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14-3-3 proteins are relevant to cancer biology as they are key regulators of major cellular processes such as proliferation, differentiation, senescence and apoptosis. So far, the sigma isoform (14-3-3 σ ) has most directly been implicated in carcinogenesis and was recognized as a tumour-suppressor gene. The other six members of the mammalian 14-3-3 gene family likely behave as oncogenes, although direct evidence supporting this view is largely circumstantial. In this report, we show that knockdown of 14-3-3ζ induces at least two isoform-specific phenotypes that are consistent with a potential oncogenic activity during tumorigenesis. Firstly, downregulation of 14-3-3ζ sensitized cells to stress-induced apoptosis and JNK/p38 signalling and secondly, it enforced cell–cell contacts and expression of adhesion proteins. Apparently, the zeta isoform restrains both cell adhesion and the cellular propensity for apoptosis, two activities that are also restrained during carcinogenesis. The assumption that 14-3-3ζ has oncogenic properties was substantiated with a web-based meta-analysis (Oncomine), revealing that 14-3-3ζ is overexpressed in various types of carcinomas. As the highly conserved human 14-3-3 gene family encodes proteins with either tumour-promoting or tumour-suppressing activities, we infer that the cellular balance between the various 14-3-3 isoforms is crucial for the proper functioning of cells.

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 (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–B56 γ 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–B56 δ 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 B56 γ and B56 δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death.

The chicken anemia virus protein apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Here, we show that, in vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial, and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. 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 predominantly in the cytoplasm, whereas in transformed and malignant cells it was located in the nucleus, suggesting that the localization of apoptin is related to its activity. These properties make apoptin a potential agent for the treatment of a large number of tumors, also those lacking p53 and/or overexpressing Bcl-2.

14-3-3 proteins are relevant to cancer biology as they are key regulators of major cellular processes such as proliferation, differentiation, senescence and apoptosis. So far, the sigma isoform (14-3-3[sigma]) has most directly been implicated in carcinogenesis and was recognized as a tumour-suppressor gene. The other six members of the mammalian 14-3-3 gene family likely behave as oncogenes, although direct evidence supporting this view is largely circumstantial. In this report, we show that knockdown of 14-3-3[zeta] induces at least two isoform-specific phenotypes that are consistent with a potential oncogenic activity during tumorigenesis. Firstly, downregulation of 14-3-3[zeta] sensitized cells to stress-induced apoptosis and JNK/p38 signalling and secondly, it enforced cell-cell contacts and expression of adhesion proteins. Apparently, the zeta isoform restrains both cell adhesion and the cellular propensity for apoptosis, two activities that are also restrained during carcinogenesis. The assumption that 14-3-3[zeta] has oncogenic properties was substantiated with a web-based meta-analysis (Oncomine), revealing that 14-3-3[zeta] is overexpressed in various types of carcinomas. As the highly conserved human 14-3-3 gene family encodes proteins with either tumour-promoting or tumour-suppressing activities, we infer that the cellular balance between the various 14-3-3 isoforms is crucial for the proper functioning of cells.

The critical concentrations pertaining to the liquid crystal formation of pUC18 plasmid in saline solutions were obtained from 31P nuclear magnetic resonance, polarized light microscopy, and phase equilibrium experiments. The transition is strongly first order with a broad gap between the isotropic and anisotropic phase. The critical boundaries are strongly and reversibly dependent on temperature and weakly dependent on ionic strength. With polarized light microscopy on magnetically oriented samples, the liquid crystalline phase is assigned cholesteric with a pitch on the order of 4 μm. Preliminary results show that at higher concentrations a true crystal is formed. The isotropic–cholesteric transition is interpreted with lyotropic liquid crystal theory including the effects of charge, orientation entropy, and excluded volume effects. It was found that the molecular free energy associated with the topology of the superhelix is of paramount importance in controlling the width of the phase gap. The theoretical results compare favorably with the critical boundary pertaining to the disappearance of the isotropic phase, but they fail to predict the low concentration at which the anisotropic phase first appears.

With a view to determine the configuration and regularity of plectonemically supercoiled DNA, we have measured the small angle neutron scattering from pUC18 plasmid in saline solutions. Furthermore, we have derived the mathematical expression for the single chain scattering function (form factor) of a superhelical structure, including the longitudinal and transverse interference over the plectonemic pitch and radius, respectively. It was found that an interwound configuration describes the data well, provided interactions among supercoils are accounted for in the second virial approximation. The opening angle was observed to be relatively constant and close to 58°, but it was necessary to include a significant distribution in radius and pitch. For diluted supercoils with vanishing mutual interaction, the derived structural results agree with independent measurements, including the distribution in linking number deficit as determined by gel electrophoresis. With increasing plasmid concentration, prior and covering the transition to the liquid-crystalline phase, the radius and pitch are seen to decrease significantly. The latter observation shows that compaction of negatively supercoiled DNA by confinement results in a decrease in writhing number at the cost of a positive twist exerted on the DNA duplex. It is our conjecture that the free energy associated with this excess twist is of paramount importance in controlling the critical boundaries pertaining to the transition to the anisotropic, liquid-crystalline phase.