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.

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing.

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A-B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A-B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.

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.

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.

The viral death effector Apoptin reveals tumor-specific processes.

Several natural proteins, including the cellular protein TRAIL and the viral proteins E4orf4 and Apoptin, have been found to exert a tumor-preferential apoptotic activity. These molecules are potential anti-cancer agents with direct clinical applications. Also very intriguing is their possible utility as sensors of the tumorigenic phenotype. Here, we focus on Apoptin, discussing recent research that has greatly increased our understanding of its tumor-specific processes. Apoptin, which kills tumor cells in a p53- and Bcl-2-independent, caspase-dependent manner, is biologically active as a highly stable, multimeric complex consisting of 30 to 40 monomers that form distinct superstructures upon binding cooperatively to DNA. In tumor cells, Apoptin is imported into the nucleus prior to the induction of apoptosis; this contrasts with the situation in primary or low-passage normal cell cultures where nuclear translocation of Apoptin is rare and inefficient. Apoptin contains two autonomous death-inducing domains, both of which exhibit a strong correlation between nuclear localization and killing activity. Nevertheless, forced nuclear localization of Apoptin in normal cells is insufficient to allow induction of apoptosis, indicating that another activation step particular to the tumor or transformed state is required. Indeed, a kinase activity present in cancer cells but negligible in normal cells was recently found to regulate the activity of Apoptin by phosphorylation. However, in normal cells, Apoptin can be activated by transient transforming signals conferred by ectopically expressed SV40 LT antigen, which rapidly induces Apoptin's phosphorylation, nuclear accumulation and the ability to induce apoptosis. The region on LT responsible for conferring this effect has been mapped to the N-terminal J domain. In normal cells that do not receive such signals, Apoptin becomes aggregated, epitope-shielded and is eventually degraded in the cytoplasm. Finally, Apoptin interacts with various partners of the human proteome including DEDAF, Nmi and Hippi, which may help to regulate either Apoptin's activation or execution processes. Taken together, these recent advances illustrate that elucidating the mechanism of Apoptin-induced apoptosis can lead to the discovery of novel tumor-specific pathways that may be exploitable as anti-cancer drug targets.

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'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.

Stable transplantation results of magnetically retracted islets: a novel method.

AIMS/HYPOTHESIS: Large quantities of pure viable donor islets are necessary for clinical transplantation. At present, low yields and low viability of pancreatic islets after transplantation necessitate the use of multiple donors for a single recipient. In this study an improved method for obtaining large quantities of pure viable islets of Langerhans for transplantation was developed in the rat. METHODS: Islets of Langerhans were isolated from Albino Oxford rats. The donor pancreata were perfused in situ with iron oxide, which resulted in entrapment of iron particles in the capillaries of the islets. Subsequently, the islets were isolated by magnetic retraction. Islets obtained with this method were compared with islets obtained by density gradient-isolated islets with respect to yields, purity, and insulin production capacity. Islets isolated with the magnetic retraction method were transplanted under the renal capsule of streptozotocin-induced diabetic recipients. Blood-glucose levels in the recipients were monitored for 2 months after transplantation. RESULTS: This method yielded more pure and viable islets than the conventional protocol. No contamination of exocrine tissue was observed after isolation. Furthermore, the islets isolated by magnetic retraction stained strongly positive for insulin during the entire observation period in vitro, and produced high amounts of insulin upon a challenge with glucose. The islets that were obtained by this new protocol were suitable for safe and effective transplantation. CONCLUSIONS/INTERPRETATION: We have shown that both the quantity and quality of islets obtained with this method were sufficient to induce insulin independence in a diabetic recipient using islets from only one donor.

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.

The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells.

The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome c is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.

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.

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene.

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843-5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vectors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.

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.

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.