High-resolution and super-resolution techniques become more frequently used in thick, inhomogeneous samples. In particular for imaging life cells and tissue in which one wishes to observe a biological process at minimal interference and in the natural environment, sample inhomogeneities are unavoidable. Yet sample-inhomogeneities are paralleled by refractive index variations, for example between the cell organelles and the surrounding medium, that will result in the refraction of light, and therefore lead to sample-induced astigmatism. Astigmatism in turn will result in positional inaccuracies of observations that are at the heart of all super-resolution techniques. Here we introduce a simple model and define a figure-of-merit that allows one to quickly assess the importance of astigmatism for a given experimental setting. We found that astigmatism caused by the cell’s nucleus can easily lead to aberrations up to hundreds of nanometers, well beyond the accuracy of all super-resolution techniques. The astigmatism generated by small objects, like bacteria or vesicles, appear to be small enough to be of any significance in typical super-resolution experimentation.

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.

Cellular force generation is essential for tissue morphogenesis and cell proliferation. The balance of forces is in particular needed for successful chromosome condensation, separation and ultimately cell division. Although cellular force exertion has been studied extensively during interphase, its characterization during mitosis is still lacking. Recently, a fascinating study already showed the importance of the mechanical outside-in coupling during mitosis [1]. Here, we directly measured the inside-out force exerted during mitosis with GFP-labeled H2B to follow the cell cycle and GFP-labeled tubulin to examine the role of microtubules. Using micropillars of different heights, we quantified force exertion of dividing HeLa cells. Before prophase, we observed cell polarization where in the direction of polarization pulling forces were exerted. Polarization corresponded to the direction of division, as was expected from stretch-induced directionality of division [1]. Surprisingly, we consistently found radial outward forces during the subsequent phases in mitosis. This pushing force increased greatly in prophase, slowed in increase in metaphase and reached its peak at the start of anaphase. These displacement-controlled forces increase 5-fold over these phases. During anaphase and telophase, the forces decreased rapidly until the two daughter cells spread into their normal interphase conformation. Experiments with GFP-labeled tubulin demonstrated that pushing forces were present where microtubules were present, emanating from the centrosomes. From these observations, we developed a model for the mechanical balancing act through which pushing forces mediated by microtubules ensure proper cell division. Reference 1. Fink et al. (2011), Nature Cell Biology.

The outermost layer of our skin functions as a barrier to protect us from physical, chemical, and biological environmental insults. This protective function is mediated by the epidermal cornified cell envelope (CE) which serves both as a mechanical and permeability barrier. Recently we have discovered that the CE constitutes also a first-line antioxidant shield which relies greatly on cysteine residues in CE precursor proteins. Here we describe methods and protocols to study the cysteine-mediated antioxidant function of the CE at the level of the whole organ (the skin), individual cells (keratinocytes), or isolated proteins (SPRR family).

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.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Folic acid-modified mesoporous silica nanoparticles are designed for cellular and nuclear targeted drug delivery. Camptothecin is efficiently delivered to human cancer cells resulting in apoptosis without premature leakage. The folic acid moiety is an integral part of a rotaxane structure, which locks the drug molecules in the pores, preventing premature release. Upon folate receptor-mediated cell uptake, esterases cleave the rotaxane valves and camptothecin is released, resulting in apoptosis. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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.

Protection against injurious external insults and loss of vital fluids is essential for life and is in all organisms, from bacteria to plants and humans, provided by some form of barrier. Members of the small proline-rich (SPRR) protein family are major components of the cornified cell envelope (CE), a structure responsible for the barrier properties of our skin. These proteins are efficient reactive oxygen species (ROS) quenchers involved not only in the establishment of the skin's barrier function but also in cell migration and wound healing. Here, a proteomic analysis of in vivo SPRR-interacting proteins confirmed their function in CE-formation and ROS-quenching and also revealed a novel unexpected role in DNA-binding. Direct in vitro and in vivo evidence proved that the DNA-binding capacity of SPRRs is regulated by the oxidation state of the proteins. At low ROS levels, nuclear SPRR is able to bind DNA and prevent ROS-induced DNA damage. When ROS levels increase, SPRR proteins multimerize and form an effective antioxidant barrier at the cell periphery, possibly to prevent the production or infiltration of ROS. At even higher ROS exposure, DNA-binding is restituted. A molecular model explaining how the intracellular oxidation state of SPRRs likely influences their selective protective function is provided.

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.

The cornified cell envelope (CE) is a specialized structure assembled beneath the plasma membrane of keratinocytes in the outermost layers of the epidermis. It is essential for the physical and permeability properties of the barrier function of the skin. Our skin is continuously exposed to atmospheric oxygen and threatened by reactive oxygen species (ROS). Here, we identify the CE as a first line of antioxidant defense and show that the small proline-rich (SPRR) family of CE precursor proteins have a major role in ROS detoxification. Cysteine residues within these proteins are responsible for ROS quenching, resulting in inter- and intramolecular S-S bond formation, both in isolated proteins and purified CEs. The related keratinocyte proline-rich protein is also oxidized on several cysteine residues within the CE. Differences in antioxidant potential between various SPRR family members are likely determined by structural differences rather than by the amount of cysteine residues per protein. Loricrin, a major component of the CE with a higher cysteine content than SPRRs, is a weak ROS quencher and oxidized on a single cysteine residue within the CE. It is inferred that SPRR proteins provide the outermost layer of our skin with a highly adaptive and protective antioxidant shield.

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I:C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1β, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocyte's inflammatory response, and contributes to the understanding of virus persistence.

Abstract Background The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. Results Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. Conclusions This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

The uppermost layer of our skin, the epidermis, is formed largely of keratinocytes which constitute the skin's major barrier function and the first line of defence against environmental physical, chemical and biological agents. The subsequent layer, the dermis, which is mainly formed by fibroblasts, has a more supportive function, containing large amounts of collagen, blood vessels and nerve endings and is less directly affected by external insults. Hence it is likely that keratinocytes and fibroblasts have evolved different strategies to cope with the dangers of the environment. Mouse models with various genetic backgrounds in genome care-taking systems, such as DNA repair processes, are well suited to study differences between these two cell types and their implications for cancer and aging. In this chapter we describe a simple procedure to establish long-term keratinocyte and fibroblast cultures from, respectively, the epidermis and dermis of normal or NER-deficient newborn mice. The importance of the external O(2) pressure during the establishment and maintenance of these matched cultures is discussed.

Wound healing is a complex dynamic process characterised by a uniform flow of events in nearly all types of tissue damage, from a small skin scratch to myocardial infarction. Reactive oxygen species (ROS) are essential during the healing process at multiple stages, ranging from the initial signal that instigates the immune response, to the triggering of intracellular redox-dependent signalling pathways and the defence against invading bacteria. Excessive ROS in the wound milieu nevertheless impedes new tissue formation. Here we identify small proline-rich (SPRR) proteins as essential players in this latter process, as they directly link ROS detoxification with cell migration. A literature-based meta-analysis revealed their up-regulation in various forms of tissue injury, ranging from heart infarction and commensal-induced gut responses to nerve regeneration and burn injury. Apparently, SPRR proteins have a far more widespread role in wound healing and tissue remodelling than their established function in skin cornification. It is inferred that SPRR proteins provide injured tissue with an efficient, finely tuneable antioxidant barrier specifically adapted to the tissue involved and the damage inflicted. Their recognition as novel cell protective proteins combining ROS detoxification with cell migration will provide new venues to study and manage tissue repair and wound healing at a molecular level.

Recently, a select number of viral and cellular proteins sharing a unique property have been identified, namely apoptin, HAMLET, TRAIL, MDA7, E4ORF4, NS1 and Brevinin-2R, where all have the remarkable ability to selectively kill transformed and/or tumour cells, while causing no or only minor cytotoxicity in normal and non-transformed cells.Research worldwide is now starting to unravel the molecular signalling pathways by which each of these proteins exerts its tumour-selective function, and the first (pre)clinical studies are very promising.The workshop 'Proteins Killing Tumour Cells' brought together, for the first time, researchers working on this novel class of intriguing proteins.Participants from 14 different countries shared their views on (possible common) mechanisms behind tumour-specific apoptosis induction and strategies to implement this knowledge in the clinic.

The avian virus-derived protein apoptin induces p53-independent apoptosis in a tumor-specific way. Apoptin acts as a multimeric complex and forms superstructures upon binding to DNA. In tumor cells, apoptin is phosphorylated and mainly nuclear, whereas in normal cells it is unphosphorylated, cytoplasmic, and becomes readily neutralized. Interestingly, apoptin phosphorylation, nuclear translocation, and apoptosis can transiently be induced in normal cells by cotransfecting SV40 large T oncogene, indicating that apoptin recognizes early stages of oncogenic transformation. In cancer cells, apoptin appears to recognize survival signals, which it is able to redirect into cell death impulses. Apoptin targets include DEDAF, Nur77, Nmi, Hippi, and the potential drug target APC1. Apoptin-transgenic mice and animal tumor models have revealed apoptin as a safe and efficient antitumor agent, resulting in significant tumor regression. Future antitumor therapies could use apoptin either as a therapeutic bullet or as an early sensor of druggable tumor-specific processes.