Apoptosis-inducing proteins in chicken anemia virus and TT virus.

Torque teno viruses (TTVs) share several genomic similarities with the chicken anemia virus (CAV). CAV encodes the protein apoptin that specifically induces apoptosis in (human) tumor cells. Functional studies reveal that apoptin induces apoptosis in a very broad range of (human) tumor cells. A putative TTV open reading frame (ORF) in TTV genotype 1, named TTV apoptosis inducing protein (TAIP), it induces, like apoptin, p53-independent apoptosis in various human hepatocarcinoma cell lines to a similar level as apoptin. In comparison to apoptin, TAIP action is less pronounced in several analyzed human non-hepatocarcinoma-derived cell lines. Detailed sequence analysis has revealed that the TAIP ORF is conserved within a limited group of the heterogeneous TTV population. However, its N-terminal half, N-TAIP, is rather well conserved in a much broader set of TTV isolates. The similarities between apoptin and TAIP, and their relevance for the development and treatment of diseases is discussed.

Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor.

Specificity is a prerequisite for systemic gene therapy of hepatocarcinoma. In vitro, the tumor-specific viral death effector Apoptin selectively induces apoptosis in malignant hepatic cells. Intratumoral treatment of xenografted subcutaneous hepatomas with Apoptin results in tumor regression. Here, we report a systemic delivery vehicle containing the Apoptin gene linked to asialoglycoprotein (Asor), which targets asialoglycoprotein receptor (ASGPR) present only on the surface of hepatocytes. In vitro, the protein-DNA complex Asor-Apoptin induced apoptosis in HepG2 hepatocarcinoma cells but not in normal L-02 hepatocytes. Non-hepatocyte-derived tumorigenic human A549 cells lacking the membrane ASGPR were not affected by Asor-Apoptin. In vivo systemic delivery of Asor-Apoptin via the tail vein into mice bearing in situ hepatocarcinoma resulted in specific and efficient distribution of Apoptin in both hepatocarcinoma cells and normal hepatocytes. Five days after injection of Asor-Apoptin, the in situ hepatocarcinomas showed significant signs of regression, whereas the surrounding normal hepatocytes did not. Systemically delivered Asor-LacZ expressing non-apoptotic LacZ gene did not inhibit tumor growth. Our data reveal that systemic delivery of Asor-Apoptin specifically induces apoptosis in malignant hepatocytes and thus constitutes a powerful and safe therapeutics against hepatocarcinomas.

Fibroblast-like synoviocytes from patients with rheumatoid arthritis are more sensitive to apoptosis induced by the viral protein, apoptin, than fibroblast-like synoviocytes from trauma patients.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) show characteristics of transformation. Because the chicken anemia virus protein, apoptin, induces apoptosis solely in transformed cells, it was investigated whether FLS from patients were more sensitive to apoptin-induced apoptosis than FLS from normal joints obtained from trauma patients. METHODS: FLS were transduced with maltose-binding protein (MBP)-apoptin recombinant protein or MBP as a control protein by microinjection. After 24 hours, cells were fixed and stained with immunofluorescence to detect apoptin or MBP and the number of dead cells was assessed. Furthermore, phosphorylation of apoptin was analysed in FLS from patients with RA and from trauma patients by in vitro kinase assay. RESULTS: FLS from patients with RA were significantly more sensitive to apoptin-induced apoptosis than FLS from trauma patients (p = 0.0263). Furthermore, MBP-apoptin induced more apoptosis than MBP in RA FLS (p = 0.004). No phosphorylation of apoptin was observed in FLS from patients with RA. DISCUSSION: FLS from patients with RA are more sensitive to apoptin-induced apoptosis than normal FLS, which is consistent with a transformed phenotype of these cells. However, given the lack of phosphorylation of apoptin in RA FLS the mechanism of action of apoptin seems to differ between tumour cells and RA FLS. This study indicates that apoptin may help to identify a new therapeutic pathway against hyperplasia of the synovium and joint destruction in RA.

The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes.

Apoptin, a protein derived from chicken anemia virus, has previously been shown to induce apoptosis in a p53-independent and Bcl-2-stimulated manner in transformed and tumorigenic human cells but not in normal diploid human cells, suggesting that it is a potential agent for tumor therapy. Here we report that Apoptin can induce apoptosis in UV-C-irradiated diploid skin fibroblasts from individuals with various hereditary cancer-prone syndromes that are characterized by a germ-line mutation in a tumor suppressor gene. The same effect is found when these cells are irradiated with X-rays. In contrast, diploid skin fibroblasts from healthy donors or from individuals with DNA repair disorders are not responsive to Apoptin-induced apoptosis upon UV-C or X-ray irradiation. After transfection of untreated cells, Apoptin is found predominantly in the cytoplasm, whereas in UV-C-exposed Apoptin-responsive cancer-prone cells, it migrates to the nucleus, where it causes rapid apoptosis. Apoptin remains localized in the cytoplasm after UV-C treatment of diploid cells from healthy individuals. The induction of apoptosis by Apoptin in cancer-prone cells with a germ-line mutation in a tumor suppressor gene is UV dose-dependent and transient, just like many other UV-induced processes. These results suggest that Apoptin may be used as a diagnostic tool for detection of individuals with an increased risk for hereditary cancer and premalignant lesions.

Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells.

Previously, we have shown that the chicken anemia virus-derived VP3 ("apoptin") protein induces apoptosis in chicken mononuclear cells. Here, we report that apoptin also induces apoptosis in human osteosarcoma cells, regardless of whether they expressed wild-type, mutant p53, or no p53 at all. Moreover, the nuclear location of apoptin appears to be important for its optimal induction of apoptosis. The fact that apoptin can induce p53-independent apoptosis in human tumor cells makes apoptin a potential candidate for treatment of frequently occurring types of cancer cells that do not contain functional p53.

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing.

Apoptin, a protein from chicken anemia virus without an apparent cellular homologue, can induce apoptosis in mammalian cells. Its cytotoxicity is limited to transformed or tumor cells, making Apoptin a highly interesting candidate for cancer therapy. To elucidate Apoptin's mechanism of action, we have searched for binding partners in the human proteome. Here, we report that Apoptin interacts with DEDAF, a protein previously found to associate with death effector domain (DED)-containing pro-apoptotic proteins, and to be involved in regulation of transcription. Like Apoptin, after transient overexpression, DEDAF induced apoptosis in various human tumor cell lines, but not in primary fibroblasts or mesenchymal cells. DEDAF-induced cell death was inhibited by the caspase inhibitor p35. Together with the reported association of DEDAF with a DED-containing DNA-binding protein in the nucleus and the transcription regulatory activity, our findings may provide a clue for the mechanism of Apoptin's actions in mammalian cells.

Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro.

Apoptin, a small protein encoded by chicken anemia virus (CAV) was expressed in various human hematologic malignant cell lines derived from leukemias and lymphoma. Three of these cell lines contain bcl-2 or BCR-ABL proteins, known to block apoptosis induced by chemotherapeutic compounds. By immunofluorescence and propidium-iodide staining apoptin was shown to induce apoptosis in all analysed cell lines. Early after expression, apoptin exhibited a fine-granular distribution in the still intact nucleus. Later, apoptin became aggregated and the nucleus segmented. The data with truncated apoptin indicate that for optimal induction of apoptosis apoptin has to be located in the nucleus.

Apoptin acts as a tumor-specific killer: potentials for an anti-tumor therapy.

Apoptin, a protein encoded by an avian virus, induces apoptosis in a tumor-specific way, acts p53-independently and is even stimulated by the anti-apoptotic protein Bcl-2. Activation of upstream caspases is not required, whereas the activation of downstream caspases is involved in rapid Apoptin-induced cell death. Yet, in a caspase-3-negative human breast cancer cell line, Apoptin can induce apoptosis, but delayed. These features indicate that Apoptin can induce apoptosis via multiple pathways in tumor cells when other agents might fail. Apoptin is biologically active as a highly stable, multimeric complex, consisting of 30 to 40 monomers and forms cooperatively distinct superstructures upon binding to DNA. In tumor cells, Apoptin is imported into the nucleus prior to the induction of apoptosis; this contrasts with the situation in primary, normal cell cultures where nuclear import of Apoptin is very rare. Apoptin contains two different domains that induce apoptosis independently, and for both domains, a strong correlation exists between nuclear localization and killing activity. Apoptin is regulated by a kinase activity present in cancer cells but negligible in normal cells. Apoptin interacts with various partners of the human proteome such as DEDAF, which when overexpressed induces apoptosis in various human tumor cell lines but not in primary human cells, similar to Apoptin. In normal cells, Apoptin becomes aggregated, epitope shielded and eventually degraded in the cytoplasm. Furthermore, Apoptin-transgenic mice and other animal models have revealed Apoptin as a safe and efficient anti-tumor agent. These in vitro and in vivo tumor-specific features of Apoptin imply that it can form the basis of future anti-tumor therapies.

Identification of the promoter region of chicken anemia virus (CAV) containing a novel enhancer-like element.

The single promoter region in the cloned genome [Noteborn et al., J. Virol. 65 (1991) 3131-3139] of chicken anemia virus (CAV) in chicken T-cells was analysed via CAT assays. A unique region containing four or five near-perfect direct repeats (DR) of 21 bp with one 12-bp insert was proven to be the main transcription-activation element, with enhancer-like characteristics. PCR studies revealed that CAV isolates from across the world all contained this promoter sequence. Electrophoretic mobility-shift assays (EMSA) showed that individual DR units, as well as the 12-bp insert, can bind to nuclear factors of chicken T-cells. Competition assays revealed that the DR units bound to factors other than the 12-bp insert. A synthetic oligodeoxyribonucleotide containing an SP1-box (5'-GGGCGG) could compete with factors binding to the 12-bp insert. Purified human SP1 was shown to have very strong affinity for the 12-bp insert.

Chicken anemia virus strains with a mutated enhancer/promoter region share reduced virus spread and cytopathogenicity.

Plasmid pCAV/E contains an infectious cloned double-stranded CAV (chicken anemia virus) DNA genome (Noteborn et al., J. Virol. 65 (1991) 3131-3139). We have constructed mutated CAV genomes by introducing mutations into the CAV promoter/enhancer region of pCAV/E. Various mutated CAV strains were functional and had a smaller cytopathogenic effect in chicken T cells than wild-type CAV. In particular, mutations within the '12-bp insert' of the promoter/enhancer region had this effect. PCR and sequence analysis showed that the CAV mutants were stable under cell-culture conditions. Southern-blot analysis showed that all replication DNA intermediates were normally formed by the CAV mutants. All viable mutant CAV strains were able to produce a neutralizing conformational epitope, which implies that they can trigger the required protective immune response. These features make these mutant CAV strains potential candidates for the development of an attenuated CAV vaccine.

Transcription of the chicken anemia virus (CAV) genome and synthesis of its 52-kDa protein.

This paper describes the expression of the chicken anemia virus (CAV) genome, a recently characterized single-stranded circular-DNA virus of a new type [Noteborn et al., J. Virol. 65 (1991) 3131-3139]. The major transcript from the CAV genome is an unspliced mRNA of about 2100 nucleotides (nt). Its transcription start point and poly(A)-addition site are located at nt 354 and 2317 of the CAV sequence, respectively. In vitro translation experiments provide evidence that the major CAV open reading frame encodes a 52-kDa protein by using the fifth AUG as a start codon of the unspliced CAV mRNA.

Apoptin's functional N- and C-termini independently bind DNA.

Apoptin induces apoptosis specifically in tumour cells, where Apoptin is enriched in the DNA-dense heterochromatin and nucleoli. In vitro, Apoptin interacts with dsDNA, forming large nucleoprotein superstructures likely to be relevant for apoptosis induction. Its N- and C-terminal domains also have cell-killing activity, although they are less potent than the full-length protein. Here, we report that both Apoptin's N- and C-terminal halves separately bound DNA, indicating multiple independent binding sites. The reduced cell killing activity of both truncation mutants was mirrored in vitro by a reduced affinity compared to full-length Apoptin. However, none of the truncation mutants cooperatively bound DNA or formed superstructures, which suggests that cooperative DNA binding by Apoptin is required for the formation of nucleoprotein superstructures. As Apoptin's N- and C-terminal fragments not only share apoptotic activity, but also affinity for DNA, we propose that both properties are functionally linked.

Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin.

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell-specific, p53-independent, Bcl-2-insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)-transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1-induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector.

Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system.

Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VP1L) and the 1.8 kb coding region for VP2 (AcB19VP2L), placed under the control of the polyhedrin promoter. The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. AcB19VP1L-infected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence. The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique. VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa beta-galactosidase B19 fusion protein expressed in E. coli. Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots. VP2 did not react with the fusion protein-specific mAbs. The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

Apoptin specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals: a review.

Tumor formation is caused by an imbalance between cell replication and apoptosis, which is a physiological form of cell death. For instance, UV damage can result in tumor formation due to mutations of the tumor-suppressor gene p53, a major apoptosis-inducing protein. Over-expression of the proto-oncogene Bcl-2, due to chromosomal translocation, can also inhibit apoptosis resulting in, e.g., lymphomas and leukemias. Anti-tumor therapies are often based on induction of apoptosis mediated via p53 and/or inhibited by Bcl-2, which explains the frequently poor results of anti-tumor treatment. The avian-virus-derived protein 'Apoptin', induces apoptosis in a p53-independent way, is stimulated by Bcl-2 and is insensitive to BCR-ABL, another inhibitor of chemotherapeutic agents. Apoptin induces apoptosis in human transformed/tumorigenic cells but not in normal diploid cells. Co-synthesis of SV40 large T antigen and Apoptin results in induction of apoptosis, illustrating that the establishment of a stable transformed state is not required. UV-irradiation causes an aberrant SOS-response in primary diploid cells from cancer-prone individuals and renders such cells susceptible to Apoptin-induced apoptosis. All these features make Apoptin a potential candidate as a therapeutic and diagnostic tool in cancer treatment.

BCL-2 stimulates Apoptin-induced apoptosis.

Apoptin, a protein encoded by an avian virus, induces apoptosis in various cultured human tumorigenic and/or transformed cell lines, e.g. in leukemia, lymphoma or EBV-transformed B cells. In such cells, Apoptin induces p53-independent apoptosis, and the proto-oncogene Bcl-2 accelerates this effect. The latter is surprising for, in general, Bcl-2 is known to inhibit e.g., p53-induced apoptosis. On the other hand, in normal non-transformed human cells, Apoptin is unable to induce apoptosis, even when Bcl-2 is over-expressed. In normal cells, Apoptin is found predominantly in the cytoplasm, whereas in tumor cells it is located in the nucleus. Cellular-localization studies showed that Apoptin is not located in mitochondria, indicating once more that Bcl-2 does not interfere with Apoptin in normal cells. In animal models Apoptin appears to be a safe and efficient anti-tumor agent. These data, in continuation with the observations that Apoptin is specifically stimulated by Bcl-2 in tumor cells, does not need p53, and is not inhibited by BCR-ABL in these cells, imply that Apoptin holds the promise of being the basis for anti-tumor therapy.

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens.

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin.

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.