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Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status
(2014)
Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 µF/cm2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7–4.0 µF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.
The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.
Biodiversity, a multidimensional property of natural systems, is difficult to quantify partly because of the multitude of indices proposed for this purpose. Indices aim to describe general properties of communities that allow us to compare different regions, taxa, and trophic levels. Therefore, they are of fundamental importance for environmental monitoring and conservation, although there is no consensus about which indices are more appropriate and informative. We tested several common diversity indices in a range of simple to complex statistical analyses in order to determine whether some were better suited for certain analyses than others. We used data collected around the focal plant Plantago lanceolata on 60 temperate grassland plots embedded in an agricultural landscape to explore relationships between the common diversity indices of species richness (S), Shannon's diversity (H'), Simpson's diversity (D-1), Simpson's dominance (D-2), Simpson's evenness (E), and Berger-Parker dominance (BP). We calculated each of these indices for herbaceous plants, arbuscular mycorrhizal fungi, aboveground arthropods, belowground insect larvae, and P.lanceolata molecular and chemical diversity. Including these trait-based measures of diversity allowed us to test whether or not they behaved similarly to the better studied species diversity. We used path analysis to determine whether compound indices detected more relationships between diversities of different organisms and traits than more basic indices. In the path models, more paths were significant when using H', even though all models except that with E were equally reliable. This demonstrates that while common diversity indices may appear interchangeable in simple analyses, when considering complex interactions, the choice of index can profoundly alter the interpretation of results. Data mining in order to identify the index producing the most significant results should be avoided, but simultaneously considering analyses using multiple indices can provide greater insight into the interactions in a system.
Background
Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs.
Methods
Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC).
Results
RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease.
Conclusions
A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.
Climatic extreme events can cause the shift or disruption of plant-insect interactions due to altered plant quality, e.g. leaf carbon to nitrogen ratios, and phenology. However, the response of plant-herbivore interactions to extreme events and climatic gradients has been rarely studied, although climatic extremes will increase in frequency and intensity in the future and insect herbivores represent a highly diverse and functionally important group. We set up a replicated climate change experiment along elevational gradients in the German Alps to study the responses of three plant guilds and their herbivory by insects to extreme events (extreme drought, advanced and delayed snowmelt) versus control plots under different climatic conditions on 15 grassland sites. Our results indicate that elevational shifts in CN (carbon to nitrogen) ratios and herbivory depend on plant guild and season. CN ratios increased with altitude for grasses, but decreased for legumes and other forbs. In contrast to our hypotheses, extreme climatic events did not significantly affect CN ratios and herbivory. Thus, our study indicates that nutritional quality of plants and antagonistic interactions with insect herbivores are robust against seasonal climatic extremes. Across the three functional plant guilds, herbivory increased with nitrogen concentrations. Further, increased CN ratios indicate a reduction in nutritional plant quality with advancing season. Although our results revealed no direct effects of extreme climatic events, the opposing responses of plant guilds along elevation imply that competitive interactions within plant communities might change under future climates, with unknown consequences for plant-herbivore interactions and plant community composition.
Background
VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) – the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1.
Results
In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme.
Conclusions
The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.
Background. Up to 75% of crop species benefit at least to some degree from animal pollination for fruit or seed set and yield. However, basic information on the level of pollinator dependence and pollinator contribution to yield is lacking for many crops. Even less is known about how insect pollination affects crop quality. Given that habitat loss and agricultural intensification are known to decrease pollinator richness and abundance, there is a need to assess the consequences for different components of crop production. Methods. We used pollination exclusion on flowers or inflorescences on a whole plant basis to assess the contribution of insect pollination to crop yield and quality in four flowering crops (spring oilseed rape, field bean, strawberry, and buckwheat) located in four regions of Europe. For each crop, we recorded abundance and species richness of flower visiting insects in ten fields located along a gradient from simple to heterogeneous landscapes. Results. Insect pollination enhanced average crop yield between 18 and 71% depending on the crop. Yield quality was also enhanced in most crops. For instance, oilseed rape had higher oil and lower chlorophyll contents when adequately pollinated, the proportion of empty seeds decreased in buckwheat, and strawberries' commercial grade improved; however, we did not find higher nitrogen content in open pollinated field beans. Complex landscapes had a higher overall species richness of wild pollinators across crops, but visitation rates were only higher in complex landscapes for some crops. On the contrary, the overall yield was consistently enhanced by higher visitation rates, but not by higher pollinator richness. Discussion. For the four crops in this study, there is clear benefit delivered by pollinators on yield quantity and/or quality, but it is not maximized under current agricultural intensification. Honeybees, the most abundant pollinator, might partially compensate the loss of wild pollinators in some areas, but our results suggest the need of landscape-scale actions to enhance wild pollinator populations.
In recent years three-dimensional (3D) super-resolution fluorescence imaging by single-molecule localization (localization microscopy) has gained considerable interest because of its simple implementation and high optical resolution. Astigmatic and biplane imaging are experimentally simple methods to engineer a 3D-specific point spread function (PSF), but existing evaluation methods have proven problematic in practical application. Here we introduce the use of cubic B-splines to model the relationship of axial position and PSF width in the above mentioned approaches and compare the performance with existing methods. We show that cubic B-splines are the first method that can combine precision, accuracy and simplicity.
Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates the turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-dependent substrate ubiquitination is antagonized by the Usp28 deubiquitinase. Here, we show that Usp28 preferentially antagonizes autocatalytic ubiquitination and stabilizes Fbw7, resulting in dose-dependent effects in Usp28 knockout mice. Monoallelic deletion of Usp28 maintains stable Fbw7 but drives Fbw7 substrate degradation. In contrast, complete knockout triggers Fbw7 degradation and leads to the accumulation of Fbw7 substrates in several tissues and embryonic fibroblasts. On the other hand, overexpression of Usp28 stabilizes both Fbw7 and its substrates. Consequently, both complete loss and ectopic expression of Usp28 promote Ras-driven oncogenic transformation. We propose that dual regulation of Fbw7 activity by Usp28 is a safeguard mechanism for maintaining physiological levels of proto-oncogenic Fbw7 substrates, which is equivalently disrupted by loss or overexpression of Usp28.
Clinical prognosis of metastasized colorectal carcinoma (CRC) is still not at desired levels and novel drugs are needed. Here, we focused on the multi-tyrosine kinase inhibitor E7080 (Lenvatinib) and assessed its therapeutic efficacy against human CRC cell lines in vitro and human CRC xenografts in vivo. The effect of E7080 on cell viability was examined on 10 humanCRCcell lines and humanendothelial cells (HUVEC). The inhibitory effect of E7080 on VEGF-induced angiogenesis was studied in an ex vivo mouse aortic ring angiogenesis assay. In addition, the efficacy of E7080 against xenografts derived fromCRC cell lines and CRC patient resection specimenswithmutated KRASwas investigated in vivo. Arelatively low cytotoxic effect of E7080 on CRC cell viabilitywas observed in vitro. Endothelial cells (HUVEC)weremore susceptible to the incubation with E7080. This is in line with the observation that E7080 demonstrated an anti-angiogenic effect in a three-dimensional ex vivo mouse aortic ring angiogenesis assay. E7080 effectively disrupted CRC cell-mediated VEGF-stimulated growth of HUVEC in vitro. Daily in vivo treatment with E7080 (5 mg/kg) significantly delayed the growth of KRAS mutated CRC xenografts with decreased density of tumor-associated vessel formations and without tumor regression. This observation is in line with results that E7080 did not significantly reduce the number of Ki67-positive cells in CRC xenografts. The results suggest antiangiogenic activity of E7080 at a dosage thatwas well tolerated by nudemice. E7080 may provide therapeutic benefits in the treatment of CRC with mutated KRAS.