TSC2 regulates tumor susceptibility to TRAIL-mediated T-cell killing by orchestrating mTOR signaling.

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we use whole-genome CRISPR-Cas9 knockout data to uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocyte (CTL) killing in human melanoma cells. TSC2-depleted tumor cells had disrupted mTOR regulation following CTL attack, which was associated with enhanced cell death. Wild-type tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity, circumventing apoptosis, and necroptosis. TSC2 ablation strongly augmented tumor cell sensitivity to CTL attack in vitro and in vivo, suggesting one of its functions is to critically protect tumor cells. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptor expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockade. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

DBP7 and YRF1-6 Are Involved in Cell Sensitivity to LiCl by Regulating the Translation of PGM2 mRNA.

Lithium chloride (LiCl) has been widely researched and utilized as a therapeutic option for bipolar disorder (BD). Several pathways, including cell signaling and signal transduction pathways in mammalian cells, are shown to be regulated by LiCl. LiCl can negatively control the expression and activity of PGM2, a phosphoglucomutase that influences sugar metabolism in yeast. In the presence of galactose, when yeast cells are challenged by LiCl, the phosphoglucomutase activity of PGM2p is decreased, causing an increase in the concentration of toxic galactose metabolism intermediates that result in cell sensitivity. Here, we report that the null yeast mutant strains DBP7∆ and YRF1-6∆ exhibit increased LiCl sensitivity on galactose-containing media. Additionally, we demonstrate that DBP7 and YRF1-6 modulate the translational level of PGM2 mRNA, and the observed alteration in translation seems to be associated with the 5'-untranslated region (UTR) of PGM2 mRNA. Furthermore, we observe that DBP7 and YRF1-6 influence, to varying degrees, the translation of other mRNAs that carry different 5'-UTR secondary structures.

Lithium chloride sensitivity connects the activity of PEX11 and RIM20 to the translation of PGM2 and other mRNAs with structured 5'-UTRs.

Lithium chloride (LiCl) is a widely used and extensively researched drug for the treatment of bipolar disorder (BD). As a result, LiCl has been the subject of research studying its toxicity, mode of action, and downstream cellular responses. LiCl has been shown to influence cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3 in mammalian cells. LiCl's significant downstream effects on the translational pathway necessitate further investigation. In yeast, LiCl is found to lower the activity and alter the expression of PGM2, a gene encoding a sugar-metabolism enzyme phosphoglucomutase. When phosphoglucomutase activity is reduced in the presence of galactose, intermediates of galactose metabolism aggregate, causing cell sensitivity to LiCl. In this study, we identified that deleting the genes PEX11 and RIM20 increases yeast LiCl sensitivity. We further show that PEX11 and RIM20 regulate the expression of PGM2 mRNA at the translation level. The observed alteration of translation seems to target the structured 5'-untranslated region (5'-UTR) of the PGM2 mRNA.

The Potential Predictors in Chemotherapy Sensitivity.

Monitoring of patient and tumor during chemotherapy is important to determine whether the chemotherapy is effective to the patient. Variants affect drug enzyme activities and altered enzyme activities can be potential predictors for chemotherapeutic agents including cyclophosphamide and paclitaxel. Response to chemotherapy is primarily based on somatic mutations but germline variants may predict cancer cell sensitivity to chemotherapeutic agents. Furthermore, patient's genetic variation of immune system was reported to be associated with drug response and toxicity. Recently, the somaric and germilne genomic variation influences the pharmacokinetics of chemotherapy and these variation can be biomarkers for chemotherapy.

Beta-cell sensitivity to insulinotropic gut hormones is reduced after gastric bypass surgery.

OBJECTIVE: Postprandial hyperinsulinaemia after Roux-en Y gastric bypass (GB) has been attributed to rapid nutrient flux from the gut, and an enhanced incretin effect. However, it is unclear whether surgery changes islet cell responsiveness to regulatory factors. This study tested the hypothesis that ß-cell sensitivity to glucagon like-peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) is attenuated after GB. DESIGN: Ten non-diabetic subjects with GB, and 9 body mass index (BMI)-matched and age-matched non-surgical controls (CN) with normal glucose tolerance had blood glucose clamped at ~7.8 mM on three separate days. Stepwise incremental infusions of GLP-1 (15, 30, 60, 120 and 300 ng/LBkg/h), GIP (75, 150, 300, 600 and 1200 ng/LBkg/h) or saline were administered from 90 to 240 min and insulin secretion measured. RESULTS: GB subjects had similar fasting glucose levels but lower fasting insulin compared with CN, likely due to increased insulin clearance. The average insulin secretion rates (ISRs) to 7.8 mM glucose were ~30% lower in GB relative to CN subjects. However, incretin-stimulated ISRs, adjusted for insulin sensitivity and glucose-stimulated insulin secretion, were even more attenuated in the GB subjects, by threefold to fourfold (AUCISR(90-240 min) during GLP-1 and GIP: 47±8 and 44±12 nmol in GB and 116±16 and 161±44 in CN; p<0.01). CONCLUSION: After GB, the sensitivity of insulin secretion to both glucose and incretins is diminished.

Visual Quantitative Detection of Circulating Tumor Cells with Single-Cell Sensitivity Using a Portable Microfluidic Device.

Immunocytological technologies, molecular technologies, and functional assays are widely used for detecting circulating tumor cells (CTCs) after enrichment from patients' blood sample. Unfortunately, accessibility to these technologies is limited due to the need for sophisticated instrumentation and skilled operators. Portable microfluidic devices have become attractive tools for expanding the access and efficiency of detection beyond hospitals to sites near the patient. Herein, a volumetric bar chart chip (V-Chip) is developed as a portable platform for CTC detection. The target CTCs are labeled with aptamer-conjugated nanoparticles (ACNPs) and analyzed by V-Chip through quantifying the byproduct (oxygen) of the catalytic reaction between ACNPs and hydrogen peroxide, which results in the movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Thus, the CTC number is decoded into visually quantifiable information and a linear correlation can be found between the distance moved by the ink and number of cells in the sample. This method is sensitive enough that a single cell can be detected. Furthermore, the clinical capabilities of this system are demonstrated for quantitative CTC detection in the presence of a high leukocyte background. This portable detection method shows great potential for quantification of rare cells with single-cell sensitivity for various applications.

Phylogenetic analysis and phenotypic characterisatics of two Tibet EV-C96 strains.

BACKGROUND: Enterovirus C96 (EV-C96) is a newly named type of enterovirus belonging to species C, and the prototype strain (BAN00-10488) was firstly isolated in 2000 from a stool specimen of a patient with acute flaccid paralysis in Bangladesh. In this study, we report the genomic and phenotypic characteristics of two EV-C96 strains isolated from individuals from the Tibet Autonomous Region of China. METHODS: Human rhabdomyosarcoma (RD), human laryngeal epidermoid carcinoma (HEp-2), and human cervical cancer (Hela) cells were infected with the Tibet EV-C96 strains, and enterovirus RNA in the cell culture was detected with a real time RT-PCR-based enterovirus screening method. The temperature sensitivity of Tibet EV-C96 strains were assayed on a monolayer of RD cells in 24-well plates. Full-length genome sequencing was performed by a 'primer-walking' strategy, and the evolutionary history of EV-C96 was studied by maximum likelihood analysis. RESULTS: Strain 2005-T49 grew in all three kinds of cells, and it was not temperature sensitive. In contrast, none of the three cells produced CPE for strain 2012-94H. Phylogenetic analysis of the two Tibetan viruses, other EV-C96 strains, and EV-C prototypes showed that EV-C96 strains were grouped into three clusters (Cluster1-3) based on their VP1 sequences, which may represent three genotypes. Phylogenetic trees based on the P2 and P3 regions highlighted the difference between Chinese EV-C96 strains and the EV-C96 prototype strain BAN-10488. All Chinese strains formed a cluster separate from BAN-10488, which clustered with CV-A1/CV-A22/CV-A19. CONCLUSIONS: There is genetic variability between EV-C96 strains which suggest that at least few genetic lineages co-exist and there has been some degree of circulation in different geographical regions for some time. Some recombination events must have occurred during EV-C96 evolution as EV-C96 isolates cluster with different EV-C prototype strains in phylogenetic trees in different genomic regions. However, recombination does not seem to have occurred frequently as EV-C96 isolates from different years and locations appear to cluster together in all genomic regions analysed. These findings expand the understanding of the characterization of EV-C96 and are meaningful for the surveillance of the virus.

Pharmacogenetic Predictors of Response.

Pharmacogenetics attempts to predict treatment response using a patient's "germline" genome as the biomarker of interest. This chapter on pharmacogenetic predictors of breast cancer response is divided into four sections. The first introduces readers to genetic variation and describes how variation in the germline genome can affect biology or pharmacology. The second section introduces the translational pathway for pharmacogenetic research and discusses the specific challenges to identifying pharmacogenetic predictors of breast cancer response. The third section is divided into three subsections, each of which discusses a distinct category of pharmacogenetic response predictors; pharmacokinetics, cancer cell sensitivity, and effector cell activation. Within each subsection a specific pharmacogenetic association is described in detail; CYP2D6-tamoxifen, BRCA-PARP inhibitors, and FCGRA-trastuzumab, respectively, followed by a general discussion of other less well-established examples or areas for further research. The chapter concludes with a summary of the current status of pharmacogenetic predictors of breast cancer response and a few predictions for the future of this field.

Comet assay measures of DNA damage are predictive of bladder cancer cell treatment sensitivity in vitro and outcome in vivo.

Bladder cancer patients suffer significant treatment failure, including high rates of recurrence and poor outcomes for advanced disease. If mechanisms to improve tumour cell treatment sensitivity could be identified and/or if tumour response could be predicted, it should be possible to improve local-control and survival. Previously, we have shown that radiation-induced DNA damage, measured by alkaline Comet assay (ACA), correlates bladder cancer cell radiosensitivity in vitro. In this study we first show that modified-ACA measures of cisplatin and mitomycin-C-induced damage also correlate bladder cancer cell chemosensitivity in vitro, with essentially the same rank order for chemosensitivity as for radiosensitivity. Furthermore, ACA studies of radiation-induced damage in different cell-DNA substrates (nuclei, nucleoids and intact parent cells) suggest that it is a feature retained in the prepared nucleoids that is responsible for the relative damage sensitivity of bladder cancer cells, suggestive of differences in the organisation of DNA within resistant vs. sensitive cells. Second, we show that ACA analysis of biopsies from bladder tumours reveal that reduced DNA damage sensitivity associates with poorer treatment outcomes, notably that tumours with a reduced damage response show a significant association with local recurrence of non-invasive disease and that reduced damage response was a better predictor of recurrence than the presence of high-risk histology in this cohort. In conclusion, this study demonstrates that mechanisms governing treatment-induced DNA damage are both central to and predictive of bladder cancer cell treatment sensitivity and exemplifies a link between DNA damage resistance and both treatment response and tumour aggression.

Analysis of the Adherent Cell Response to the Substrate Stiffness Using Tensegrity.

Cell's shape is dependent on the cytoskeleton mechanical properties. Hybrid models were developed that combine the discrete structure for the cytoskeleton and continuum parts for other cell organelles. Tensegrity-based structures that consist of tensile and compression elements are useful models to understand the cytoskeleton mechanical behavior. In this study, we are looking to examine the reaction of the cell to a variety of substrate stiffnesses and explain the relationship between cell behavior and substrate mechanical properties. However, which tensegrity structure is appropriate for modeling a living cell? Is the structure's complexity play a major role? We used two spherical tensegrities with different complexities to assess the impact of the structure on the cell's mechanical response versus substrate's stiffness. Six- and twelve-strut tensegrities together with membrane, cytoplasm, nucleoskeleton, and nucleus envelope were assembled in Abaqus package to create a hybrid cell model. A compressive load was applied to the cell model and the reaction forces versus deflection curves were analyzed for number of substrate stiffness values. By analyzing the difference due to two different tensegrities it became clear that the lower density structure is a better choice for modeling stiffer cells. It was also found that the six-strut tensegrity is sensitive to higher range of substrate stiffness.

In vitro screening of topical formulation excipients for epithelial toxicity in cancerous and non-cancerous cell lines.

Chemical excipients used in topical formulations may be toxic to living skin cells. Here, we compared the in vitro toxicity of some common solubilizing excipients against human melanoma cells, human keratinocytes (HaCaT) and primary skin fibroblasts (FB) as examples of cancerous, immortalized and primary human skin cells, often used as experimental models representative of in vivo conditions. Two distinct endpoint assays (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV)) were used. The mechanism of cell death after excipient exposure was assessed through Reactive Oxygen Species (ROS) production, cell membrane integrity and cell cycle progression. Results showed that the surfactants, Labrasol®, Labrafil® and Transcutol®, were less toxic than Triton X-100 (a model irritant) in all cell types whereas the oil, Labrafac®, was non-toxic. The human melanoma WM164 cell line showed the greatest sensitivity toward cytotoxicity after chemical exposure, while the other cell lines were more resistant. The relative excipient cytotoxicity responses observed in the MTT and CV assays were comparable and similar trends were seen in their estimated 50 % inhibitory concentration (IC50) values. DNA fragmentation by flow cytometry after exposing the cells to IC50 concentrations of the excipients showed negligible apoptotic populations. ROS production was increased in all cell types after toxic exposure; however, ROS elevation did not lead to apoptosis. The toxicity profiles of each excipient are not only relevant to their use in formulating safe topical products but also in the potential synergistic efficacy in the topical treatment of melanoma.

Features of hemodynamic and immunological parameters in patients with recurrent uveitis complicated by hypertension, Fuchs heterochromic uveitis and Posner-Schlossman syndrome.

Introduction: Uveitis is a disease that manifests with increased vascular permeability and occlusion, with some ischemia and inflammatory mediators. It is characterized by a wide range of pathological processes, including inflammation, increased vascular permeability and occlusion, local ischemia and cell alteration by inflammatory mediators, and is characterized by the presence of complications. Aim: To study the state of ocular hemodynamics by rheoophthalmography, as well as the immune status in patients with idiopathic recurrent anterior uveitis complicated by intraocular hypertension, Fuchs heterochromic uveitis, Posner-Schlossman syndrome, during the relapse period. Materials and methods: 93 patients with idiopathic recurrent anterior uveitis were included in this study, 8 patients with Fuchs' uveitis, and 6 patients with Posner-Schlossman syndrome. According to clinical signs, relapse and remission were considered. The control group (healthy volunteers of the same age) consisted of 27 people. In this regard, 5 groups of subjects were formed. The mean age of the patients was (M ± SD) 39.2 ± 14.6 years. According to the Median (range), the duration of the disease in patients was 2033 (350-3285) days, intraocular hypertension being recorded at P0 > 20 mm Hg. Statistical analysis was carried out in spreadsheets using STATISTICA 8.0 (StatSoft.Inc) program. Quantitative indicators were evaluated according to the correspondence to the normal distribution and to the Kolmogorov-Smirnov criterion. With a normal distribution, arithmetic means (M) and standard deviations (SD), limits of the 95% confidence interval (95% CI) and Student's t-test were calculated. Results: The volumetric blood filling of the eye according to the rheoophthalmographic indicator RQ during the period of remission of uncomplicated and complicated by hypertension anterior uveitis was reduced by 32.4%-40.5%, respectively, compared with the norm. During the period of relapse, RQ was significantly higher by 28% (p<0.05) than in remission, in the group of uncomplicated uveitis, and in the group of uveitis with increased IOP, no significant differences between the periods of remission and relapse were observed, which reflected the ischemic process in the relapse period. Volumetric blood filling in Fuchs and Posner-Schlossman syndromes in the acute period did not differ from the norm. Cellular immunity in the groups of uncomplicated and complicated by intraocular hypertension idiopathic uveitis, as well as with Fuchs and Posner-Schlossman syndromes, had a higher level of CD4 helper lymphocytes and a lower level of CD8 suppressor lymphocytes, which reflected higher values of the immunoregulatory index. The increase in the immunoregulatory index is most pronounced in Fuchs and Posner-Schlossman syndromes. Discussion: In the presented study, the incidence of idiopathic recurrent anterior uveitis complicated by intraocular hypertension was 9,9% among all cases of idiopathic recurrent anterior uveitis in one-time period. According to literature, this complicated form of uveitis occurs in 11,5%-46,1% of cases. Most often (up to 92% of cases), the anterior chamber angle was open. Conclusions: Different activity of the mechanisms regulating the balance of cellular and humoral immunity, sensitivity of T-cells to eye antigens in idiopathic anterior uveitis, Fuchs and Posner-Schlossman syndromes was assumed. Peculiarities of eye hemodynamics in these forms of uveitis were also revealed. Abbreviations: IOP = intraocular pressure, IOHS = inflammatory ocular hypertension syndrome, HSV = herpes simplex virus, CMV = cytomegalovirus, OCT = optical coherence tomography, OD = right eye, OS = left eye.

Dendrobium officinale leaf polysaccharides ameliorated hyperglycemia and promoted gut bacterial associated SCFAs to alleviate type 2 diabetes in adult mice.

The present study aimed to explore the possible mechanisms underlying Dendrobium officinale leaf polysaccharides of different molecular weight to alleviate glycolipid metabolic abnormalities, organ dysfunction and gut microbiota dysbiosis of T2D mice. An ultrafiltration membrane was employed to separate two fractions from Dendrobium officinale leaf polysaccharide named LDOP-A and LDOP-B. Here, we present data supporting that oral administration of LDOP-A and LDOP-B ameliorated hyperglycemia, inhibited insulin resistance, reduced lipid concentration, improved ß-cell function. LDOP-A with lower molecular weight exhibited improved effect on diabetes than LDOP-B, concurrent with increased levels of colonic short-chain fatty acids (SCFAs) i.e., butyrate, decreased ratio of Firmicutes to Bacteroidetes phyla, and increased abundance of the gut beneficial bacteria i.e., Lactobacillus, Bifidobacterium and Akkermansia. These results suggest that LDOP-A possesses a stronger effect in ameliorating T2D than LDOP-B which may be related to the distinct improved SCFAs levels produced by the change of intestinal flora microstructure.

Comparative sensitivity study of primary cells, vero, OA3.Ts and ESH-L cell lines to lumpy skin disease, sheeppox, and goatpox viruses detection and growth.

Lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox (GTPV) virus have been usually grown on primary cells for diagnosis, production and titration purposes. The use of primary cells present several inconvenient, heavy preparation, heterogeneous cell population, non-reproducible viral titration and presence of potential endogenous contaminants. Therefore investigating sensitivity of candidate continuous cell lines is needed. In this study, we compared the above Capripox viruses (CaPVs) sensitivity of primary cells of four origin (heart, skin, testis and kidney), with three cell lines (Vero, OA3.Ts and ESH-L). We tested sensitivity for virus isolation, replication cycle and titration, revealed by cytopathic effect (CPE), immunoenzymatic staining and immunofluorescence. Our results show that ESH-L cells and primary fetal heart cells present the highest sensitivity for CaPVs growth and detection. Vero cells can replicate those viruses but without showing any CPE while the titer obtained on OA3.Ts is lower than primary and ESH-L cells. ESH-L cells are an effective alternative to primary cells use for growing Capripoxviruses and their diagnosis.

Value of long non-coding RNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy.

BACKGROUND: Esophageal cancer is a common digestive tract tumor that is generally treated with radiotherapy. Poor responses to radiotherapy in most patients generally result in local radiotherapy failure, so it is essential to find new radiosensitizers that can enhance the response of cancer cells to radiotherapy and improve the survival of esophageal cancer patients with radiation resistance. The long non-coding RNA (lncRNA) Rpph1 is highly expressed in human gastric cancer tissues, and represses breast cancer cell proliferation and tumorigenesis. However, the expression of lncRNA Rpph1 in esophageal cancer and its relationship with radio-sensitivity has not been studied. AIM: To explore the value of lncRNA Rpph1 in esophageal cancer and its effect on cancer cell sensitivity to radiotherapy. METHODS: Eighty-three patients with esophageal cancer admitted to Qilu Hospital of Shandong University and 90 healthy participants who received physical examinations were collected as research participants. The expression of Rpph1 was determined by qRT-PCR. siRNA-NC and siRNA-Rpph1 were transfected into esophageal cancer cell lines, and cells without transfection were designated as the blank control group. Cell survival was tested by colony formation assays, and the levels of proteins related to apoptosis and epithelial-mesenchymal transitions were determined by Western blot assays. Cell proliferation was assessed by MTT assays, cell apoptosis by flow cytometry, and cell migration by wound-healing assays. Changes in cell cycle distribution were monitored. RESULTS: Rpph1 was highly expressed in esophageal carcinoma, making it a promising marker for the diagnosis of esophageal cancer. Rpph1 could also be used to distinguish different short-term responses, T stages, N stages, and clinical stages of esophageal cancer patients. The results of 3-year overall survival favored patients with lower Rpph1 expression over patients with higher Rpph1 expression (P < 0.05). In vitro and in vivo experiments showed that silencing Rpph1 expression led to higher sensitivity of esophageal cancer cells to radiotherapy, stronger apoptosis in esophageal cancer cells induced by radiotherapy, higher expression of Bax and caspase-3, and lower expression of Bcl-2 (Bax, caspase-3, and Bcl-2 are apoptosis-related proteins). Additionally, silencing Rpph1 attenuated radiation-induced G2/M phase arrest, and significantly inhibited the expression of proteins involved in cell proliferation, migration, and epithelial-mesenchymal transition regulation in esophageal cancer cells. CONCLUSION: Rpph1 is highly expressed in esophageal cancer. Silencing Rpph1 expression can promote cell apoptosis, inhibit cell proliferation and migration, and increase radio-sensitivity.

Differentiated mitochondrial function in mouse 3T3 fibroblasts and human epithelial or endothelial cells in response to chemical exposure.

Mouse 3T3 fibroblasts are commonly used for in vitro toxicity testing; however, their sensitivity to stimuli is not well defined. To assess the sensitivity of the 3T3 cell line, the study compared the changes in mitochondrial membrane potential (MMP) occurring after exposure to eight chemicals known to demonstrate pro-apoptotic activity (glycerol, isopropanol, ethanol, paracetamol, propranolol, cobalt chloride, formaldehyde and atropine). Five cell lines were used as follows: mouse 3T3 fibroblasts, human epithelial cells (A549, Caco-2 and HepG2) and human endothelial cells (HMEC-1). Cell sensitivity was assessed based on the total area under and over the dose-response curves (AUOC) in relation to baselines. The 3T3 fibroblasts had the highest AUOC values and were the most sensitive to the action of all the examined chemicals, with the exception of formaldehyde. Significant changes in MMP between the 3T3 cell line and other cells were observed after cell treatment with atropine (A549, Caco-2 or HMEC-1 cells vs 3T3 cells, P < 0.05), propranolol (A549 vs 3T3 cells, P < 0.01; HepG2 vs 3T3 cells, P < 0.05), cobalt chloride (A549 cells vs 3T3 cells, P < 0.01) or ethanol (HMEC-1 vs 3T3, P < 0.05). Formaldehyde appeared the most toxic compound for Caco-2 cells (Caco-2 vs 3T3 cells, P < 0.05). The surface areas (AUOC) calculated for each other chemical and obtained for HepG2, Caco-2, A549 and HMEC-1 did not differ significantly between cell lines. We postulate that mouse 3T3 fibroblasts demonstrate significantly higher relative sensitivity to many agents with toxic potential.

A Brief Guide to Performing Pharmacological Studies In Vitro: Reflections from the EORTC-PAMM Course "Preclinical and Early-phase Clinical Pharmacology".

One aim of cell-based in vitro assays is to identify the best drug candidate to develop using the best tumor cell model. This is challenging in every anticancer drug discovery process. Briefly, we summarize the parameters to be taken into account when performing in vitro cell assays, in order to obtain reliable and reproducible results, which was fundamentally discussed by lecturers at the educational course on preclinical and early-phase clinical pharmacology studies, at the 40th Winter Meeting of the Pharmacology and Molecular Mechanisms Group of the European Organization for Research and Treatment of Cancer. Moreover, specific cellular sensitivity tests are described. In addition to monolayer in vitro cell models for the screening of new potential candidate drugs, three-dimensional tumor/cell tissue models are emerging as new pre-clinical tools that more closely reflect the in vivo microenvironment. Therefore, the use of different in vitro models for drug screening can enhance the predictability and reliability of pre-clinical drug-discovery phases and target validation.

Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence.

VeroNectin-4 is a highly sensitive cell line that can be used for the isolation and titration of Peste des Petits Ruminants virus.

Peste des Petits Ruminants virus (PPRV) is a member of the Morbillivirus subgroup of the family Paramyxoviridae, and is one of the most contagious diseases of small ruminants throughout Africa and the rest of the world. Different cell lines have previously been used to isolate PPRV but with limited success. Thus, to improve the isolation of Morbilliviruses, human, canine, and goat homologues of the lymphocyte receptor signaling lymphocyte activation molecule (SLAM) have been introduced into cells that can support virus replication. However, the amino acid sequence of SLAM varies between species, and often requires adaptation of a particular virus to different versions of the receptor. The protein sequence of Nectin-4 is highly conserved between different mammals, which eliminate the need for receptor adaptation by the virus. Cell lines expressing Nectin-4 have previously been used to propagate measles and canine distemper viruses. In this study, we compared infections in Vero cells expressing canine SLAM (VeroDogSLAM) to those in Vero cells expressing Nectin-4 (VeroNectin-4), following inoculations with wild-type strains of PPRV. Virus isolation using VeroNectin-4 cells was successful with 23% of swabbed samples obtained from live infected animals, and was 89% effective using post-mortem tissues of infected sheep. By contrast, only 4.5% efficiency was observed from swab samples and 67% efficiency was obtained in virus isolation from post-mortem tissues using VeroDogSLAM cells. The average incubation period for virus recovery from post-mortem tissues was 3.4 days using VeroNectin-4 cells, compared with 5.5 days when using VeroDogSLAM cells. The virus titers of PPRV obtained from VeroNectin-4 cells were also higher than those derived from VeroDogSLAM cells. A comparison of the growth kinetics for PPRV in the two cell lines confirmed the superiority of VeroNectin-4 cells for PPR diagnostic purposes and vaccine virus titration.

Advances in the understanding of Fanconi Anemia Complementation Group D2 Protein (FANCD2) in human cancer.

Fanconi anemia (FA) is a rare human genetic disease, resulting from dysfunction in any of 17 known complementation proteins: FANC-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, Q & S, and other unknowns. Besides the severe bone marrow failure, an extremely high incidence of cancer as well as many other clinic symptoms associated with FA patients, FA cells are known of insufficiency in homologous recombination, DNA mismatch repair, nucleotide excision repair, translesion DNA synthesis, and other molecular defects, leading to genome instability. Those similar molecular and cellular/tissue features show that all FA proteins function in one common signaling pathway, namely, the FA pathway. The monoubiquitination of FANCD2 is the central step of the FA pathway activation upon DNA damage or during DNA replication. The molecular functions of FANCD2 emerge as a very attractive filed of investigation in cancer research. Herein, we review the recent progresses in FANCD2 functions at these rapidly progressed aspects.