MYC is a metastasis gene for non-small-cell lung cancer.

Metastasis is a process by which cancer cells learn to form satellite tumors in distant organs and represents the principle cause of death of patients with solid tumors. NSCLC is the most lethal human cancer due to its high rate of metastasis. Lack of a suitable animal model has so far hampered analysis of metastatic progression. We have examined c-MYC for its ability to induce metastasis in a C-RAF-driven mouse model for non-small-cell lung cancer. c-MYC alone induced frank tumor growth only after long latency at which time secondary mutations in K-Ras or LKB1 were detected reminiscent of human NSCLC. Combination with C-RAF led to immediate acceleration of tumor growth, conversion to papillary epithelial cells and angiogenic switch induction. Moreover, addition of c-MYC was sufficient to induce macrometastasis in liver and lymph nodes with short latency associated with lineage switch events. Thus we have generated the first conditional model for metastasis of NSCLC and identified a gene, c-MYC that is able to orchestrate all steps of this process. Potential markers for detection of metastasis were identified and validated for diagnosis of human biopsies. These markers may represent targets for future therapeutic intervention as they include genes such as Gata4 that are exclusively expressed during lung development

Oxidative and nitrative alpha-synuclein modifications and proteostatic stress: implications for disease mechanisms and interventions in synucleinopathies

Alpha-synuclein (ASYN) is a major constituent of the typical protein aggregates observed in several neurodegenerative diseases that are collectively referred to as synucleinopathies. A causal involvement of ASYN in the initiation and progression of neurological diseases is suggested by observations indicating that single-point (e.g., A30P, A53T) or multiplication mutations of the gene encoding for ASYN cause early onset forms of Parkinson's disease (PD). The relative regional specificity of ASYN pathology is still a riddle that cannot be simply explained by its expression pattern. Also, transgenic over-expression of ASYN in mice does not recapitulate the typical dopaminergic neuronal death observed in PD. Thus, additional factors must contribute to ASYN-related toxicity. For instance, synucleinopathies are usually associated with inflammation and elevated levels of oxidative stress in affected brain areas. In turn, these conditions favor oxidative modifications of ASYN. Among these modifications, nitration of tyrosine residues, formation of covalent ASYN dimers, as well as methionine sulfoxidations are prominent examples that are observed in post-mortem PD brain sections. Oxidative modifications can affect ASYN aggregation, as well as its binding to biological membranes. This would affect neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), ASYN's degradation within a cell and, possibly, the transfer of modified ASYN to adjacent cells. Here, we propose a model on how covalent modifications of ASYN link energy stress, altered proteostasis, and oxidative stress, three major pathogenic processes involved in PD progression. Moreover, we hypothesize that ASYN may act physiologically as a catalytically regenerated scavenger of oxidants in healthy cells, thus performing an important protective role prior to the onset of disease or during aging

Retroviral Infection of Syrian Hamster BHK Cells Depends on Age and Susceptibility toward Sialidase

AbstractInfection with recombinant retroviruses is a standard method to efficiently manipulate murine or human cells genetically. Due to differences in glycosylation of the hamster homologue to the murine ecotropic receptor and the absence of an amphotropic receptor, murine retroviruses are unable to infect hamster cells. Here we describe an isolated cell clone of the Baby Hamster Kidney cell line, BHK-21, that can be successfully infected with ecotropic murine retroviruses. This finding prompted us to investigate the possibility of changing the infectability of other cell strains by manipulating the receptors of these cell linesin vitro.We found that treatment of other BHK cell clones with sialidase fromC. Perfringensmakes these cell clones also permissive to murine ecotropic retroviruses. Long term cultivation of three tested cell clones increased this susceptibility. Although the resulting infectants show a higher rate of infectability, they still require the manipulation with sialidase for this superinfection to be efficient

SUIKER : Quantification of antigens in cell organelles, neurites and cellular sub-structures by imaging

Quantification of fluorescence colocalization and intensity of strongly overlapping cells, e.g., neuronal cultures, is challenging for programs that use image segmentation to identify cells as individual objects. Moreover, learning to use and apply one of the large imaging packages can be very time- and/or resource-demanding. Therefore, we developed the free and highly interactive image analysis program SUIKER (program for SUperImposing KEy Regions) that quantifies colocalization of different proteins or other features over an entire image field. The software allows definition of cellular subareas by subtraction ("punching out") of structures identified in one channel from structures in a second channel. This allows, e.g., definition of neurites without cell bodies. Moreover, normalization to live or total cell numbers is possible. Providing a detailed manual that contains image analysis examples, we demonstrate how the program uses a combination of colocalization information and fluorescence intensity to quantify carbohydrate-specific stains on neurites. SUIKER can import any multichannel histology or cell culture image, builds on user-guided threshold setting, batch processes large image stacks, and exports all data (including the settings, results and metadata) in flexible formats to be used in Excel.publishe

Food for thought ... considerations and guidelines for basic test method descriptions in toxicology

The development and application of new test systems and test methods is central to the progress of in vitro toxicology. In order to live up to the future challenges, it is important to use the vast knowledge of adjoining fields, such as cell biology or developmental biology, and to attract specialists from such areas to develop new methods. Not all of them bring with them the necessary experience and training required for the development of toxicological test systems. Thus, promising new biological test systems sometimes still require additional considerations to become successful. Sometimes even the referees of scientific journals and their editors seem to lack judgement guidelines for minimum performance standards. Here we provide a list of points to be considered for the establishment of a test method. The chapters range from the explanation of the meaning of positive controls, performance standards or signal-noise ratios to a discussion of statistical considerations, suitable solvents and data display formats. The considerations are simple and expressed in a non-mathematical format, with a strong focus on plausibility and common sense. The major intention of this article is to provide a compilation of important issues requiring consideration. Whether they apply to a specific system and whether action is required must be determined by individual judgement

Uncoupling of ATP-depletion and cell death in human dopaminergic neurons

The mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP(+)) is the toxicologically relevant metabolite of 1-methyl-4-phenyltetrahydropyridine (MPTP), which causes relatively selective degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic LUHMES cells were used to investigate whether ATP-depletion can be uncoupled from cell death as a downstream event in these fully post-mitotic human neurons. Biochemical assays indicated that in the homogeneously differentiated cell cultures, MPP(+) was taken up by the dopamine transporter (DAT). MPP(+) then triggered oxidative stress and caspase activation, as well as ATP-depletion followed by cell death. Enhanced survival of the neurons in the presence of agents interfering with mitochondrial pathology, such as the fission inhibitor Mdivi-1 or a Bax channel blocker suggested a pivotal role of mitochondria in this model. However, these compounds did not prevent cellular ATP-depletion. To further investigate whether cells could be rescued despite respiratory chain inhibition by MPP(+), we have chosen a diverse set of pharmacological inhibitors well-known to interfere with MPP(+) toxicity. The antioxidant ascorbate, the iron chelator desferoxamine, the stress kinase inhibitor CEP1347, and different caspase inhibitors reduced cell death, but allowed ATP-depletion in protected cells. None of these compounds interfered with MPP(+) accumulation in the cells. These findings suggest that ATP-depletion, as the initial mitochondrial effect of MPP(+), requires further downstream processes to result in neuronal death. These processes may form self-enhancing signaling loops, that aggravate an initial energetic impairment and eventually determine cell fate

CaFFEE : A program for evaluating time courses of Ca2+ dependent signal changes of complex cells loaded with fluorescent indicator dyes

Quantification of changes in intracellular free Ca2+ concentrations [Ca2+]i is fundamental to the understanding of the physiology of single cells in response to both environmental and endogenous stimuli. Here we present easy to use freeware that allows especially the evaluation of [Ca2+]i signals in complex and mixed cultures. The program CaFFEE (Calcium Fluorescent Flash Evaluating Engine) enables the user to evaluate the response of hundreds of cells to treat-ments that influence [Ca2+]i. CaFFEE processes large quantities of image data, automatically identifies individual cells in mixed, heterogeneous populations, and evaluates their fluorescence signal. All data are exported in spreadsheet format, and data on thousands of cells can be batch-processed. Moreover, the program optimizes the visual representation of time-lapse image data for user-guided data exploration (setting of parameters for semi-automated data processing). The freeware allows the standardized and transparent processing of imaging data independent of the platform used to generate the data.publishe

Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson’s disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant β-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog β-synuclein (βS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, βS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, βS, and Δ3 might therefore play a crucial role in neurodegenerative disease.publishe

Profiling of Human Neural Crest Chemoattractant Activity as a Replacement of Fetal Bovine Serum for In Vitro Chemotaxis Assays

Fetal bovine serum (FBS) is the only known stimulus for the migration of human neural crest cells (NCCs). Non-animal chemoattractants are desirable for the optimization of chemotaxis as-says to be incorporated in a test battery for reproductive and developmental toxicity. We con-firmed here in an optimized transwell assay that FBS triggers directed migration along a con-centration gradient. The responsible factor was found to be a protein in the 30-100 kDa size range. In a targeted approach, we tested a large panel of serum constituents known to be chem-otactic for NCCs in animal models (e.g., VEGF, PDGF, FGF, SDF-1/CXCL12, ephrins, endothelin, Wnt, BMPs). None of the corresponding human proteins showed any effect in our chemotaxis assays based on human NCCs. We then examined, whether human cells would produce any fac-tor able to trigger NCC migration in a broad screening approach. We found that HepG2 hepa-toma cells produced chemotaxis-triggering activity (CTA). Using chromatographic methods and by employing the NCC chemotaxis test as bioassay, the responsible protein was enriched by up to 5000-fold. We also explored human serum and platelets as a direct source, independent of any cell culture manipulations. A CTA was enriched from platelet lysates several thousand-fold. Its temperature and protease sensitivity suggested also a protein component. The capacity of this factor to trigger chemotaxis was confirmed by single-cell video-tracking analysis of migrating NCCs. The human CTA characterized here may be employed in the future for the setup of assays testing for the disturbance of directed NCC migration by toxicants.publishe

Specific Attenuation of Purinergic Signaling during Bortezomib-Induced Peripheral Neuropathy In Vitro

Human peripheral neuropathies are poorly understood, and the availability of experimental models limits further research. The PeriTox test uses immature dorsal root ganglia (DRG)-like neurons, derived from induced pluripotent stem cells (iPSC), to assess cell death and neurite damage. Here, we explored the suitability of matured peripheral neuron cultures for the detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A two-step differentiation protocol, involving the transient expression of ectopic neurogenin-1 (NGN1) allowed for the generation of homogeneous cultures of sensory neurons. After >38 days of differentiation, they showed a robust response (Ca2+-signaling) to the P2X3 ligand α,β-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at ≥5 nM, while 50–200 nM was required in the PeriTox test to identify neurite damage and cell death. A 24 h treatment with low nM concentrations of bortezomib led to moderate increases in resting cell intracellular Ca2+ concentration but signaling through transient receptor potential V1 (TRPV1) receptors or depolarization-triggered Ca2+ influx remained unaffected. We interpreted the specific attenuation of purinergic signaling as a functional cell stress response. A reorganization of tubulin to form dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomicin, and MG-132 showed similar stress responses. Thus, the model presented here may be used for the profiling of new proteasome inhibitors in regard to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signaling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons