Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells.

Metabolism in cells adapts quickly to changes in nutrient availability and cellular differentiation status, including growth conditions in cell culture settings. The last decade saw a vast increase in three-dimensional (3D) cell culture techniques, engendering spheroids and organoids. These methods were established to improve comparability to in vivo situations, differentiation processes and growth modalities. How far spheroids mimic in vivo metabolism, however, remains enigmatic. Here, to our knowledge, we compare for the first time metabolic fingerprints between cells grown as a single layer or as spheroids with freshly isolated in situ tissue. While conventionally grown cells express elevated levels of glycolysis intermediates, amino acids and lipids, these levels were significantly lower in spheroids and freshly isolated primary tissues. Furthermore, spheroids differentiate and start to produce metabolites typical for their tissue of origin. 3D grown cells bear many metabolic similarities to the original tissue, recommending animal testing to be replaced by 3D culture techniques.

Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

TNFα sensitizes hepatocytes to FasL-induced apoptosis by NFκB-mediated Fas upregulation.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.

Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells.

Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-xL. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.

How do viruses control mitochondria-mediated apoptosis?

There is no doubt that viruses require cells to successfully reproduce and effectively infect the next host. The question is what is the fate of the infected cells? All eukaryotic cells can "sense" viral infections and exhibit defence strategies to oppose viral replication and spread. This often leads to the elimination of the infected cells by programmed cell death or apoptosis. This "sacrifice" of infected cells represents the most primordial response of multicellular organisms to viruses. Subverting host cell apoptosis, at least for some time, is therefore a crucial strategy of viruses to ensure their replication, the production of essential viral proteins, virus assembly and the spreading to new hosts. For that reason many viruses harbor apoptosis inhibitory genes, which once inside infected cells are expressed to circumvent apoptosis induction during the virus reproduction phase. On the other hand, viruses can take advantage of stimulating apoptosis to (i) facilitate shedding and hence dissemination, (ii) to prevent infected cells from presenting viral antigens to the immune system or (iii) to kill non-infected bystander and immune cells which would limit viral propagation. Hence the decision whether an infected host cell undergoes apoptosis or not depends on virus type and pathogenicity, its capacity to oppose antiviral responses of the infected cells and/or to evade any attack from immune cells. Viral genomes have therefore been adapted throughout evolution to satisfy the need of a particular virus to induce or inhibit apoptosis during its life cycle. Here we review the different strategies used by viruses to interfere with the two major apoptosis as well as with the innate immune signaling pathways in mammalian cells. We will focus on the intrinsic mitochondrial pathway and discuss new ideas about how particular viruses could activately engage mitochondria to induce apoptosis of their host.

Design, Repeatability, and Comparison to Literature Data of a New Noninvasive Device Called "Rotameter" to Measure Rotational Knee Laxity.

The present paper deals with the design, the repeatability, and the comparison to literature data of a new measuring device called "Rotameter" to characterize the rotational knee laxity or the tibia-femoral rotation (TFR). The initial prototype P1 of the Rotameter is shortly introduced and then modified according to trials carried out on a prosthetic leg and on five healthy volunteers, leading therefore to an improved prototype P2. A comparison of results obtained from P1 and P2 with the same male subject shows the enhancements of P2. Intertester and intratester repeatability of this new device were shown and it was observed that rotational laxities of left and right knees are the same for a healthy subject. Moreover, a literature review showed that measurements with P2 presented lower TFR values than other noninvasive devices. The measured TFR versus torque characteristic was quite similar to other invasive devices, which are more difficult to use and harmful to the patient. Hence, our prototype P2 proved to be an easy-to-use and suitable device for quantifying rotational knee laxity. A forthcoming study will validate the Rotameter thanks to an approach based on computed tomography in order to evaluate its precision.

Use of a Computed Tomography Based Approach to Validate Noninvasive Devices to Measure Rotational Knee Laxity.

The purpose of this study is to validate a noninvasive rotational knee laxity measuring device called "Rotameter P2" with an approach based on Computed Tomography (CT). This CT-approach using X-rays is hence invasive and can be regarded as a precise reference method that may also be applied to similar devices. An error due to imperfect femur fixation was observed but can be neglected for small torques. The most significant estimation error is due to the unavoidable soft tissues rotation and hence flexibility in the measurement chain. The error increases with the applied torque. The assessment showed that the rotational knee angle measured with the Rotameter is still overestimated because of thigh and femur displacement, soft tissues deformation, and measurement artefacts adding up to a maximum of 285% error at +15 Nm for the Internal Rotation of female volunteers. This may be questioned if such noninvasive devices for measuring the Tibia-Femoral Rotation (TFR) can help diagnosing knee pathologies and investigate ligament reconstructive surgery.

Dominant negative effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 4 on TRAIL receptor 1 signaling by formation of heteromeric complexes.

The cytokine TNF-related apoptosis-inducing ligand (TRAIL) and its cell membrane receptors constitute an elaborate signaling system fulfilling important functions in immune regulation and tumor surveillance. Activation of the death receptors TRAILR1 and TRAILR2 can lead to apoptosis, whereas TRAILR3 and TRAILR4 are generally referred to as decoy receptors, which have been shown to inhibit TRAIL-induced apoptosis. The underlying molecular mechanisms, however, remain unclear. Alike other members of the TNF receptor superfamily, TRAIL receptors contain a pre-ligand binding assembly domain (PLAD) mediating receptor oligomerization. Still, the stoichiometry of TRAIL receptor oligomers as well as the issue of whether the PLAD mediates only homotypic or also heterotypic interactions remained inconclusive until now. Performing acceptor-photobleaching FRET studies with receptors 1, 2, and 4, we demonstrate interactions in all possible combinations. Formation of dimers was shown by chemical cross-linking experiments for interactions of TRAILR2 and heterophilic interactions between the two death receptors or between either of the death receptors and TRAILR4. Implications of the demonstrated receptor-receptor interactions on signaling were investigated in suitable cellular models. Both apoptosis induction and activation of the transcription factor NFκB were significantly reduced in the presence of TRAILR4. Our experimental data combined with mathematical modeling show that the inhibitory capacity of TRAILR4 is attributable to signaling-independent mechanisms, strongly suggesting a reduction of signaling competent death receptors through formation heteromeric receptor complexes. In summary, we propose a model of TRAIL receptor interference driven by PLAD-mediated formation of receptor heterodimers on the cell membrane.

A minimal mathematical model for the initial molecular interactions of death receptor signalling.

Tumor necrosis factor (TNF) is the name giving member of a large cytokine family mirrored by a respective cell membrane receptor super family. TNF itself is a strong proinflammatory regulator of the innate immune system, but has been also recognized as a major factor in progression of autoimmune diseases. A subgroup of the TNF ligand family, including TNF, signals via so-called death receptors, capable to induce a major form of programmed cell death, called apoptosis. Typical for most members of the whole family, death ligands form homotrimeric proteins, capable to bind up to three of their respective receptor molecules. But also unligated receptors occur on the cell surface as homomultimers due to a homophilic interaction domain. Based on these two interaction motivs (ligand/receptor and receptor/receptor) formation of large ligand/receptor clusters can be postulated which have been also observed experimentally. We use here a mass action kinetics approach to establish an ordinary differential equations model describing the dynamics of primary ligand/receptor complex formation as a basis for further clustering on the cell membrane. Based on available experimental data we develop our model in a way that not only ligand/receptor, but also homophilic receptor interaction is encompassed. The model allows formation of two distict primary ligand/receptor complexes in a ligand concentration dependent manner. At extremely high ligand concentrations the system is dominated by ligated receptor homodimers.

The transmembrane domains of TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 co-regulate apoptotic signaling capacity.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.

DNA damage in storage cells of anhydrobiotic tardigrades.

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 +/- 1.98% DNA in tail, compared to 0.44 +/- 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 +/- 6.41% of DNA was found in the comet tail. After ten months, 23.66 +/- 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.