Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-05-02
2003-03-04
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S069100, C436S064000
Reexamination Certificate
active
06528247
ABSTRACT:
The present invention relates to the field of cancer diagnosis and treatment, as well as to the field of determining the transforming capability of suspected tumorigenic or tumor promoting agents (the two terms will be used interchangeably herein). 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 (hereinafter all referred to as apoptin or apoptin-like activity) can be applied according to the invention. Apoptin is a protein orginally found in Chicken Anemia Virus (Noteborn et al., 1991) and was originally called VP3. The apoptotic activity of this protein was discovered by the group of the present inventors (Noteborn et al., 1994). As stared above the present invention makes use of the apoptosis inducing effect of apoptin.
Apoptosis is an active and programmed physiological process for eliminating superfluous, excessively damaged or malignant celles (Earnshaw, 1995, Duke et al., 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus and fragmentation of the cytoplasm, condensation and cleavage of DNA into domain-sized fragments, in most cases followed by internucleosomal degradation. The apoptotic cells become fragmented into membrane-enclosed apoptotic bodies. Finally, neighbouring cells and/or macrophages will rapidly phagocytose these dying cells (Wyllie et al., 1980, White, 1996). Cells grown under tissue-culture conditions and cells from tissues can be analysed for signs of apoptosis with agents staining chromosomal DNA, as e.g. DAPI or propidium iodide, which stains normal DNA (chromatic) strongly and regularly, but apoptotic chromatin weakly and/or irregularly (Noteborn et al., 1994, Telford et al., 1992).
The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995, White 1996, Levine, 1997). Changes in cell survival rate play an important role in human pathogenesis, e.g. in cancer development, which is caused by enhanced proliferation and/or by decreased cell death (Kerr et al., 1994, Paulovich, 1997). A variety of chemotherapeutic agents and radiation have been demonstrated to induce apoptosis in tumor cells which, in many instances is mediated via the tumor supressor protein p53 (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 poor response to cancer therapy. Transforming proteins of DNA tumor viruses inactivate p53 indirectly or by directly binding to it (Teodoro, 1997). An example of such an agent is the large T-antigen of the DNA tumor virus SV40. In certain hemopoietic tumors, a high expression level of the Bcl-2-oncogene is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry 1994, Sachs and Lotem, 1997). For such cancers, that are resistant to many cytotoxic agents, alternative anti-tumor therapies are under development based on induction of apoptosis (Thompson, 1995 and Paulovch et al., 1997).
Apoptin is a small protein derived from the chicken anemia virus (CAV; Noteborn and De Boer, 1995, Noteborn et al., 1991, Noteborn et al., 1994), which can induce apoptosis in human malignant and transformed cell lines, 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 e.g. by the transforming genes of SV40, they become susceptible to apoptosis by apoptin. (Danen-van Ooschot, 1997 and Noteborn, 1996). 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 localized predominantly in the cytoplasm, whereas in transformed or malignant cells, it was located in the nucleus, suggesting that the localization of apoptin is related to its death-inducing activity (Danen-van Oorschot et al. 1997).
Furthermore, we have established that apoptin can induce apoptosis in the absence of functional p53 (Zhuang et al., 1995a), and cannot be inhibited by Bcl-2, Bcr-abl (Zhuang et al., 1995), the Bcl-2-associating protein BAG-1 and the caspase-inhibitor cow-pox protein CrmA (Danen-Van Oorschot, 1997, Noteborn, 1996). Finally, it appears that cells that are only immortalized and thus minimally transformed, can also be killed by apoptin. Therefore, apoptin is an extremely potent anti-tumor agent, also for tumors that are not or less susceptible to (chemo)therapeutic agents due to the lack of functional p53, (over)-expression of Bcl-2 or other apoptosis-inhibiting genes. The fact that apoptin does not induce apoptosis in normal human cells, suggests that a toxic effect of apoptin treatment in vivo will be very low. In addition, it appears, that even premalignant, minimally transformed cells, may be sensitive to the death-inducing effect of apoptin. Knowing that apoptin is quite safe in normal cells, but that as soon as a cell becomes transformed and/or immortalized (the terms may be used interchangeably herein) the present inventors designed some uses based on this finding. Thus the invention provides a method for determining the transforming capability of a possible transforming agent, comprising providing a non-transformed cell with inducible apoptin-like apoptotic activity, exposing said cell to said transforming agent and determining the localization of said apoptotic activity within said cell or determining the induction of apoptosis in said cell. It is to be understood that in the above apoptotic activity also refers to the entity having said activity. It is preferred to provide said cell with said apoptotic activity by transducing said cell with a recombinant nucleic acid molecule encoding said activity. Apoptin-like activity is herein defined as any (preferably proteinaceous) substance having similar activity as VP3 or apoptin of chicken anemia virus. Specifically included in said definition are allelic variants, derivatives and/or fragments of apoptin, wherein derivatives are defined as having amino acid replacements which do not result in the loss of all apoptotic activity. It is to be understood that similar activity means that the kind of activity is the same although the amount may differ. The methods according to the invention are especially suitable in applications whereby said possible transforming agent is a proteinaceous substance. This allows for said proteinaceous substance to be co-expressed in said non-transformed cell with said apoptotic activity. The exemplified proteinaceous substance is the large T-antigen of SV40 or a functional equivalent thereof. The invention also provides modifications on the Apoptin gene resulting in changes on the apoptin protein enabling apoptin to enter the nucleus in non-transformed and transformed/tumorigenic cells, resulting in the induction of apoptosis. The apoptin protein is enlarged with a nuclear localization signal of SV40. Specifically included in said definition of apoptin are allelic variants, derivatives and/or fragments of apoptin, wherein derivatives are defined as having amino acid replacements which do not result in the loss of all apoptotic activity. This allows for the apoptin protein to be expressed in non-transformed cells with said apoptotic activity. Apoptin fragments with said apoptotic activity but not able to enter the nucleus of non-transformed or transformed cells by its own sequences, are able to enter the nucleus by means of the modifications and induce apoptosis.
The invention further provides a method for determining the predisposition of a cell to become a tumor cell, by providing said cell with inducible apoptin-like apoptotic activity and subjecting said cell to relatively mild tumorigenic activity and determining apoptosis in said cell and/or determining the localization of said apoptotic activity in said cell. In this case the suspected transforming agent as discussed hereinbefor
Caputa Anthony C.
Leadd B.V.
Nickol Gary B.
TraskBritt
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