Biomedical subjects

Thomas G Cotter

Publications and source records attributed to Thomas G Cotter.

6 recordsLinked to original sources

PubMed2003

Caspase-independent photoreceptor apoptosis in mouse models of retinal degeneration.

Apoptosis is the mode of cell death in retinitis pigmentosa, a group of retinal degenerative disorders primarily affecting rod photoreceptors. Although caspases have been demonstrated to play a central role in many incidences of apoptosis, accumulating evidence suggests that they may not be required for all forms of apoptotic cell death. The present study examined the mechanism of cell death in two in vivo models of photoreceptor apoptosis: the retinal degeneration (rd) mouse, a naturally occurring mutant model, and N-methyl-N-nitrosourea-induced retinal degeneration. Specifically, we examined the activation status of caspase-9, -8, -7, -3, and -2 and determined the caspase requirements for cytochrome c release, DNA fragmentation, and apoptosis-associated proteolysis of specific caspase substrates. We show that apoptosis in both in vivo models is independent of caspase-9, -8, -7, -3, and -2 activation. DNA fragmentation occurs in the absence of caspase-mediated ICAD (inhibitor of caspase-activated DNase) proteolysis, suggesting that an alternative endonuclease is responsible for DNA cleavage in these models. Importantly, we show that apoptosome activation is prevented because of an absence of mitochondrial cytochrome c release. Experiments performed using a cell-free system indicate that cytochrome c-dependent proteolysis and activation of caspase-9 can be restored in a neonatal cell-free system. However, we found that cytochrome c-dependent proteolysis and activation of caspase-9 could not be restored in an adult cell-free system because of an age-related decrease in the expression of Apaf-1 in the normal developing mouse retina. In the rd mouse, however, this age-related downregulation of apoptotic proteins was not observed, highlighting a critical feature of this model and the prevention of cytochrome c release as an apical event in caspase-independent apoptosis in this system.

Animals↗

PubMed2003

Targeting inflammatory diseases via apoptotic mechanisms.

Induction of apoptosis in immune cells is a crucial mechanism used by the body to produce immune resolution. The homeostatic mechanisms employed are currently being identified and, to date, studies have highlighted some of the signals that regulate the immune response. The exposure of phosphatidylserine on the surface of an apoptotic neutrophil is sufficient to limit the immune response in acute inflammation, whereas apoptosis of key effector cells can limit the response in chronic inflammation. Other therapeutic approaches that are being investigated include the inhibition of apoptosis by blocking the caspase cascade. This approach will be of particular relevance for the treatment of inflammatory central nervous system diseases and sepsis. An alternative approach being examined is forced resolution, whereby apoptosis is induced in effector cells, principally T cells, through activation-induced cell death mediated by Fas receptors. Inhibitors of this mechanism have been identified and targeted in several studies.

Animals↗

PubMed2002

Regulation and measurement of oxidative stress in apoptosis.

Cells are constantly generating reactive oxygen species (ROS) during aerobic metabolism. As a consequence, each cell is equipped with an extensive antioxidant defence system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cell's natural antioxidant defences. There is a growing consensus that oxidative stress and the redox state of a cell plays a pivotal role in regulating apoptosis, a tightly controlled form of cell death in which a cell partakes in its own demise. More recently, a role for reactive nitrogen species (RNI) as both positive and negative regulators of cell death has been established. This review describes the major sources of ROS and RNI in a cell, the control of cell death by these species and the role of antioxidants as regulators of oxidative stress and apoptosis. Finally, the various methods that can be employed in establishing a role for both ROS and RNI in apoptosis will be discussed with particular emphasis on their intracellular detection.

Animals↗

PubMed2002

Erythrocyte survival is promoted by plasma and suppressed by a Bak-derived BH3 peptide that interacts with membrane-associated Bcl-X(L).

Erythrocytes have a defined lifespan in vivo, and the signals that maintain their survival in circulation or trigger their death are unknown. Here, we investigated the control of erythrocyte survival and death in an in vitro culture system where erythrocytes survived for 10 days in serum-free medium in the presence or absence of bovine serum. Death of the cells in culture was correlated with increased exposure of phosphatidylserine and increased levels of intracellular calcium. Cell death could be suppressed by supplementing the medium with human plasma or serum, resulting in a doubling of the lifespan to 20 days. Freshly isolated erythrocytes and cultured erythrocytes were both found to express Bcl-X(L) and, to a lesser extent, Bak in membrane protein extracts. Treatment of the cells with a Bak-derived BH3 peptide fused to the internalization sequence of the antennapedia protein, which has previously been shown to enter cells by diffusion and antagonize Bcl-X(L), resulted in substantial cell death in erythrocyte cultures. BH3-induced death was accompanied by an immediate increase in accumulation of intracellular calcium and could be suppressed by plasma, but not by the caspase inhibitor zVAD. A BH3 peptide mutated at amino acid 78 of full-length Bak required for heterodimerization with Bcl-X(L) had no effect on cell viability or calcium levels. We conclude that the BH3 peptide accelerates erythrocyte death through antagonization of Bcl-X(L). The data suggest that erythrocyte survival is promoted by survival factors in plasma and by membrane-associated Bcl-X(L.)

Cell Culture Techniques↗

PubMed2002

p53 expression in K562 cells is associated with caspase-mediated cleavage of c-ABL and BCR-ABL protein kinases.

The chimaeric BCR-ABL oncoprotein is the molecular hallmark of chronic myeloid leukaemia (CML). Expression of Bcr-Abl has been associated with arrested differentiation as well as resistance to apoptosis. The downstream pathway involved in apoptosis resistance has been extensively studied, whereas the role of Bcr-Abl in cell differentiation is largely unclear. A recent report has shown that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage-committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. In accordance with this model, downregulation of c-Abl and Bcr-Abl has been observed during differentiation in different systems, although the mechanism is still largely unknown. To investigate the relationship between erythroid differentiation and c-Abl and Bcr-Abl levels, we induced differentiation in K562 cells using a temperature-inducible p53 mutant (p53Val1335). It was found that p53-induced erythroid differentiation in K562 cells required caspase activity. During this process, caspase-dependent cleavage of c-Abl and Bcr-Abl tyrosine kinases was observed, suggesting a new mechanism for the downregulation of the kinases during erythroid differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Between-group analysis of microarray data.

MOTIVATION: Most supervised classification methods are limited by the requirement for more cases than variables. In microarray data the number of variables (genes) far exceeds the number of cases (arrays), and thus filtering and pre-selection of genes is required. We describe the application of Between Group Analysis (BGA) to the analysis of microarray data. A feature of BGA is that it can be used when the number of variables (genes) exceeds the number of cases (arrays). BGA is based on carrying out an ordination of groups of samples, using a standard method such as Correspondence Analysis (COA), rather than an ordination of the individual microarray samples. As such, it can be viewed as a method of carrying out COA with grouped data. RESULTS: We illustrate the power of the method using two cancer data sets. In both cases, we can quickly and accurately classify test samples from any number of specified a priori groups and identify the genes which characterize these groups. We obtained very high rates of correct classification, as determined by jack-knife or validation experiments with training and test sets. The results are comparable to those from other methods in terms of accuracy but the power and flexibility of BGA make it an especially attractive method for the analysis of microarray cancer data.

Algorithms↗