7 research outputs found

    Microarray analysis distinguishes differential gene expression patterns from large and small colony Thymidine kinase mutants of L5178Y mouse lymphoma cells

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    BACKGROUND: The Thymidine kinase (Tk) mutants generated from the widely used L5178Y mouse lymphoma assay fall into two categories, small colony and large colony. Cells from the large colonies grow at a normal rate while cells from the small colonies grow slower than normal. The relative proportion of large and small colonies after mutagen treatment is associated with a mutagen's ability to induce point mutations and/or chromosomal mutations. The molecular distinction between large and small colony mutants, however, is not clear. RESULTS: To gain insights into the underlying mechanisms responsible for the mutant colony phenotype, microarray gene expression analysis was carried out on 4 small and 4 large colony Tk mutant samples. NCTR-fabricated long-oligonucleotide microarrays of 20,000 mouse genes were used in a two-color reference design experiment. The data were analyzed within ArrayTrack software that was developed at the NCTR. Principal component analysis and hierarchical clustering of the gene expression profiles showed that the samples were clearly separated into two groups based on their colony size phenotypes. The Welch T-test was used for determining significant changes in gene expression between the large and small colony groups and 90 genes whose expression was significantly altered were identified (p < 0.01; fold change > 1.5). Using Ingenuity Pathways Analysis (IPA), 50 out of the 90 significant genes were found in the IPA database and mapped to four networks associated with cell growth. Eleven percent of the 90 significant genes were located on chromosome 11 where the Tk gene resides while only 5.6% of the genes on the microarrays mapped to chromosome 11. All of the chromosome 11 significant genes were expressed at a higher level in the small colony mutants compared to the large colony mutants. Also, most of the significant genes located on chromosome 11 were disproportionally concentrated on the distal end of chromosome 11 where the Tk mutations occurred. CONCLUSION: The results indicate that microarray analysis can define cellular phenotypes and identify genes that are related to the colony size phenotypes. The findings suggest that genes in the DNA segment altered by the Tk mutations were significantly up-regulated in the small colony mutants, but not in the large colony mutants, leading to differential expression of a set of growth regulation genes that are related to cell apoptosis and other cellular functions related to the restriction of cell growth

    The application of gene expression profiling in the characterization of physiological effects of genetically modified feed components in rats

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    The study was conducted to evaluate the adequacy of expression profiling for the characterization of potential physiological side effects of genetically modified (GM) feed components. Feeding experiments with rats fed either GM or non-GM feed components were conducted and a comparative expression profiling, using DNA-chip-technology, was done. As a prerequisite for these expression studies data analysis parameters were optimized. Diet-associated expression differences between the two feeding groups were observed in spleen, small intestine and liver. It was shown that expression profiling provides great sensitivity in monitoring physiological reactions of an organism to such diets.In der vorliegenden Arbeit wurde die Eignung von "Genexpressionsprofiling" zur Charakterisierung physiologischer Nebeneffekte genetisch veränderter (GV) Futtermittel untersucht. In Fütterungsversuchen wurden Ratten entweder GV- oder Nicht-GV-Futterkomponenten verabreicht und ihre Expressionsprofile mittels DNA-chip-Technologie verglichen. Als Vorraussetzung für diese Expressionsanalysen erfolgte vorab eine Optimierung der Datenauswertung. In Milz, Leber und Dünndarm ergaben sich Diät assoziierte Expressionsunterschiede zwischen den Fütterungsgruppen. Insgesamt zeigte sich, dass „Genexpressionsprofiling“ eine hohe Sensitivität zur Erfassung physiologischer Reaktionen eines Organismus auf solche Diäten ermöglicht

    Validation and application of human in vitro models for investigating bronchial response to cigarette smoke

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    Cigarette smoke (CS) is the single most deadly and preventable cause of death. It profoundly affects smokers’ lungs, by altering genes expression profiles, epigenetic modifications, causes DNA damage and changes cell function and morphology [1]. The respiratory tract is the main lung compartment exposed to CS. The bronchial epithelium lining the airway tracts subjected to chronic smoking often leads to incurable diseases such as lung cancers or chronic obstructive pulmonary disease (COPD). Due to its complicated nature and profound impact on human health, the response to CS has been extensively studied in both in vivo and in vitro settings. While CS effects can be investigated in numerous distinct experimental set-ups, little has been done in terms of standardization or validation of these methods. Here, a comprehensive direct comparison between different in vitro CS exposures has been established and analyzed. The cell-delivered dose and the expression profile of genes typically upregulated among smokers were assessed between models, also in relation to expression profile in human lungs. Three surprisingly dissimilar models, namely acute submerged basal cells exposure to cigarette smoke extract (CSE), chronic basolateral CSE ex-posure and acute whole cigarette smoke exposure, yielded responses that were substantially better than any other investigated experimental set-up. Despite the cell-delivered doses varying substantially between these three models, each of them significantly upregulated at least six of out 10 analyzed genes in the primary human bronchial epithelial cells (phBECs). Conclusions from validation study helped choosing the right model, which was later used in the next study, in the proteomic differential expression analysis. Chronic basolateral CSE exposure was the only model that successfully upregulated seven out of 10 genes typically upregulated in smokers. Results of the proteomic analysis further validated the physiological relevance of the model by identifying activation of the molecular pathways characteristic for the CS exposure, such as activation of xenobiotic metabolism pathways and inhibited sirtuin 1 pathway [2]. Interestingly, by using advanced pathway analysis software, a new potential ferroptotic regulator was found, namely nuclear factor 1 (NUPR1). Overall, this study reported a first evidence of critical ferroptosis repressor being aberrantly changed by the CS in phBECs derived from healthy donors. The final topic of this study addressed the question whether the differentiated phBECs from ex- and current smokers exhibit transient and persistent changes caused by smoking that can be seen in vivo. Here, the advantage was taken from proteomic study performed on bronchoalveolar lavage samples, which were derived from never-, ex- and current smokers. After identifying sev-eral genes which expression changes were either transient or persistent after smoking cessation, the basal expression levels of these genes was analyzed on transcript level in differentiated phBECs in vitro, derived also from never-, ex- and current smokers. Surprisingly, the in vitro anal-ysis revealed lower constitutive expression of analyzed genes in phBECs from patients from his-tory of smoking, which did not reflect changes seen in BALF study. Taken together, this thesis presents a successful validation of the several CS exposure models on phBECs. One of them, namely chronic basolateral CSE exposure, was further validated by the proteomic analysis, which, for the first time, revealed NUPR1, a crucial ferroptosis regulator [3], as a CS-regulated gene. This study establishes practical technique of validating CS exposure models, which can be used in in vitro studies, despite possibly different basal genes expressions of CS-regulated genes

    Development of the high throughput mammalian PIG-A gene mutation assay in vitro.

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    The field of genetic toxicology has recently undergone reform which has limited or banned the use of animal models within a number of different industries (cosmetics). Consequently, greater emphasis has been placed on developing novel, highly sensitive, in vitro test systems which can generate robust data to aid regulatory hazard and risk assessment.The main aims of this project were i) to develop a highly sensitive and specific, high throughput mammalian in vitro PIG-A gene mutation assay to enable quantitative dose response modelling and further investigate the potential use of in vitro data within human health assessment, ii) Investigate the genotype to phenotype relationship, a potentially delaying step within future OECD guideline drafting for the current in-vivo Pig-a mutation assay and iii) help develop and optimise a preliminary comprehensive human PIG-A bio­monitoring platform.During in-vitro and ex-vivo PIG-A assay development, flow cytometry was the fundamental technique utilised. Multiple additional laser excitation platforms were evaluated for use, including Amnis ImageStream ™ and laser scanning confocal. Proteomic as well as genomic techniques were used during the supplementary investigations surrounding assay development, with microbiological groundings throughout.The finalised in-vitro assay protocol was established within human, metabolically active, MCL-5 cells. Using the refined assay design, proof of principle experimentations were able to show the potential for future quantitative work and the general promise with this novel approach. The genotype to phenotype relationship validation is currently still on-going following the preliminary work described herein and recent publications. The ex-vivo human PIG-A assay platforms were shown to require further optimisation in terms of sensitivity, excluding red blood cells, but showed good aptitude for future use.Currently it looks promising that further refinement could lead to a comprehensive high content, high-throughput assay system with the potential to be used within future hazard and risk assessmen

    Skin Tissue Models

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    Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio
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