MXPA97009860A - Methods and uses for apopt - Google Patents

Abstract

The present invention relates to apoptin can not induce apoptosis in normal human cells. In addition, the invention discloses that when normal cells are transformed, they become susceptible to apoptosis induced by apoptin. The invention discloses that apoptin induces a different type of p53 from apoptosis in various human tumor cells, and can not be blocked by a variety of apoptosis inhibiting agents. The invention comprises an antitumor agent, which specifically kills tumor cells and not normal cells. It also provides the induction of cell death by gene therapy. Apoptin can induce apoptosis in animal, non-human tumor cells. The invention also discloses that in normal cells apoptin was found predominantly in the cytoplasm, whereas in tumor cells, it was localized in the nucleus. In addition, the invention comprises a diagnostic test for the determination of the activity does not transform

Description

METHODS AND USES FOR APOPTINA BRIEF DESCRIPTION OF THE INVENTION The present invention relates to methods and uses for apoptin or derivatives or functional fragments thereof, by which apoptin represents viral protein 3 (VP3) of chicken anemia virus. It also provides novel derivatives of said apoptin. Apoptin itself and its apoptosis-inducing activity have been described previously (see below). However, in these previous publications, apoptin was not different from any other apoptosis-inducing agent. It has now been found that apoptin is of course very different from other apoptosis-inducing agents, and can therefore be applied in different methods and for different uses. While conventional apoptosis-inducing agents induce apoptosis in any cell in which they are present, it has now been found that apoptin induces apoptosis only in transformed cells or in tumor cells. Thus, the invention in one modality provides oral anti-oral therapies. The application REF: 26486 of apoptin as antitumor therapy will cause little toxicity for apoptin, will induce cell death to a greater degree in 'tumor cells, and very reduced, if any, in non-malignant, untransformed cells. The invention also discloses that apoptin can not be inhibited by various anti-apoptosis inhibitors. In particular, apoptin acts distinct from the p53 apoptotic pathway; which is known to be a necessary element in the mediation of apoptosis, which is triggered by a variety of agents (chemo) -therapies. The invention further discloses the finding that apoptin can induce apoptosis in various types of mammalian tumor cells. The invention further relates to the differences in the location of apoptin in cells susceptible to apoptosis induced by apoptin namely the transformed and malignant human cells, versus the cells insensitive to apoptosis induced by apotin, namely the normal human cells. The differential site is used as the test and diagnosis to analyze if a cell is normal or if it has been transformed and / or is malignant.

BACKGROUND OF THE INVENTION Jeurissen et al. (1992b) observed a number of phenomena in chicken thymuses free of specific pathogens, inoculated with chicken anemia virus (CAV), which were absent in control chicken thymuses. Ten days after infection, the entire cortex contained cells whose chromatin had condensed in areas adjacent to the nuclear membrane. Electron density bodies more or less spherical, sporadically in the cytoplasm of the epithelial cells were observed. On day 13 after infection, it was severely diminished in thymocytes, whereas in epithelial cells, many of them contained electronically dense bodies and other non-lymphoid cells were also present. The DNA isolated from the thymuses of chickens infected with various isolates in the CAV field, showed the typical ladder transformation of the oligonucleous break in an electropherogram. These observed morphological biochemical and cellular changes were also observed in the lymphoblastoid cells of birds, cultured, infected with CAV.

The above phenomena are characteristic of the physiological process of programmed cell death or apoptosis. Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and breakage of DNA into size-adjusted fragments in most cells, followed by internucleosomal degradation. Finally, apoptotic cells are fragmented into membrane-enclosed bodies, which are rapidly phagocytosed by surrounding cells. Therefore, apoptosis causes much less tissue destruction than necrosis, the non-physiological type of cell death (Wyllie et al., 1980, Arends and Wyllie, 1991). Apoptosis is a cascade of events. In general, the apoptotic process can be divided into several stages.

Stage 1. Shooting of apoptosis.

Many different external and internal agents can trigger the apoptotic process.

Stage 2. Factors that can mediate the apoptotic trigger.

A major role in this stage is played for example by the p53 tumor suppressor protein.

Stage 3. Increase of the apoptotic signal by factors such as Ba.

Stage 4. Activation of the members of the serine protease family, ICE.

As soon as these specific proteases are activated, the point of no return has been exceeded. The known inhibitors of apoptosis as growth factors (inhibitors in stage 2), Bcl-2 (stage 3) or crmA (stage 4) are known as inhibitors of apoptosis at different stages during the decision stage of the apoptotic process.

Stage 5. Execution of the apoptotic signal. For example, DNA becomes condensed and fragmented. (White, 1996).

Shortly after infection of the cultured mononuclear cells, chicken, the apoptin protein, encoded by CAV (also called VP3) is co-placed with the cellular chromatin. Long after the infection, apoptin forms aggregates, the cells become apoptotic, for example cellular DNA is fragmented and, as a result, it becomes condensed (Noteborn et al. 1994). The immuno-gold electron microscopy showed that apoptin is present in the apoptotic structures. In vitro, the expression of apoptin in transformed lymphoblastoid T cells, chicken and myeloid cells, induced apoptosis in these cells. These data indicate that apoptin can promote or trigger apoptotic guidance in cells infected with CAV (Noteborn et al., 1994, Noteborn and Koch, 1995). Apoptin is a small protein only of 121 amino acids in length, which is rather basic and rich in proline (Noteborn et al., 1991). Apoptin is located strictly within cellular chromatin structures. The truncation of the C-terminal base stretch of apoptin results in a reduced nuclear localization and significantly reduced apoptotic activity (Noteborn et al., 1994). The small size and rather basic character of apoptin may allow interaction with the histone and / or non-histone proteins within the chromatin structure. Apoptosis is an active and programmed physiological process for the elimination of superfluous, altered or malignant cells (Earnshaw, 1995). The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995 and White, 1996). 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 increased cell proliferation, but also by decreased cell death (Kerr et al 1994 ). It has been shown that a variety of chemotherapeutic compounds and radiation induce apoptosis in tumor cells, in many cases via wild-type p53 (Thomposon, 1995, Bellamy et al., 1995, Steller, 1995, Kaufman, 1989, McDonell. et al., 1995, Lowe et al. and Fisher, 1994). Many tumors, however, acquire a mutation in p53 during their development, frequently correlating with poor response to cancer therapy (Hooper, 1994). For many (leukemic) tumors, a high level of expression of the Bcl-2 proto-oncogene is associated with strong resistance to the various chemotherapeutic agents that induce apoptosis (Hoc enberry, 1994, Kerr et al. 1994, Sachs and Lotem, 1993). Apoptin can induce apoptosis in line of human malignant cells (Noteborn and Koch, 1994). It has been established that apoptosis induced by apoptin occurs in the absence of functional p53 (Zhuang et al., 1995a), and can not be blocked by Bcl-2 and BCR-ABL (Zhuang et al. nineteen ninety five) . Therefore, apoptin is useful for the destruction of tumor cells, which have become resistant to the therapeutic induction (chemo) of apoptosis, due to the lack of functional p53 and (on) -expression of Bcl- 2 and BCR-ABL.

DETAILED DESCRIPTION OF THE INVENTION The invention describes the apoptotic activity of apoptin in malignant and transformed (eg human) cells versus normal cells. Apoptin can not induce apoptosis in primary T cells, in endothelial and smooth muscle cells. When normal cells are transformed they become susceptible to apoptin. In normal cells, apoptin was found predominantly in the cytoplasm, whereas in tumor cells it was located in the nucleus. Apoptin can be used for the reduction of specifically transformed / align cells. Therefore, apoptin is a very potent antitumor agent. The expression of apoptin can be used for the induction of apoptosis in specific (human) tumor cells. Apoptosis does not induce or at least not significantly apoptosis in normal cells, indicating that the toxicity of the apoptin treatment will not be low. The invention describes that apoptin is capable of inducing apoptosis in transformed, non-immortalized cells, which implies that the apoptotic activity of apoptin becomes available during early transformation events in cells. Therefore, apoptin can be included in other therapies (for example, genotherapies or chemotherapy) to prevent the treated cells from undergoing transformation and even malignancy. Apoptin can be expressed (transiently) in tumors by transfection of DNA. The expression of apoptin in (tumor) cells can also take place by infecting cells with vectors (retroviruses) containing a coding sequence for apoptin. The administration at. Non-viral component cells (e.g., liposomes or transferrin-derived vectors) that contain apoptin proteins and / or the coding sequence for apoptin, is an additional possibility for the expression / presence of apoptin and induction of apoptosis in tumor cells. In addition, evidence has been provided that apoptin is distinct from the apoptotic pathway induced by p53. In addition to the fact that apoptin does not need functional p53, apoptin can not be blocked by inhibitors of the p53 apoptotic pathway, such as Bcl-2 (and Bcl-2-like) proteins and the crmA protein of cattle pox. , which blocks the activities of proteases similar to ICE. These inhibitors interfere with the different stages of the apoptosis cascade induced by p53. Apoptin can induce apoptosis completely independent of the apoptotic pathway blocked by these apoptotic inhibitors, or act downstream of them. These data imply that apoptin is a very potent inducer of tumor cell apoptosis, which can overcome the blockages (all analyzed) of antiapoptotic activity in transformed and malignant cells. Therefore, apoptin is a potent antitumor agent for a wide variety of tumors (cells). The differential localization of apoptin in normal versus transformed / malignant cells can be used as a diagnostic test to distinguish whether a cell has become trans / malignant. In addition to the induction of apoptosis in human tumor cells, evidence has also been provided that apoptin can induce apoptosis in other mammalian tumor cells. The invention can be used for cancer treatment in various mammalian systems. Thus, the invention provides a vehicle that distributes a tumoricidal substance or a gene encoding a tumoricidal substance, primarily but not exclusively as a tumor cell, characterized in that the tumoricidal substance is apoptin or a functional equivalent thereof. In the prior art, many vehicles have been described for distribution of cytotoxic agents or precursors therefor. A major problem in the distribution of cytotoxic agents is that they are harmful or dangerous to all cells, and not only for the tumor cells (which will be interchangeably called transformed or malignant or tumor cells, in the present). Therefore, many different ways have been investigated to trigger or promote the cytotoxic substance towards the tumor cells. Although many portions of direction to the target are known today, all suffer from the drawback of not being completely specific for the tumor target. Therefore, the use of this cytotoxic substance has not met with great success. At the time of this discovery, it was thought that apoptin suffered from the same inconvenience, since it could require a highly specific target address portion. It has now been found that this is not necessary for apoptin. Apoptin has only a significant effect on tumor cells and not on normal cells. Thus, even if the targeting portion or any other means of distribution of apoptin (or a functional equivalent) is not very specific, this will hardly result in any toxicity to normal tissue. Thus, the invention provides a conjugate for target-directed tumor therapy, comprising a target targeting portion that has binding affinity for a molecule associated primarily, but not exclusively, with the surface of a tumor cell, and apoptin or a functional equivalent thereof. A functional equivalent of apoptin is any fragment or derivative having the same type of activity possibly in different amounts. A targeting portion is well defined in the art as a molecule with a specific binding activity for a target or target molecule. This should preferably be able to perform internalization or introduction. The target molecule may be an antigen or an epitope, in which case the targeting portion is an antibody or a fragment, or a derivative thereof. The target molecule can be a receptor, in which case the targeting portion is a ligand for said receptor. These are just examples of suitable combinations. The union between the target address portion and the apoptin has only the requirement that it must allow the functions of both partners to operate. In this way, this can be a chemical link (labile), or it can be a fusion protein. The conjugate may even be a liposome coated with the targeting portions, and filled with apoptin (or a gene encoding it) etc. The invention also comprises a vehicle that distributes a gene encoding apoptin, to a tumor cell, using gene therapy. Gene therapy has many well-known methods for distributing genes to cells, using viruses such as adenovirus or retrovirus. The person skilled in the art will know how to select the correct vehicle. Because apoptin functions only to induce apoptosis in transformed cells, this or its gene can be used as a safety measure in other gene therapy regimens other than tumor therapy, such as those that rectify the deficiencies of heritable diseases. . This is then provided in a gene delivery vehicle together with the gene of interest, and if the cell in which the gene of interest is inserted becomes malignant, then it will be susceptible to the action of apoptin. Thus, the invention provides a vehicle for the delivery of a nucleic acid of interest to a target cell, said carrier further comprising a gene encoding apoptin or a functional equivalent thereof. Since this is the first truly feasible medical use of apoptin, such use is also part of the present invention. Thus, apoptin or a functional equivalent thereof is provided for the use of a method for the removal of cells from a population of target cells, whereby the method is mainly, but not completely specific for the cells of the target population, whereby apoptin or its functional equivalent is the cytotoxic agent, as well as apoptin or a functional equivalent thereof for use in a method for removing cells from a population of target cells, whereby the cells of said population are not sensitive to the other agents that induce apoptosis. The latter is possible because apoptin can not be inhibited by the mechanisms that inhibit the other substances that induce apoptosis. If apoptin is to be used to remove cells that are not transformed, then this can be achieved by providing apoptin or a functional equivalent thereof, provided with a nuclear localization signal. Since apoptin needs to be in the nucleus, this will result in apoptosis. Without such a signal apoptin could remain outside the nucleus and have no significant effect (as described herein). Thus, apoptin with a core localization signal can be used in a method for the removal of cells from a population of target cells. Nuclear localization (NLS) signals are well known in the art. A fusion gene coding for an NLS and an apoptin could also be provided.

Because apoptin has a different localization in normal cells compared to transformed cells, it is possible to use apoptin as a diagnostic tool that distinguishes between those two cell types. Thus, the invention also provides a method for distinguishing between transformed and / or malignant and / or tumor cells, and normal cells, comprising the steps of providing said cells with viral protein 3 (VP3; apoptin) and detect the location of said viral protein in the cells. Localized apoptin can be detected, for example, with antibodies. The invention will be explained in more detail based on the following experimental part. This is for illustration purposes only, and should not be considered as a limitation on the scope of protection.

EXPERIMENTAL PART Isolation of human primary cells Human primary T cells were isolated from 6 normal blood donors by Ficol centrifugation, and developed in RPMI medium containing 6% human serum, and 0.8 μg per ml of phytohemagglutinin. After 3 days the medium was refreshed and 300 units per ml of Interleukin-2 were added. The human smooth muscle primary cells (SMC) and the vascular endothelial cells (HUVEC) were isolated from umbilical cords. The SMCs were developed in CM199 medium supplemented with ECGF and heparin, and HUVEC in DMEM medium containing 10% fetal calf serum.

DNA plasmids All CAV DNA sequences are originally derived from plc-20H / CAV-EcorRI plasmid DNA (Noteborn and De Boer, 1990). All the steps of cloning with the plasmid DNAs were in principle carried out according to the methods by Maniatis et al. (1982). The expression plasmid pCMV-fs, formerly called pCMV-VP3, contains the CAV DNA sequences coding for apoptin, exclusively (nucleotides 427-868), the plasmid pCMV-tr encodes a truncated apoptin which lacks the 11 C-terminal amino acids (Zhuang et al 1995a), and pCMV-des codes for desmin, a structural protein found in muscle cells, that does not induce apoptosis (Menke et al, unpublished data). The expression plasmids pCMV-BAG-1 (Noteborn, MHM, unpublished data), pCMV-Bcl-2 (Zhuang et al., 1995b) and pCMV-crmA (Noteborn, MHN, unpublished data) express the anti-apoptosis proteins BAG-1, Bcl-2 and the crmA protein of cattle pox, which carry proteases similar to ICE, respectively. The pCMV-ElB21K expression plasmids express the ElB adenoviral protein 21kDa and pCMV-p53, the p53 tumor suppressor protein (Zhuang et al., 1995). All transiently expressed genes are under the regulation of the cytomegalovirus promoter. human, normal, transformed and malignant urea and human fibroblasts.

Human keratinocytes were isolated from the foreskin and developed in the presence of a layer of mouse 3T3 fibroblasts, lethally irradiated with iJ "Cs. The primary cultures of human epidermal keratinocytes (FSK-1) were initiated in complete medium as described (Rheinwald and Green, 1975) with minor modifications The human normal, foreskin diploid fibroblasts VH10 (Klein, 1990) were developed in DMEM supplemented with 10% fetal calf serum.The NW18 tumorigenic fibroblasts transformed with SV40 (Weissman and Stanbridge, 1983) were developed in MEM medium supplemented with 8% fetal calf serum, tumorigenic keratinocytes, SCC-15 (Rheinwald and Beckett, 1981), derived from squamous cell carcinoma, were cultured in DMEM / F12 (3: 1) containing 5% fetal calf serum, 0.4 μg per ml hydrocortisone and 1 μm isoproterenol. The fibroblasts transformed with SV40, precrisis (Pre) and postcrisis (Post), as described by B. Klein et al. (1990) were developed in MEM medium with 8% fetal calf serum. The keratinocyte strain spontaneously transformed HaCat (Boukamp et al., (1988) was a gift from Professor Dr. Fusenig, DKFZ, Heidelber, Germany.

HaCat cells were grown in DMEM medium supplemented with 10% fetal calf serum. The SVK14 strain of keratinocytes transformed with SV40 (Taylor Papadi itriou et al., 1982) was cultured in the same medium as the SCC-15 cells.

DNA infections Plasmid DNA was purified by centrifugation in a CsCl gradient and column chromatography on Sephacril s500 (Pharmacia). Primary human T cells stimulated in phytohemagglutinin were transfected in the presence of DEAE-dextran, as described (Noteborn et al. 1994). Crip mouse cells and human Hep3B, VH10, Pre- and Post-, and NW18 cells, HUVECs and SMCs were transfected with plasmid DNA by calcium phosphate precipitation, as described (Graham and Van der Eb, 1973). The FSK-1 HaCaT, SVK14 and SSC-15 cells were transfected with DOTAP [D. Fischer, unpublished results].

Immunofluorescence The T cells were developed in suspension and fixed on microscope glass slides. All other cells were developed on glass microscope slides, coated. Cells were fixed with 80% acetone for 10 minutes at room temperature, and used for direct immunofluorescence as described (Noteborn et al. 1994). To demonstrate the presence and / or cellular localization of apoptin (truncated) in transfected cells, mouse monoclonal antibodies (MAbs) CVI-CAV-85.1- (85.1) (Noteborn et al. 1991) and CVI-CAV-111.3 were used. (11.3; Koch, unpublished data) and for human desmin, mouse MAb 33 (Monosan, Uden, The Netherlands) was used. Goat anti-mouse antibodies, labeled with fluorescein isothiocyanate (Jackson Immunoresearch Laboratories Inc., West Grove PA) were used as second antibodies. The nuclear DNA was stained with 2,4-diamidino-2-phenylindole (DAPI) or propidium iodide (PI).

Stable transfection of VH10 cell DNA Normal, human diploid VH10 fibroblasts were stably transfected with pCMV-fs, expressing the full-size apoptin or pCMV-neo-Bam, the empty plasmid without the CAV sequences coding for apoptin. The stable clones were selected with the G418 medium, developed on microscope glass slides, and fixed with 30% acetone. The expression of apoptin was analyzed by direct immunofluorescence using MAb 85.1.

RESULTS AND DISCUSSIONS Apoptine expression in mouse tumor cells in vitro It has been examined whether apoptin can induce apoptosis also in mammalian tumor cells of non-human origin. Therefore, cells from the Crip line of mouse tumor cells (Danos 1988) were transfected with the pCMV-fs DNA that purifies for apoptin. Three days after transfection, the cells were fixed. By means of immunofluorescence and Tinsion with PI, the cells were examined for the expression of apoptin, and to see if they became apoptotic. Three days after transfection, 54% of the mouse tumor cells containing apoptin had already become apoptotic. These results indicate that apoptin can induce apoptosis in different mammalian tumor cell lines The apoptotic pathway induced by apoptin in Hep 3B cells is different from the apoptotic pathway induced by P53- Recently, apoptin has been shown to induce apoptosis in osteosarcoma cells without the presence of wild-type p53 (Zhuan et al., 1995, Noteborn and Koch, 1994). Chiou et al. (1994) and Debbas and White (1993) have provided evidence that Bcl-2 and 21B ElB protein of adenovirus 5 (Ad 5) block induced / p53-mediated apoptosis by downstream action from p53 . It has been examined whether the ElB protein of 21kd of Ad 5 and Bcl-2 can inhibit the p53 independent pathway of apoptosis induced by apoptin, compared to p53-dependent apoptosis. For that purpose, the effect of the coexpression of these proteins with apoptin or p53 in the Hep3B cell line of human hepatoma was studied. Hep3B cells were co-transfected with pCMV-fs, which codes for apoptin and pCMV-neo-Ba DNA (negative control), pCMV-ElB21 DNA, which encodes the 12K ElB protein of Ad5 or pCMV-Bcl -2 (Zhuang et al. 1995b), which codes for human Bcl-2. The number of cells expressing apoptin was selected by direct immunofluorescence and by tinsion with DAPI, which is weak and / or regular when the cells have become apoptotic. Surprisingly, at various time points after transfection, the percentage of Hep3B cells expressing apoptin, which became apoptotic, was similar to Hep3B cells containing apoptin and E1B-21K or apoptin and Bcl-2. For brevity, only the data obtained six days after the transfection are given in figure 1. To illustrate that E1B-21K or Bcl-2 of course have an anti-apoptotic effect of Hep3B cells, it was examined whether these two proteins could inhibit apoptosis caused by overexpression of p53 in Hep3B cells. Compared to transfection in Hep3B cells with the pCMV-p53 DNA encoding wild-type p53 (Baker et al., 1990) and pCMV-neo-Bam, cotransfection with the pCMV-p53 and pCMV-ElB21 plasmids, or pCMV-Bcl2 resulted in a significant reduction of apoptosis induced by p53, as analyzed by immunofluorescence and tinsion with PI (Figure 2). Thus, the absence of the effect of the ElB 21K and Bcl-2 proteins on apoptosis induced by apoptin can not be explained by the non-functionality of the proteins expressed in Hep3B cells so that they can inhibit the induced apoptotic pathway. by p53. The fact that ElB 21K and Bcl-2 could still negatively influence the apoptotic pathway regulated by p53 in Hep3B cells, although the apoptosis induced by apoptin could not be inhibited, indicates that the p53-dependent apoptotic pathway, and inducible for apoptin and independent of p53, they are so different at least within Hep3B cells. The fact that in a large number of tumors apoptosis can not be induced in a variety of chemotherapeutic agents seems to be related to the impaired functions of p53 (Lowe et al., 1993). Therefore, the induction of an apoptotic pathway independent of p53 is a useful procedure as an alternative for tumor therapy.

Characterization of apoptotic pathway induced by apoptin in Saos-2 cells The apoptotic pathway induced by apoptin appears to be different from that induced by p53 (see above). Bcl-2, known to be involved in tumor formation via inhibition of the apoptotic pathway, may not block apoptosis induced by apoptin, but could block the p53 pathway. Recently, it has been reported by others that Bcl-2 and Bag-1 (Takayama et al., 1995) could be jointly required for the inhibition of so-called Bcl-2 independent apoptosis, as seems to be the case for apoptosis induced by Apoptin To examine whether Bag-1 could negatively influence apoptosis induced by apoptin alone or together with Bcl-2, cotransfections of Saos-2 cells, pCMV-fs and pCMV-Bcl-2 or pCMV-Bag were carried out. -1, or pCMV-Bcl-2 and pCMV-Bag-1. In parallel, similar cotransfection experiments, control, with p53 were carried out. Bag-1 or a combination of Bag-1 and Bcl-2 did not inhibit apoptosis induced by apoptin, whereas apoptosis induced by p53 was significantly induced by Bag-1 or Bag-1 and Bcl-2 (Figure 3). It is concluded that the apoptotic pathway induced by apoptin is independent of Bcl-2-like proteins or acts downstream of them. Using double immunofluorescence microscopy and video intensified fluorescence, we analyzed whether apoptin could regulate Bax expression. Bax is a cellular protein, which can induce apoptosis and associate proteins Bcl-2 and other, still unknown, cellular proteins. p53 upregulates the expression of Bax, which results in the induction of apoptosis (Oltvai et al. 1993). Evidence has been found that apoptin can not over regulate the level of Bax protein, while in a similar experiment it was shown that p53 can do so. This is another argument that apoptin does not seem to act through the apoptotic pathway of p53. The proteins similar to (ICE) the enzyme of the beta conversion of interleukin-1 are known to be the last or one of the last steps in the decisive cascade of the apoptotic process (Kumar 1995). To examine whether ICE-like proteins may play a role in apoptosis induced by apoptin, crmA inhibitor of ICE was co-expressed with apoptin. Evidence was obtained that the inhibition of ICE-like proteins, due to the expression of the bovine pox protein crmA, does not result in the inhibition of apoptosis induced by apoptin. However, in a parallel experiment it was shown that the expression of crmA could reduce the apoptosis induced by p53 (Figure 4). Therefore, it is concluded that apoptin is very close to or even beyond the point of no return within the decisive cascade of apoptosis. In addition, apoptosis induced by apoptin does not use or only uses a very small part of the apoptotic pathway induced by p53.

Expression of apoptin in human [normal] cells Three types of human primary cells, for example vascular endothelial cells (HUVEC), smooth muscle cells (HSMC) and T cells, were transiently transfected with a plasmid coding for full size apoptin (pCMV-fs). Cells expressing apoptin were selected via direct immunofluorescence with MAb 85.1 [8] or 111.3. The induction of apoptosis in apoptin-positive cells was analyzed with the help of DAPI or PI, which stained the intact nuclei regularly, but the apoptotic cells irregularly and / or weakly (Telford 1992). Five days after transfection, only about 20% of the primary cells expressing apoptin were abnormally stained with DAPI or PI (data not shown). Menke and colleagues (unpublished data) have reported for another system of apoptosis, that this low percentage of abnormal cells-DAPI is not due to a specific agent inducing apoptosis, but due to transfection events. Also, the experiments shown below prove this statement. In similar experiments, 60-90% of the malignant cells containing apoptin were apoptotic (Zhuang et al., 1995, 1995a, b). The location of apoptin in these primary cells also differed from the location in the tumor cells. In all normal cells, apoptin was found in the cytoplasm, and not in the nucleus as has been observed for various lines of tumor cells (Zhuang et al., 1995, 1995a, b). These results suggest that apoptin can not induce apoptosis in several non-tumorigenic, non-transformed, human, cultured cells, and that the cellular localization of apoptin is important for its apoptotic activity.

Expression of apoptin in normal human cells and their malignant derivatives The presence for the expression of apoptin in lines of tumor cells has been examined, versus that of the normal cells from which they have been generated. To that end, diploid skin fibroblasts and keratinocytes from normal individuals and their tumorigenic counterparts were transiently transfected as pCMV-fs, which express the full-size apoptin, pCMV-tr, which express the truncated apoptin or a plasmid that code for desmin (pCMV-des). Desmin has no apoptotic activity (Menke et al., Unpublished data), and is used as a negative control. Five days after transfection, the percentage of VH10 positive fibroblasts to apoptin (Figure 5) and FSK-1 keratinocytes (Figure 6), which have become apoptotic, were not above 15%. This level of aberrance of cells stained with DAPI was similar for cells containing truncated apoptin or desmin. The low level of apoptosis in these apoptin-positive cells may be due not to the specific induction of apoptin in death cells, but to transfection events. To examine whether tumorigenic fibroblasts and keratinocytes were susceptible to apoptin, NW18 and SCC-15 were transfected with plasmids coding for apoptin (full-length or truncated) or desmin. Apoptin, and to a lesser degree, truncated apoptin could induce apoptosis in NW-18 (Figure 5) and SCC-15 (Figure 6) which are tumor cells. Up to 65-75% of these apoptin-positive cells were apoptotic at five days after transfection, which is the similar interval as reported for cell death induced by apoptin in osteosarcoma cell lines (Zhuang et al. , 1995a). The level of abnormal cells DES-positive NW-18 and SCC-15, stained with DAPI, was not significantly higher than that between normal fibroblasts and keratinocytes (Figure 5 and 6). The differential activity of apoptin in normal and tumor cells could not be explained by different proportions of proliferation, since these are similar to the VH10 fibroblasts and the osteosarcoma Saos-2 cells. The present observations show that apoptin does not induce apoptosis in normal fibroblasts and keratinocytes, but in its tumorigenic derivatives.

Apoptin expression in human transformed cells Is the tumorigenicity required by apoptin sufficient to induce cell death, or is it the mere transformation of a cell? To answer this question we have examined the effect of apoptin on fibroblasts and transformed, non-tumorigenic keratinocytes. Apoptin, truncated apoptin and desmin were transiently expressed in fibroblasts transformed with SV40, before (Pre) after (post) immortalization, in SVK14 keratinocytes transformed with SV40 and immortalized, and in spontaneously transformed HaCaT keratinocytes. Apoptin was able to induce apoptosis in those types of transformed cells, to a degree similar to that in tumor cells (Figures 5 and 6). The percentage of apoptine-containing cells (truncated) which had become apoptotic was significantly higher than in the desmin-positive cells. These data imply that apoptin can induce apoptosis in malignant and transformed cells. Some chemotherapeutic agents and radiation can induce apoptosis in transformed cells, but they can not do so in non-transformed cells (Thompson, 1995, McDonell et al., 1995). The transformation seems to cause changes that make a cell more sensitive to apoptotic stimuli. It is known that some other proteins kill tumor cells specifically. The structural protein of parvovirus is NS-1 induces cell lysis specifically in neoclassical cells (VanAcker and Rommelaere, 1995). Also, an adenovirus expressing bcl-xs has been constructed, it induces apoptosis in neuroblast cells and breast and colon carcinoma, but not in normal human hematopoietic cells, or in the human K562 leukemia cell line (Clarke et al. collaborators, 1995) Stable transfection of the VH10 cell with a plasmid coding for apoptin To exclude that apoptin has a minor but significant apoptotic activity in normal VH10 cells, these cells were stably transfected with pCMV-fs or with pCMV-neo-Bam as a control. A similar amount of colonies was obtained in both transfections. The resulting cells, stably transfected with pCMV-fs, expressed apoptin, which was localized in the perinuclear region (data not shown). Therefore, it can be concluded that apoptin can not induce apoptosis or inhibit the growth of normal VH10 cells in any other way.

Cellular apoptin localization in normal versus transformed and malignant cells Apart from apoptotic activities other than apoptin in malignant and transformed cells versus normal cells, differences in apoptin cellular localization have also been observed in these cell types. In transformed and malignant cells, before the apoptotic and morphological changes are detectable, apoptin is distributed as fine granules, mainly in the nucleus. After the cells had undergone apoptosis, apoptin was added to the nucleus. In contrast, the location of apoptin in normal fibroblasts and keratinocytes is mainly in the cytoplasm, they are concentrated around the nucleus, either as small granules and as larger aggregates. In the primary HUVECs HSMCs and T cells, apoptin was also localized in these characteristic perinuclear structures. By far, in all lines of malignant cells and transformed and studied, apoptin had a nuclear location, while in all normal cells analyzed in this way, did not have it. Others have proposed that the transformed cells have undergone loss-of-function mutations, as a result of which a normally functioning inhibitor has been changed. Due to these changes, the nuclear transport of proteins can be promoted or prevented in cancer cells (Csermely et al., 1995). It is possible that in normal cells apoptin is associated with and / or modified by one or more cellular factors, resulting in its placement within the perinuclear structures. The loss of such (functional) factors in malignant cells may allow apoptin to enter the nucleus. Apoptin hosts not only putative nuclear import sequences (Noteborn et al., 1991, 1994, 1995, Zhuang et al., 1995, 1995a, b) but also an amino acid region that resembles nuclear export signals (position 33). -46: IRIGIAGITITLSL), similar to that of the protein-kinase inhibitor (PKI) and the HIV-Rev protein (Wen et al., 1995, Fischer, et al., 1995, Gerace, 1995).

It may be that this nuclear export signal, potential, can not be recognized in the various malignant and transformed cell lines, analyzed. The results described here indicate that the nuclear localization of apoptin is important for its ability to induce apoptosis. This is in agreement with the observation that truncated apoptin, which has a reduced apoptotic activity (Figure 5 and 6) is partially in the cytoplasm. Electron microscopy studies with chicken mononuclear cells revealed that apoptin is co-localized with cellular chromatin (Noteborn and collaborators, 1994). The interaction of apoptin with chromatin could result in the unwinding of its superstructure. The last phenomenon has been described for rat ventral prostate cells, which became apoptotic after castration of rats (Kyprianou and Isaacs, 1989).

Apoptin as an antitumor agent The present results indicate that apoptin is an antitumor agent. First, apoptin is specifically active in malignant and transformed cells, but, at least in vitro, not in the normal cells tested. Therefore, the toxic effect of apoptin treatment can be very low. Second, apoptin induces apoptosis independently of p53. The fact that several chemotherapeutic agents lose their capacity to induce apoptosis in a large number of tumors seems to be related to an interruption and deterioration of p53 function. Therefore, the induction of a p53-independent apoptotic pathway may be a useful procedure as an alternative candidate for tumor therapy. In addition, apoptin apparently is not blocked by Bcl-2, which is known to be involved in the development of, for example, leukemic tumors, and which can inhibit apoptosis induced by chemotherapeutic agents. In addition, it has been observed that BAG-1 and the protein related to Bcl-2, is not able to block the apoptosis induced by apoptin.

Description of the figures Figure 1 shows the effect of the expression of ElB-12kD and Bcl-2 on the induction of apoptosis by VP3 in Hep3B p53-minus cells. The percentage given is that of apoptin-positive cells, which are apoptotic 6 days after transfection. Cells were co-transfected with 2.5 μg of pCMV-VP3 and 5 μg pCMV-neo-Bam (open bars), 5 μg of pCMV-ElB21 (shaded bars), or 5 μg of plasmid DNA pCMV-Bcl-2 (line bars) discontinuous). At least 3 independent experiments were carried out. By experiment, at least 200 cells positive for apoptin were examined.

Figure 2 shows the effect of the E1B-21K and Bcl-2 proteins on the induction of apoptosis by p53 in the Hep3B cell line. The cells were co-transfected with 2.5 μg of pCMV-p53 and Éμg of pCMV-neo-Bam, 5 μg of pCMV-ElB21K, or 5 μg of the plasmid DNA of pCMV-Bcl-2. Two independent transfections were carried out. Cells were analyzed 4 or 5 days after transfection. The percentage given is that of p53-positive cells, which are apoptotic. By experiment, at least 200 p53 positive cells were examined. Figure 3 shows the effect of Bcl-2 and Bag-1 expression on apoptosis induced by p53 or apoptosis induced by apoptin. Saos-2 cells were cotransfected with 2.5 μg of pCMV-fs, and 5 μg of pCMV-Bcl-2, pCMV-Bag-1 or pCMV-Bag-1 and pCMV-Bcl-2. In a parallel experiment, the cells were cotransfected with 2.5 μg pCMV-p53 and 5 μg pCMV-Bcl-2, pCMV-Bag-1 or pCMV-Bag-1 and pCMV-Bcl-2. As controls, pCMV-p53 or pCMV-fs were cotransfected with pCMV-neo-Bam. At least, 3 independent experiments of both series were carried out. Cells were harvested 4 days after transfection. The percentage is given of the p53 or apoptin positive cells, which have become apoptotic. By experiment, at least 200 cells have been examined. Figure 4 shows the effect of ICE inhibitor crmA on the induction of apoptosis induced by p53 or apoptin. The Saos-2 cells were cotransfected with 2.5 μg of pCMV-fs and 5 μg of pCMV-crmA (+ crmA) or pCMV-neo-Bam (-crmA). In parallel, the cells were cotransfected with 2.5 μg pCMV-p53 and 5 μg of pCMV-crmA or pCMV-neo-Bam. Two independent experiments were carried out. Cells were harvested 5 days after transfection. The percentage of cells positive to p53 or apoptin is given, which have become apoptotic. By experiment, at least 200 positive cells were examined. Figure 5 shows the activity of apoptin in normal human fibroblasts versus transformed or malignant fibroblasts. The percentage of cells that were abnormally stained with DAPI, is given as a measure relative to apoptosis in normal VH10 fibroblasts versus transformed (Pre, Post) and tumor (NW18), transiently transfected with pCMV-fs, pCMV-tr or pCMV-des. Cells were fixed five days after transfection and analyzed by direct immunofluorescence. The results are the means of at least three independent experiments. In each experiment, at least 200 full size, or apoptine positive or desmin, truncated cells were examined. The cells were fixed 5 days after transfection and analyzed by indirect immunofluorescence. Figure 6 shows the activity of apoptin in normal versus transformed or malignant keratinocytes. The percentage of cells that were abnormally stained with DAPI is given as a relative measure for apoptosis in normal keratinocytes (FSK-1) versus transformed (SVK14, HaCAT) and tumor (SCC-15), transiently transfected with pCMV-fs , pCMV-tr, or pCMV-des. Cells were fixed five days after transfection and analyzed by indirect immunofluorescence. The results are the means of at least three independent experiments. In each experiment, at least 200 full size or positive truncated apoptin or desmin cells were examined.

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It is noted that in relation to this date, the best method known to 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 (9)

1. A method for distinguishing between transformed and / or malignant and / or tumor cells and normal cells, characterized in that it comprises the steps of providing the cells with a viral protein 3 (VP3; apoptin) and the detection of viral protein localization in said cell.

2. A vehicle for distribution of nucleic acid of interest to a target cell, the vehicle is characterized in that it comprises a gene encoding apoptin or a functional equivalent thereof.

3. A conjugate for target-directed tumor therapy, characterized in that it comprises a targeting position, which has binding affinity for a molecule associated principally but not exclusively with the surface of a tumor cell and apoptin or a functional equivalent of the same

4. A vehicle for the delivery of a tumoricidal substance or a gene encoding a tumoricidal substance to a molecule associated principally but not exclusively with a tumor cell, characterized in that the tumoricidal substance is apoptin or a functional equivalent thereof.

5. Apoptin or a functional equivalent thereof, characterized in that it is for use in a method for the elimination of cells from a target cell population, by which it is principal but not completely, specific to the cell of the target population, with which apoptin or its functional equivalent is the cytotoxic agent.

6. Apoptin or a functional equivalent thereof, characterized in that it is for use in a method for the elimination of cells from a target cell population, whereby the cells of said population are not sensitive to other agents inducing apoptosis.

7. Apoptin or a functional equivalent thereof, characterized in that it is provided with a nuclear localization signal. 35

8. The apoptin or a functional equivalent according to claim 7, characterized in that it is for the use of a method for the removal of cells from a population of target cells.

9. A nucleic acid, characterized in that it encodes an apoptin or a functional equivalent thereof, according to claim 8.