it comprises subjecting the cell to a method according to claim 5 or 6.

9. The use of a nucleic acid encoding apoptin or a derivative or functional fragment thereof in the preparation of a medicament for the prophylactic treatment of subsets of cells in a person, such a subset of cells is prone to cancer.

10. The use according to claim 9, characterized in that the nucleic acid is provided in the form of a vehicle for the delivery of the genes.

11. A set or diagnostic test set for carrying out a method according to any of claims 1-4, characterized in that it comprises a non-transformed cell transduced with a nucleic acid encoding apoptin or a derivative or functional fragment thereof .

12. A set or set of diagnostic tests for carrying out a method according to any of claims 5-8, characterized in that it comprises a nucleic acid encoding apoptin or a derivative or functional fragment thereof, capable of transducing a cell eukaryotic and capable of being expressed in such a cell.

13. The set or diagnostic test set according to claim 12, characterized in that it comprises a means for subjecting a cell to a mild tumorigenic activity.

14. The method according to claim 4, characterized in that the proteinaceous substance is the SV40 large T antigen or a functional equivalent thereof.