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Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the ‘3Rs’ and the desire to phase-out animal research, in vitro models are needed. One potential approach uses clinostat-cultured 3D HepG2–C3A liver-mimetic spheroids. They take 18 days to recover in vivo physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human in vivo toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized).

[Image: see text] Colorectal cancer is one of the leading causes of cancer-related deaths. A main problem for its treatment is resistance to chemotherapy, requiring the development of new drugs. The success rate of new candidate cancer drugs in clinical trials remains dismal. Three-dimensional (3D) cell culture models have been proposed to bridge the current gap between in vitro chemotherapeutic studies and the human in vivo, due to shortcomings in the physiological relevance of the commonly used two-dimensional cell culture models. In this study, LS180 colorectal cancer cells were cultured as 3D sodium alginate encapsulated spheroids in clinostat bioreactors. Growth and viability were evaluated for 20 days to determine the ideal experimental window. The 3- (4,5- dimethylthiazol- 2- yl)-2,5-diphenyltetrazolium bromide assay was then used to establish half maximal inhibitory concentrations for the standard chemotherapeutic drug, paclitaxel. This concentration was used to further evaluate the established 3D model. During model characterization and evaluation soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase, glucose consumption, and P-glycoprotein (P-gp) gene expression were measured. Use of the model for chemotherapeutic treatment screening was evaluated using two concentrations of paclitaxel, and treatment continued for 96 h. Paclitaxel caused a decrease in cell growth, viability, and glucose consumption in the model. Furthermore, relative expression of P-gp increased compared to the untreated control group. This is a typical resistance-producing change, seen in vivo and known to be a result of paclitaxel treatment. It was concluded that the LS180 sodium alginate encapsulated spheroid model could be used for testing new chemotherapeutic compounds for colorectal cancer.

Culturing cells in 3D is often considered to be significantly more difficult than culturing them in 2D. In practice, this is not the case: the situation is that equipment needed for 3D cell culture has not been optimised as much as equipment for 2D. Here we present a few key features which must be considered when designing 3D cell culture equipment. These include diffusion gradients, shear stress and time. Diffusion gradients are unavoidably introduced when cells are cultured as clusters. Perhaps the most important consequence of this is that the resulting hypoxia is a major driving force in the metabolic reprogramming. Most cells in tissues do not experience liquid shear stress and it should therefore be minimised. Time is the factor that is most often overlooked. Cells, irrespective of their origin, are damaged when cultures are initiated: they need time to recover. All of these features can be readily combined into a clinostat incubator and bioreactor. Surprisingly, growing cells in a clinostat system do not require specialised media, scaffolds, ECM substitutes or growth factors. This considerably facilitates the transition to 3D. Most importantly, cells growing this way mirror cells growing in vivo and are thus valuable for biomedical research.

Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell-cell adhesion while allowing gas, nutrient, signal, and metabolite exchange. Downstream analysis can become more complicated because many assays (like phase contrast microscopy) cannot be used, but their replacements have been in use for many years. Most importantly, there is a huge gain in value in obtaining data that is more representative of the organism in vivo. It is the goal of the protocols presented here to make the transition to a new dimension as painless as possible. Grown optimally, most biopsy derived organoids will retain patient phenotypes, while cell (both stem cell, induced or otherwise or immortalized) derived organoids or spheroids will recover tissue functionality.

The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations.

Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer.

Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell–cell adhesion while allowing gas, nutrient, signal, and metabolite exchange. Downstream analysis can become more complicated because many assays (like phase contrast microscopy) cannot be used, but their replacements have been in use for many years. Most importantly, there is a huge gain in value in obtaining data that is more representative of the organism in vivo. It is the goal of the protocols presented here to make the transition to a new dimension as painless as possible. Grown optimally, most biopsy derived organoids will retain patient phenotypes, while cell (both stem cell, induced or otherwise or immortalized) derived organoids or spheroids will recover tissue functionality.

Colorectal cancer remains to be one of the leading causes of death worldwide, with millions of patients diagnosed each year. Although chemotherapeutic drugs are routinely used to treat cancer, these treatments have severe side effects. As a result, the use of herbal medicines has gained increasing popularity as a treatment for cancer. In this study, two South African medicinal plants widely used to treat various diseases, Sutherlandia frutescens and Xysmalobium undulatum, were evaluated for potential activity against colorectal cancer. This potential activity for the treatment of colorectal cancer was assessed relative to the known chemotherapeutic drug, paclitaxel. The cytotoxic activity was considered in an advanced three-dimensional (3D) sodium alginate encapsulated LS180 colorectal cancer functional spheroid model, cultured in clinostat-based rotating bioreactors. The LS180 cell mini-tumors were treated for 96 h with two concentrations of each of the crude aqueous extracts or paclitaxel. S. frutescens extract markedly decreased the soluble protein content, while decreasing ATP and AK per protein content to below detectable limits after only 24 h exposure. X. undulatum extract also decreased the soluble protein content, cell viability, and glucose consumption. The results suggested that the two phytomedicines have potential to become a source of new treatments against colorectal cancer.

Ethnopharmacology relevance Traditional herbal medicines are utilized by 27 million South Africans. Xysmalobium undulatum (Uzara) is one of the most widely used traditional medicinal plants in Southern Africa. A false belief in the safety of herbal medicine may result in liver injury. Herb-induced liver injury (HILI) range from asymptomatic elevation of liver enzymes, to cirrhosis and in certain instances even acute liver failure. Various in vitro and in vivo models are available for the pre-clinical assessment of drug and herbal hepatotoxicity. However, more reliable and readily available in vitro models are needed, which are capable of bridging the gap between existing models and real human exposure. Three-dimensional (3D) spheroid cultures offer higher physiological relevance, overcoming many of the shortcomings of traditional two-dimensional cell cultures. Aims of this study This study investigated the hepatotoxic and anti-prolific effects of the crude X. undulatum aqueous extract during a sub-chronic study (21 days), in both a 3D HepG2/C3A spheroid model and the Sprague Dawley rat model. Methods HepG2/C3A spheroids were treated with a known hepatotoxin, valproic acid, and crude X. undulatum aqueous extract for 21 days with continuous evaluation of cell viability and proliferation. This was done by evaluating cell spheroid growth, intracellular adenosine triphosphate (ATP) levels and extracellular adenylate kinase (AK). Sprague Dawley rats were treated with the same compounds over 21 days, with evaluation of in vivo toxicity effects on serum chemistry. Results The results from the in vitro study clearly indicated hepatotoxic effects and possible liver damage following treatment with valproic acid, with associated growth inhibition, loss of cell viability and increased cytotoxicity as indicated by reduced intracellular ATP levels and increased AK levels. These results were supported by the increased in vivo levels of AST, ALT and LDH following treatment of the Sprague Dawley rats with valproic acid, indicative of hepatic cellular damage that may result in hepatotoxicity. The in vitro 3D spheroid model was also able to predict the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. Similarly, the results obtained from the in vivo Sprague Dawley model indicated moderate hepatotoxic potential. Conclusion The data from both the 3D spheroid model and the Sprague Dawley model were able to indicate the potential concentration dependant hepatotoxicity of the crude X. undulatum aqueous extract. The results obtained from this study also confirmed the ability of the 3D spheroid model to effectively and reliably predict the long-term outcomes of possible hepatotoxicity.

The liver is a vital organ fulfilling a central role in over 500 major metabolic functions, including serving as the most essential site for drug biotransformation. Dysfunction of the drug biotransformation processes may result in the exposure of the liver (and other organs) to hepatotoxins, potentially interacting with cellular constituents and causing toxicity and various lesions. Hepatotoxicity can be investigated on a tissue, cellular and molecular level by employing various in vivo and in vitro techniques, including novel three-dimensional (3 D) cell culturing methods. This paper reflects on the liver and its myriad of functions and the influence of drug biotransformation on liver dysfunction. Current in vivo and in vitro models used to study liver function and dysfunction is outlined, emphasizing their advantages and disadvantages. The advantages of novel in vitro 3 D cell culture models are discussed and the possibility of novel models to bridge the gap between in vitro and in vivo models is explained. Progression made in the field of cell culturing methods such as 3 D cell culturing techniques over the last decade promises to reduce the use of in vivo animal models in biotransformation and toxicological studies of the liver.

Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. Methods CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. Results Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. Conclusions 3D spheroid cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.

Fish oil (FO) and tetradecylthioacetic acid (TTA) — a synthetic modified fatty acid have beneficial effects in regulating lipid metabolism. In order to dissect the mechanisms underlying the molecular action of those two fatty acids we have investigated the changes in mitochondrial protein expression in a long-term study (50 weeks) in male Wistar rats fed 5 different diets. The diets were as follows: low fat diet; high fat diet; and three diets that combined high fat diet with fish oil, TTA or combination of those two as food supplements. We used two different proteomics techniques: a protein centric based on 2D gel electrophoresis and mass spectrometry, and LC-MS E based peptide centric approach. As a result we provide evidence that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate that fatty acid metabolism; lipid oxidation, amino acid metabolism and oxidative phosphorylation pathways are involved in fish oil and TTA action. Evidence for the involvement of PPAR mediated signalling is provided. Additionally we postulate that down regulation of components of complexes I and II contributes to the strong antioxidant properties of TTA. Biological significance This study for the first time explores the effect of fish oil and TTA — tetradecyl-thioacetic acid and the combination of those two as diet supplements on mitochondria metabolism in a comprehensive and systematic manner. We show that fish oil and TTA modulate mitochondrial metabolism in a synergistic manner yet the effects of TTA are much more dramatic. We demonstrate in a large scale that fatty acid metabolism and lipid oxidation are affected by fish oil and TTA, a phenomenon already known from more directed molecular biology studies. Our approach, however, shows additionally that amino acid metabolism and oxidative phosphorylation pathways are also strongly affected by TTA and also to some extent by fish oil administration. Strong evidence for the involvement of PPAR mediated signalling is provided linking the different metabolic effects. The global and systematic viewpoint of this study compiles many of the known phenomena related to the effects of fish oil and fatty acids giving a solid foundation for further exploratory and more directed studies of the mechanisms behind the beneficial and detrimental effects of fish oil and TTA diet supplementation. This work is already a second article in a series of studies conducted using this model of dietary intervention. In the previous study (Vigerust et al., [21]) the effects of fish oil and TTA on the plasma lipids and cholesterol levels as well as key metabolic enzymes in the liver have been studied. In an ongoing study more work is being done to explore in detail for example the link between the down regulation of the components of the respiratory chain (observed in this study) and the strong antioxidant effects of TTA. The reference diet in this study has been designed to mimic an unhealthy – high fat diet that is thought to contribute to the development of metabolic syndrome – a condition that is strongly associated with diabetes, obesity and heart failure. Fish oil and TTA are known to have beneficial effects for the fatty acid metabolism and have been shown to alleviate some of the symptoms of the metabolic syndrome. To date very little is known about the molecular mechanisms behind these beneficial effects and the potential pitfalls of the consumption of those two compounds. Only studies of each compound separately and using only small scale molecular biology approaches have been carried out. The results of this work provide an excellent starting point for further studies that will help to understand the metabolic effects of fish oil and TTA and will hopefully help to design dietary programs directed towards reduction of the prevalence of metabolic syndrome and associated diseases.

The introduction of trypsinisation in the 1950’s was a paradigm shift which helped instigate cell culture. We demonstrate here that human hepatocyte cell line HepG2–C3A needs at least 18 days after trypsinisation to re-establish key ultrastructural and physiological traits. After trypsinisation, cells start to recover these traits at similar rates in both monolayer (2D) or spheroid (3D) growth environments. While this development is restarted by trypsinisation of 2D cultures (typically after 5 days), recovery continues in 3D cultures up until 15–18 days when changes in growth rate, adenylate kinase, ATP, urea and cholesterol all suggest that spheroids undergo some type of physiological transition. Several other cell lines (e.g. Caco-2, HT 29, MDCK, MCF-10A and HepG2 used to model the small and large intestine, kidney, breast acini and liver respectively) are reported in the literature to exhibit very similar changes, on a similar timescale to those reported here. These changes may thus represent a ubiquitous recovery process after trypsinisation rather than differentiation. This would partially explain the common observation that cells grown in 3D exhibit physiological capabilities that are closer to those seen in the intact tissue or organ.

Acetaminophen (APAP) is possibly the most widely used medication globally and yet little is known of its molecular effects at therapeutic doses., Acetaminophen (APAP) is possibly the most widely used medication globally and yet little is known of its molecular effects at therapeutic doses. Using a novel approach, we have analysed the redox proteome of the hepatocellular cell line HepG2/C3A treated with therapeutic doses of APAP and quantitated both individual protein abundance and their reversible S-nitrosylation (SNO) and S-sulfenylation (SOH) modifications by mass spectrometry. APAP treatment results in a late, transient increase in ATP production and a multiplicity of alterations in protein abundance and modifications. The majority of the differentially SNO or SOH modified proteins are found in the endoplasmic reticulum and cytosol, suggesting that the source of reactive species is there. The cellular response indicates: constraint of fatty acid metabolism; reduction in ribosome construction and protein synthesis (to conserve ATP); maintenance of glutathione levels (by increased synthetic capacity); and an increased NADPH production (via the pentose phosphate pathway). This response appears to be coordinated, directly or indirectly, by the canonical Wnt and Nrf2 signalling pathways. Combined with the known role of NAPQI, these studies suggest that the physiological and toxicological responses form a continuum: therapeutic doses of APAP produce reactive species and NAPQI in the cytoplasm but result in little permanent damage. The cell mounts a multifaceted response which minimises disruption and repairs are effected within a day or two. Higher doses of APAP lead to intensified reactive species production, which increasingly disturbs mitochondrial function and eventually leads to cell death.

We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our methodology which includes isolation, spectral characterisation, stability testing, gel-based separation and mass spectrometric identification was optimised on samples collected during the Danish Galathea 3 expedition. Four corals of the Fungia, Sarcophyton and Acropora species emitting green fluorescence were tested. Each of the fluorescent extracts behaves differently under denaturing conditions but complete fluorescence loss was not observed. Optimised electrophoretic conditions yielded effective separation of active fluorescent proteins in both 1DE and 2DE. Mass spectrometric analysis of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples. The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition.

The recovery of physiological functionality, which is commonly seen in tissue mimetic three-dimensional (3D) cellular aggregates (organoids, spheroids, acini, etc.), has been observed in cells of many origins (primary tissues, embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and immortal cell lines). This plurality and plasticity suggest that probably several basic principles promote this recovery process. The aim of this study was to identify these basic principles and describe how they are regulated so that they can be taken in consideration when micro-bioreactors are designed. Here, we provide evidence that one of these basic principles is hypoxia, which is a natural consequence of multicellular structures grown in microgravity cultures. Hypoxia drives a partial metabolic reprogramming to aerobic glycolysis and an increased anabolic synthesis. A second principle is the activation of cytoplasmic glutaminolysis for lipogenesis. Glutaminolysis is activated in the presence of hypo- or normo-glycaemic conditions and in turn is geared to the hexosamine pathway. The reducing power needed is produced in the pentose phosphate pathway, a prime function of glucose metabolism. Cytoskeletal reconstruction, histone modification, and the recovery of the physiological phenotype can all be traced to adaptive changes in the underlying cellular metabolism. These changes are coordinated by mTOR/Akt, p53 and non-canonical Wnt signaling pathways, while myc and NF-kB appear to be relatively inactive. Partial metabolic reprogramming to aerobic glycolysis, originally described by Warburg, is independent of the cell's rate of proliferation, but is interwoven with the cells abilities to execute advanced functionality needed for replicating the tissues physiological performance.

Udgivelsesdato: 2008-Jul-21 Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by mass spectrometric identification of the proteins in the protein spots has become a central tool in proteomics. CMT167(H), CMT64(M) and CMT170(L) cell lines, selected from a spontaneous mouse lung adenocarcinoma, with high-, middle- or low-metastatic potential have been characterized in vivo. In this study, the comprehensive protein expression profiles of the CMT cell lines were analyzed at passages 5, 15 and 35 in order to assess the cell line stability. During the passages 5 to 15, the expression profiles of CMT cells remained reasonably stable as evidenced by only 0.7%, 3.9% and 1.1% proteins changed in CMT167(H), CMT64(M) and CMT170(L) respectively. However, the number of differentially expressed proteins were considerably increased at passage 35 in CMT64(M) and CMT170(L) while CMT167(H) remained stable. Based on our selection criteria, 22, 109 and 84 spots in CMT167(H), CMT64(M) and CMT170(L) were selected for protein identification by MS and 99 unique proteins were identified. Bioinformatics analysis indicated that most of these proteins participate in cellular metabolism. In conclusion, proteomics was found to be a useful tool for assessing differences in cell line stability. This approach provided a tool to select the best cell line and optimal subculture period for studies of cancer related phenomena and for testing the effect of potential anticancer drugs.

Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and coexisting PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

Natural products, or their synthetic derivatives are a treasure trove to find potential candidates for novel drugs for human treatment. The selection of diamonds from the huge pile of worthless stone is a critical – and difficult - stage in the discovery pipeline. Of all the factors to be considered, perhaps the most important, is that the compound should have the desired effect on the tissue in vivo. Since it is not possible (or ethical) to test all compounds in vivo one must preselect using a surrogate assay system. While animal models have the advantage of being holistic and current 3D culture systems are reductionistic, they at least can be constructed from human cell types.In this review we will consider some of the evidence demonstrating that cells grown in 3D cultures have physiological performances that mimic functions seen in human tissues significantly better than cells grown using classical 2D culture systems. We will discuss advantages and disadvantages of these new culture technologies and highlight theoretical reasons for the differences.3D cell culture technologies are more labour intensive than 2D culture systems and therefore their introduction is a trade-off between the value of obtaining data that is more relevant to the human condition against their through-put. It is already clear that future in vitro 3D systems will become more complex, using multiple cell types to more faithfully represent a particular tissue or even organ system. And one thing is sure – the diamonds are not easy to find!

Udgivelsesdato: 2006-May-26 Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets and insulinoma cells confirmed IL-1beta regulated gal-3 expression of several gal-3 isoforms and a complex in vivo expression profile during diabetes development in rats. Over-expression of gal-3 protected beta-cells against IL-1beta toxicity, with a complete blockage of JNK phosphorylation, essential for IL-1-mediated apoptosis. Mutation scanning of regulatory and coding regions of the gal-3 gene (LGALS3) identified six polymorphisms. A haplotype comprising three cSNPs showed significantly increased transmission to unaffected offspring in 257 T1D families and replicated in an independent set of 170 T1D families. In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies.

Udgivelsesdato: 2003-Mar-21 Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.

In the cell, the majority of proteins exist in complexes. Most of these complexes have a constant stoichiometry and thus can be used as internal standards. In this rapid communication we show that it is possible to calculate a correlation coefficient which reflects the reproducibility of the analytical approach used. The abundance of one subunit in a heterodimer is plotted against the abundance of the other, and this is repeated for all subunits in all heteromers found in the data set. The correlation coefficient obtained (the “heteromer score”) is a new bioinformatic tool which is independent of the method used to collect the data, requires no special sample preparation and can be used retrospectively on old datasets. It can be used for quality control, to indicate when a change becomes significant or identify complexes whose stoichiometry has been perturbed during the experiment.

Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analysed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Ultrastructurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate cycle are increased while Krebs cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased underpinning the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in G0. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicle has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo (Wrzesinski and Fey 2013, Wrzesinski et al 2013, Fey and Wrzesinski 2012). Here we describe the multitude of protein changes necessary to achieve this performance.

Primary human hepatocytes are widely used as an in vitro system for the assessment of drug metabolism and toxicity. Nevertheless a cell system with higher stability of physiological functions is required for the investigation of drugs’ mode of action, pathway analyses and biomarkers evaluations. We recently discovered that the human hepatocellular carcinoma cell line, HepG2/C3A, cultured as spheroids in a 3D system can recover their main functions after trypsinisation within about 18 days. The objective of this study was to investigate whether the spheroids’ metabolic functions remained stable after this recovery period. Therefore we evaluated physiological capabilities of the spheroids (cell survival, growth rate, glycogenesis, ATP, cholesterol and urea synthesis and drug metabolism) and the expression of key genes related tothe main liver pathways in spheroids cultured for an additional 24 days after full recovery (day 18). Here we show that after the recovery period, the 3D spheroid culture can provide a metabolically competent homeostatic cell model which is in equilibrium with its culture environment for more than 3 weeks. Sucha stable system could be used for the assessment of the drugs’ mode of action, for biomarkers evaluation and for any systems biology studies which require medium- to long-term stability of metabolic functions.

Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD50 values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD50 values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo.

Udgivelsesdato: 2008-Nov-10 Metastasis is a lethal attribute of a cancer and presents a continuing therapeutic challenge. Metastasis is a highly complex process and more knowledge about the mechanisms behind metastasis is highly desirable. Isogenic CMT cell lines were selected from a spontaneous mouse lung adenocarcinoma and characterized in vivo to have different metastatic potential. In this study, the comprehensive protein expression profiles of three of these CMT cell lines at passage 5, 15 and 35 were analyzed by 2-DE separation followed by MS identification. As a result, 82 and 40 unique proteins were found to be significantly up- or down-regulated between cell lines with different metastatic potential at passages 5 and 15, respectively. These proteins were identified by MS and most of them have previously been reported to be related to cancer development and/or metastasis. Bioinformatics analysis indicated that several of the proteins were involved in proteasome, cell-cycle and cell-communication pathways. Among them, some keratins, 14-3-3 proteins and 26S proteasome proteins were identified and their aberrant expression may be directly or indirectly involved in cancer development and metastasis. In conclusion, our comprehensive 2-DE-based proteomics studies revealed some candidate proteins, protein families and signaling pathways, which might be important in cancer development and metastasis.

Streptococcus mutans is a component of the dental plaque biofilm and a major causal agent of dental caries. Log-phase cells of the organism are known to induce an acid tolerance response (ATR) at sub-lethal pH values ( approximately 5.5) that enhances survival at lower pH values such as those encountered in caries lesions. In this study, we have employed a rod biofilm chemostat system to demonstrate that, while planktonic cells induced a strong ATR at pH 5.5, biofilm cells were inherently more acid resistant than such cells in spite of a negligible induction of an ATR. Since these results suggested that surface growth itself triggered an ATR in biofilm cells, we were interested in comparing the effects of a pH change from 7.5 to 5.5 on protein synthesis by the two cell types. For this, cells were pulse labeled with [(14)C]-amino acids following the pH change to pH 5.5, the proteins extracted and separated by two-dimensional (2D) electrophoresis followed by autoradiography and computer-assisted image analysis. A comparison between the cells incubated at pH 5.5 and the control biofilm cells revealed 23 novel proteins that were absent in the control cells, and 126 proteins with an altered relative rate of synthesis. While the number of changes in protein expression in the biofilm cells was within the same range as for planktonic cells, the magnitude of their change was significantly less in biofilm cells, supporting the observation that acidification of biofilm cells induced a negligible ATR. Mass spectrometry and computer-assisted protein sequence analysis revealed that ATR induction of the planktonic cells resulted in the downregulation of glycolytic enzymes presumably to limit cellular damage by the acidification of the external environment. On the other hand, the glycolytic enzymes in control biofilm cells were significantly less downregulated and key enzymes, such as lactate dehydrogenase were upregulated during pH 5.5 incubation, suggesting that the enhanced acid resistance of biofilm cells is associated with the maintenance of pH homeostasis by H+ extrusion via membrane ATPase and increased lactate efflux.

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.