Biomedical subjects

Beth Coyle

Publications and source records attributed to Beth Coyle.

5 recordsLinked to original sources

PubMed2005

Strategies to investigate gene expression and function in granule cells.

Studying gene expression in granule cells is a major route to understanding the factors required for many key cellular processes such as specification, proliferation, migration, differentiation, apoptosis, tumour formation and neurodegeneration. A greater understanding of these processes will not only provide insight into cerebellum development, but also diseases of the cerebellum. Granule cells can be readily grown in culture and both viral and non-viral strategies have been optimised to allow gene transfer and expression in cultured cells. However, granule cell migration and maturation are inherent parts of cerebellum development and these rely on interactions with other cells. Hence, a true picture of gene function in these cells can only be obtained when tissue context is maintained. Studies of gene function in this context can be achieved by creation of mouse models. Conditional mouse models, where loss of gene expression is restricted as far as possible to granule cells, are by far the most informative resource in this respect. Despite their obvious benefits, the production of mouse models is both costly and time-consuming and this may be further compounded by a potential lack of phenotype due to redundancy of gene function. Organotypic slice cultures, on the other hand, are a comparatively cheap and accessible model for studies of gene function where tissue context is maintained. Recent technologies have provided the means to manipulate gene expression in such systems and are beginning to yield valuable insights into the molecular regulation of cerebellum development.

Animals↗

PubMed2004

Regulation of apoptosis by peroxisome proliferators.

Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours.

Animals↗

PubMed2004

Novel strategy to study gene expression and function in developing cerebellar granule cells.

The advent of techniques for global analyses of cell biology, such as genomics and proteomics, opens the way to rapid progress in understanding the molecular control of developing tissues. However, such studies in the CNS are hindered by the complexity of this tissue. In particular, few approaches allow cells to be isolated that are enriched for specific stages of their maturation. We describe a new strategy to study gene expression and function in cerebellar granule cells. In these experiments, we have used square pulse electroporation to introduce fluorescent dye or DNA constructs into immature granule cell precursors in situ. This method only labels granule cell precursors in the superficial part of the external granule layer. Combining this labelling with fluorescent activated cell sorting (FACS) allows the transfected cells to be isolated at any time during their subsequent development, thus providing a means of analysing granule cells as they undergo maturation. This transfection method can be used to study events in the normal maturation of granule cells or the effects of introduced transgenes. Such studies can be carried out on cells purified from primary cultures or cells in situ using cerebellar slice cultures. Our strategy provides a new route to detailed analysis of the role of genes in controlling many aspects of granule cell biology. These approaches will allow recent global analyses to be more readily applied to subpopulations of cells in complex tissues.

Animals↗

PubMed2003

Early drug safety evaluation: biomarkers, signatures, and fingerprints.

When target organ toxicity arises in animal models during routine drug safety evaluation, it raises several key questions: Is this target organ toxicity related to the pharmacology? What is the mode of action (MOA)? Is the target organ toxicity relevant to humans? Pathology or prior knowledge of the compound class may provide clues on a possible MOA for toxicity. However, if this deductive approach yields no results, the inductive approach offered by new technologies can generate novel research leads. For example, toxicogenomics can generate a gene expression profile of the toxicity that can be compared with reference compounds or with other candidate drugs. Similarly, proteomic analysis of the protein profile at the toxic vs. the efficacious dose can provide clues on MOA for the toxicity and may allow differentiation of the pathways of the toxic response from those required for pharmacological activity.

Animals↗

PubMed2002

Characterization of the transforming growth factor-beta 1-induced apoptotic transcriptome in FaO hepatoma cells.

We have previously shown that transforming growth factor-beta(1) (TGF-beta(1))-induced apoptosis in FaO hepatoma cells is mediated by cytochrome c release, apoptosome formation, and caspase activation. Although TGF-beta(1) acts via the SMAD signaling pathway to initiate de novo gene transcription, little is known about the downstream gene targets that are involved in the regulation of apoptosis. Therefore, in this study, we used in-house microarrays (approximately 5500 genes) to identify pathway-specific gene clustering in TGF-beta(1)-treated cells. A total of 142 genes showed time-dependent changes in expression during TGF-beta(1)-induced apoptosis. The polycaspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone, which, on its own, had no effect on gene transcription, blocked TGF-beta(1)-induced cell death and significantly altered the expression of 261 genes, including 185 down-regulated genes. Cluster analysis identified up-regulation of early response genes (0-4 h) encoding for the extracellular matrix and cytoskeleton, including the pro-apoptotic CTGF gene, and delayed response genes (8-16 h), including pro-apoptotic genes. A second delayed response cluster (44 genes) was also observed when TGF-beta(1)-induced caspase activation was blocked by benzyloxycarbonyl-VAD-fluoromethyl ketone. This cluster included genes encoding stress-related proteins (e.g. Jun, ATF3, TAB1, and TANK), suggesting that their up-regulation may be in response to secondary necrosis. Finally, we identified an early response set of nine down-regulated genes that are involved in antioxidant defense. We propose that the regulation of these genes by TGF-beta(1) could provide a molecular mechanism for the observed elevation in reactive oxygen species after TGF-beta(1) treatment and may represent the primary mechanism through which TGF-beta(1) initiates apoptosis.

Amino Acid Chloromethyl Ketones↗