Worldwide, cervix carcinoma is among the most dangerous cancer types, and novel therapies are under development. Cancer treatments are often hampered because of lack of specificity. The chicken anemia virus-derived apoptin induces apoptosis selectively in tumor cells and leaves normal cells unharmed. Here, we have carried out in-vitro and in-vivo studies on the cytotoxic effect of apoptin in a cervix carcinoma model. Apoptin was fused to the protein transduction domain 4 (PTD4), enabling delivery of the fusion protein across cellular membranes. PTD4-apoptin protein is located in the nuclei of human cervical carcinoma HeLa cells and in the cytoplasm of normal cells L02. By MTT and flow cytometry analysis, we have proven that PTD4-apoptin protein induced apoptosis in the cervical carcinoma cells. PTD4-apoptin enhanced the level of active executioner caspase-3. Neither caspase-3 activation nor apoptin-induced accumulation of the mitochondrial outer-membrane protein Mfn-2 was affected by ectopic Bcl-2 expression. In contrast, apoptin-mediated AKT activation was inhibited by Bcl-2. In vivo, cervix carcinoma xenografts were treated for 7 days with PTD4-apoptin protein. The PTD4-apoptin treatment induced a decrease in the cervix carcinoma, whereas the PTD4-GFP protein-treated controls expanded significantly. TUNEL analysis showed that PTD4-apoptin protein induced apoptosis in cervix carcinoma cells, in contrast to the control PTD-GFP-treated ones. Our results indicate that apoptin is a potential anticancer agent for treating cervix carcinoma.

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent. Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

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.

Apoptin, a protein derived from chicken anemia virus (CAV), induces apoptosis selectively in human tumor cells as compared with normal cells. This activity depends on phosphorylation and relocation of apoptin to the nucleus of cancer cells. Here, we describe an in vitro kinase assay that allows the biochemical characterization of apoptin kinase activity in tumor cells. The kinase phosphorylates apoptin in a strictly ATP-dependent fashion and in a broad salt range. The kinase activity is present constitutively in both cytoplasm and nucleus of various human tumor cells. Q-column chromatography showed that both cytoplasmic and nuclear fractions have identical fractionation characteristics, suggesting that the same kinase is present in both cellular compartments. Kinase activity derived from positive Q-column fractions bound to amylose–maltose-binding protein (MBP)–apoptin and could be eluted with ATP only in the presence of the cofactor Mg2+. Apparently, unphosphorylated apoptin interacts with the kinase and is released only after phosphorylation has occurred, proving that our assay recognizes the genuine apoptin kinase. This is further corroborated by the finding that apoptin is phosphorylated in vitro at positions Thr108 and Thr107, in concert with earlier in vivo observations. Our assay excludes cyclin-dependent kinase 2 (CDK2) and protein kinase C beta (PKC-β), previously nominated by two separate studies as being the genuine apoptin kinase.

The small viral protein apoptin is capable of inducing apoptosis selectively in human tumor cells. In normal cells apoptin localizes in the cytoplasm where it forms aggregates, becomes epitope-shielded and eventually degraded. By inhibiting the proteasome activity with the chemical inhibitors bortezomib and Ada-Ahx3L3VS apoptin levels can be stabilized in normal cells similar to the tumor suppressor p53 protein. In contrast, proteasome inhibition in tumor cells did not affect the apoptin stability while it still stabilized p53 levels. Apparently, apoptin is degraded by proteasomal activity in normal human cells, a process that no longer takes place in tumor cells. This loss of proteasomal susceptibility appears to be specific for apoptin.

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.

−1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in −1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

Recent studies have revealed a new class of genes encoding proteins with specific anticancer activity. Upon ectopic expression, these factors cause cell death specifically in tumour cells by apoptosis, autophagy or mitotic catastrophe, yet normal cells are spared. Some of these genes or their encoded proteins are in clinical development and show promising results, and their signalling pathways are currently under intense investigation. Defining these genes as anticancer genes, we review what is known about their functions, the specific cell death signals they induce and the status of cancer therapy approaches that emulate their function. Systematic screening for such anticancer genes might lead to the identification of a repertoire of signalling pathways directed against cellular alterations that are specific for tumour cells.

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12-18 nt. Antisense oligonucleotides bearing locked nucleic acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.

PTD4-apoptin protein enters cells and harbors tumor-selective cell death activity. Dacarbazine is the mainstay of treatment for malignant melanoma. In this study, we investigated the cytotoxic effect of PTD4-apoptin protein and/or dacarbazine in mouse B16-F1 and human A875 and SK-MEL-5 melanoma cells in vitro and by means of a mouse B16-F1 melanoma model in vivo. PTD4-apoptin protein inhibits the growth of B16-F1, A875 and SK-MEL-5 melanoma cells in a dose-dependent manner, but not in normal human cell lines WI-38 and L-02. PTD4-apoptin combined with dacarbazine revealed a synergistic cytotoxic effect (coefficient of drug interaction<1) in all three different tumor cell lines. In vivo, PTD4-apoptin protein and dacarbazine alone effectively inhibited the growth of B16-F1 melanoma in C57BL/6 mice. Strikingly, combined PTD4-apoptin/dacarbazine treatment significantly increased the antitumor effect in comparison to the single treatments. As important, a combined PTD4-apoptin/dacarbazine treatment with a 50% reduction of dacarbazine revealed similar antitumor activities, without detectable hematologic side effects. A combined PTD4-apoptin/dacarbazine treatment represents a promising novel efficient and safe anticancer strategy.

Treatment of head and neck cancers is still rather poor and worldwide new treatment options are sought. Sensitizing radioresistant tumours by combining irradiation with other therapeutics to induce apoptosis are widely investigated. We examined whether chicken anaemia virus-derived apoptin protein would have a beneficial effect on irradiation of radiosensitive SCC61 and radioresistant SQD9 human head and neck squamous carcinoma cell lines. In both cell lines, concurrent exposure to irradiation and apoptin resulted in analysed mitochondrial cytochrome c release and in cleavage of caspase-3, whereas irradiation alone of SQD9 cells under identical conditions did not. Moreover, in comparison with the irradiation, only the synchronized treatment of apoptin and irradiation resulted in increased cell death in especially the radioresistant SQD9 cells, as measured by means of a colony survival assay. Our data reveal that apoptin treatment represents an effective way for enhancing radiotherapy of tumours responding poorly to radiotherapy.

Despite recent advances, the prognosis of oral squamous cell carcinoma is still poor. Therapeutic options such as radiotherapy, chemotherapy, surgery and the novel treatment option gene therapy are being investigated in animal models. Diverse models have been studied to induce oral squamous cell carcinomas. The carcinogenic 4-nitroquinoline-1-oxide (4NQO) model has proven to be successful although until now it is unknown at what time point the established tumor is a representative squamous cell carcinoma and has a suitable volume for scientific treatment. For this end we applied 4NQO 3 times a week during 16 weeks and measured the volume of tumor tissue each week until the end of the experiment at 40 weeks. Concurrent histopathology at different time points up to the end of the experiment revealed that all mice bearing oral tumors were diagnosed with squamous cell carcinoma. Immunohistochemistry with markers cyclin D1 and E-cadherin revealed that the generated mouse oral tumors showed strong similarities with the described immunopathology in human oral tumors. The 4NQO model is a suitable alternative for preclinical gene therapy experiments with primary oral tumors. Future survey of therapeutic options in the carcinogenic 4NQO model should be conducted around 40 weeks after the start of the treatment.

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.

All human cells have a genetic program that upon activation will cause cell death, named apoptosis. Cancer cells can grow due to unbalances in proliferation, cell cycle regulation and their apoptosis machinery: genomic mutations resulting in non-functional pro-apoptosis proteins or over-expression of anti-apoptosis proteins form the basis of tumor formation. Surprisingly, lessons learned from viruses show that cancer cannot be regarded simply as the opposite of apoptosis. For instance, adenovirus can only transform cells when both its anti- and pro-apoptotic proteins are produced. Oncolytic viruses are known to replicate selectively in tumor cells resulting in cell death. Proteins derived from viruses, i.e. chicken anemia virus (CAV)-derived apoptosis-inducing protein (apoptin), adenovirus early region 4 open reading frame (E4orf4) and parvovirus-H1 derived non-structural protein 1 (NS1), the human α-lactalbumin made lethal to tumor cells (HAMLET), which is present in human milk or the human cytokines melanoma differentiation-associated gene-7 (mda-7) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have all the ability to induce tumor-selective apoptosis. The tumor-selective apoptosis-inducing proteins seem to interact with transforming survival processes, which can become redirected by these proteins into cell death. Transformation-related processes have been identified, which seem to be crucial for the tumor-selectively killing activity of these proteins. For instance, the transformation-related protein phosphatase 2A (PP2A) plays a role in the induction of tumor-selective apoptosis. The proteins mda-7, TRAIL and HAMLET are already successfully tested in first clinical trials. Proteins harboring tumor-selective apoptosis characteristics represent, therefore, a therapeutic potential and a tool for unraveling tumor-related processes. Fundamental molecular and (pre)clinical therapeutic studies of the various tumor-selective apoptosis-inducing proteins apoptin, E4orf4, HAMLET, mda-7, NS1, TRAIL and related proteins will be discussed.

Apoptin protein harbors tumor‐selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)‐mediated transduction of recombinant apoptin protein. In vitro , the PTD4‐apoptin fusion protein is located in the nucleus and induces cell death in, e.g ., human hepatocarcinoma HepG2 cells. In normal human L‐02 hepatocytes, PTD4‐apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor‐selective characteristics. In vivo , liver, cervix and gastric carcinoma xenografts treated with PTD4‐apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin‐mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4‐apoptin protein has significant anticancer activity, whereas control treatment with PTD4‐enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4‐apoptin transduced xenografted tumors. As important, although PTD4‐apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy. © 2009 UICC