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C Stephen Downes

Publications and source records attributed to C Stephen Downes.

15 recordsLinked to original sources
PubMed2006

Computational methodologies for modelling, analysis and simulation of signalling networks.

This article is a critical review of computational techniques used to model, analyse and simulate signalling networks. We propose a conceptual framework, and discuss the role of signalling networks in three major areas: signal transduction, cellular rhythms and cell-to-cell communication. In order to avoid an overly abstract and general discussion, we focus on three case studies in the areas of receptor signalling and kinase cascades, cell-cycle regulation and wound healing. We report on a variety of modelling techniques and associated tools, in addition to the traditional approach based on ordinary differential equations (ODEs), which provide a range of descriptive and analytical powers. As the field matures, we expect a wider uptake of these alternative approaches for several reasons, including the need to take into account low protein copy numbers and noise and the great complexity of cellular organisation. An advantage offered by many of these alternative techniques, which have their origins in computing science, is the ability to perform sophisticated model analysis which can better relate predicted behaviour and observations.

Algorithms↗
PubMed2006

Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.

There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region-specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3 micromol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion-induced molecular changes.

Azacitidine↗
PubMed2005

Survival trees for analyzing clinical outcome in lung adenocarcinomas based on gene expression profiles: identification of neogenin and diacylglycerol kinase alpha expression as critical factors.

We present survival trees as an exploratory tool for revealing new insights into gene expression profiles in combination with clinical patient data. Survival trees partition the patient data studied into groups with similar survival outcomes and identify characteristic genetic profiles within these groups. We demonstrate the application of survival trees in a study involving the expression profiles of 3,588 genes in 211 lung adenocarcinoma patients. The survival tree identified a group of early-stage cancer patients with relatively low survival rates and another group of advanced-stage patients with remarkably good survival outcome. For both groups, the tree identified characteristic expression profiles of genes that might play a role in cancerogenesis and disease progression, notably the genes for the netrin receptor neogenin and the Ras/Rho kinase modulator diacylglycerol kinase alpha.

Adenocarcinoma↗
PubMed2005

Mathematical models of cell cycle regulation.

The cell division cycle is a fundamental process of cell biology and a detailed understanding of its function, regulation and other underlying mechanisms is critical to many applications in biotechnology and medicine. Since a comprehensive analysis of the molecular mechanisms involved is too complex to be performed intuitively, mathematical and computational modelling techniques are essential. This paper is a review and analysis of recent approaches attempting to model cell cycle regulation by means of protein-protein interaction networks.

Algorithms↗
PubMed2004

Emerging diet-related surrogate end points for colorectal cancer: UK Food Standards Agency diet and colonic health workshop report.

The UK Food Standards Agency convened a group of expert scientists to review current research investigating emerging diet-related surrogate end points for colorectal cancer (CRC). The workshop aimed to overview current research and establish priorities for future research. The workshop considered that the validation of current putative diet-related surrogate end points for CRC and the development of novel ones, particularly in the emerging fields of proteomics, genomics and epigenomics, should be a high priority for future research.

Biomarkers, Tumor↗
PubMed2003

Cell cycle checkpoint function in bladder cancer.

BACKGROUND: Cell cycle checkpoints function to maintain genetic stability by providing additional time for repair of DNA damage and completion of events that are necessary for accurate cell division. Some checkpoints, such as the DNA damage G1 checkpoint, are dependent on p53, whereas other checkpoints, such as the decatenation G(2) checkpoint, are not. Because bladder transitional cell carcinomas (TCCs) often contain numerous chromosomal aberrations and appear to have highly unstable genomes, we analyzed cell cycle checkpoint functions in a panel of TCC lines. METHODS: Cell cycle arrest was induced in normal human fibroblasts (NHF1-hTERT) and normal human uroepithelial cells (HUCs), and TCC lines and checkpoint functions were quantified using flow cytometry and fluorescence microscopy. The inducers and checkpoints were ionizing radiation (i.e., DNA damage) (G1 and G2 checkpoints), the mitotic inhibitor colcemid (polyploidy checkpoint), or the topoisomerase II catalytic inhibitor ICRF-193 (decatenation G2 checkpoint). Four of the five TCC lines expressed mutant p53. RESULTS: HUCs had an effective G1 checkpoint response to ionizing radiation, with 68% of cells inhibited from moving from G1 into S phase. By contrast, G1 checkpoint function was severely attenuated (<15% inhibition) in three of the five TCC lines and moderately attenuated (<50% inhibition) in the other two lines. NHF1-hTERT had an effective polyploidy checkpoint response, but three of five TCC lines were defective in this checkpoint. HUCs had effective ionizing radiation and decatenation G2 checkpoint responses. All TCC lines had a relatively effective G2 checkpoint response to DNA damage, although the responses of two of the TCC lines were moderately attenuated relative to HUCs. All TCC lines had a severe defect in the decatenation G2 checkpoint response. CONCLUSION: Bladder TCC lines have defective cell cycle checkpoint functions, suggesting that the p53-independent decatenation G2 checkpoint may cooperate with the p53-dependent G1 checkpoints to preserve chromosomal stability and suppress bladder carcinogenesis.

Carcinoma, Transitional Cell↗
PubMed2003

Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells.

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.

Cell Line, Tumor↗
PubMed2003

Variation in sequence-specific repair of UV damage in human pericentromeric heterochromatin of different cell lines.

Damaged nucleotides are removed from the condensed non-coding, or transcriptionally inactive regions of the genome by the relatively slow global genome repair system. Since few data are available for the repair of the pericentric heterochromatin region our aim was to study the repair of a specific sequence, known to be located in this region. We applied a PCR based method to monitor UV damage and repair in chAB4, a human pericentromeric heterochromatin sequence in 10 human cell lines. We here present evidence that excision repair of a sequence in the pericentromeric heterochomatin also varies between cell lines in a manner inconsistent with the canonical model. In some cell lines repair rates were efficient in heterochromatin, comparable to transcription coupled repair, but in some tumour-derived and repair-deficient cell lines we have detected deficient repair.

Butyrates↗
PubMed2003

Zinc supplementation has no effect on circulating levels of peripheral blood leucocytes and lymphocyte subsets in healthy adult men.

As a result of evidence documenting harmful effects of Zn supplementation on immune function and Cu status, thirty-eight men were recruited onto a Zn supplementation trial. The aim was to examine the effects of chronic Zn supplementation on circulating levels of peripheral blood leucocytes and lymphocyte subsets. Subjects (n 19) took 30 mg Zn/d for 14 weeks followed by 3 mg Cu/d for 8 weeks to counteract adverse effects, if any, of Zn supplementation on immune status resulting from lowered Cu status. A control group (n 19) took placebo supplements for the duration of the trial. Dietary intakes of Zn approximated 10 mg/d. Blood samples, taken throughout the trial, were assessed for full blood profiles and flow cytometric analyses of lymphocyte subsets. Putative indices of Cu status were also examined. Results indicate that there was no effect of Zn supplementation on circulating levels of peripheral blood leucocytes or on lymphocyte subsets. Cu status was also unaltered. Independent of supplement, there appeared to be seasonal variations in selected lymphocyte subsets in both placebo and supplemented groups. Alterations in circulating levels of B cells (cluster of differentiation (CD) 19), memory T cells (CD45RO) and expression of the intracellular adhesion molecule-1 (CD54) on T cells were observed. Findings indicated no adverse effects of Zn supplementation on immune status or Cu status and support the US upper level of Zn tolerance of 40 mg/d. The seasonal variations observed in lymphocyte subsets in the group as a whole could have implications for seasonal variability in the incidence of infectious diseases.

Adult↗
PubMed2003

Zinc supplementation has no effect on lipoprotein metabolism, hemostasis, and putative indices of copper status in healthy men.

Pharmacological doses of zinc can adversely affect body copper status. The resulting copper deficiency can impact directly upon cholesterol metabolism and a suboptimal copper status has been observed to influence markers of hemostasis (specifically fibrinogen and the copper-containing coagulation factors V and VIII). The aim of this investigation was to examine the effect of a low level of zinc supplementation, to include dietary intake, at the United States tolerable upper intake level of 40 mg/d upon indicators of lipid metabolism, hemostasis, and copper. Thirty-eight subjects were recruited onto a double-blind placebo-controlled intervention trial and randomly selected to one of two groups. Group 1 took zinc supplements (30 mg/d) for 14 wk followed by copper supplements (3 mg/d) for 8 wk (to counteract adverse effects, if any, of zinc supplementation). A second group took placebo supplements for the full duration of the trial. Estimated dietary zinc intake approximated 10 mg/d. The effect of supplement was analyzed by repeated-measures analysis of variance (anova). Results indicate that no effect of zinc supplementation on putative indices of copper status, lipoprotein metabolism, and markers of hemostasis. These results indicate that short-term low-level zinc supplementation (total intake 40 mg/d) is not detrimental to health.

Adult↗
PubMed2003

Multiclass cancer classification using gene expression profiling and probabilistic neural networks.

Gene expression profiling by microarray technology has been successfully applied to classification and diagnostic prediction of cancers. Various machine learning and data mining methods are currently used for classifying gene expression data. However, these methods have not been developed to address the specific requirements of gene microarray analysis. First, microarray data is characterized by a high-dimensional feature space often exceeding the sample space dimensionality by a factor of 100 or more. In addition, microarray data exhibit a high degree of noise. Most of the discussed methods do not adequately address the problem of dimensionality and noise. Furthermore, although machine learning and data mining methods are based on statistics, most such techniques do not address the biologist's requirement for sound mathematical confidence measures. Finally, most machine learning and data mining classification methods fail to incorporate misclassification costs, i.e. they are indifferent to the costs associated with false positive and false negative classifications. In this paper, we present a probabilistic neural network (PNN) model that addresses all these issues. The PNN model provides sound statistical confidences for its decisions, and it is able to model asymmetrical misclassification costs. Furthermore, we demonstrate the performance of the PNN for multiclass gene expression data sets. Here, we compare the performance of the PNN with two machine learning methods, a decision tree and a neural network. To assess and evaluate the performance of the classifiers, we use a lift-based scoring system that allows a fair comparison of different models. The PNN clearly outperformed the other models. The results demonstrate the successful application of the PNN model for multiclass cancer classification.

Artificial Intelligence↗
PubMed2002

ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase.

An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.

Alleles↗
PubMedDate not supplied

Elevated levels of oxidative DNA damage in lymphocytes from patients with Alzheimer's disease.

Previous studies have provided evidence of the involvement of oxidative damage in the pathogenesis of Alzheimer's disease (AD). Although the role of oxidative stress in the aetiology of the disease is still not clear, the detection of an increased damage status in the cells of patients could have important therapeutic implications. The level of oxidative damage and repair capacity in peripheral lymphocytes of AD patients and of age-matched controls was determined by the Comet assay applied to freshly isolated blood samples with oxidative lesion-specific DNA repair endonucleases. This is less prone to errors arising from oxidative artifacts than chemical analytical methods; and is therefore a relatively reliable, as well as rapid method for assay of oxidative DNA damage in cells. Statistically significant elevations (P < 0.05) of oxidized purines were observed in nuclear DNA of peripheral lymphocytes from AD patients, compared to age matched control subjects, both at basal level and after oxidative stress induced by H(2)O(2.) AD patients also showed a diminished repair of H(2)O(2) -induced oxidized purines.

Aged↗
PubMedDate not supplied

Degradation of ATM-independent decatenation checkpoint function in human cells is secondary to inactivation of p53 and correlated with chromosomal destabilization.

DNA topoisomerase II is required in the cell cycle to decatenate intertwined daughter chromatids prior to mitosis. To study the mechanisms that cells use to accomplish timely chromatid decatenation, the activity of a catenation-responsive checkpoint was monitored in human skin fibroblasts with inherited or acquired defects in the DNA damage G2 checkpoint. G2 delay was quantified shortly after a brief incubation with ICRF-193, which blocks the ability of topoisomerase II to decatenate chromatids, or treatment with ionizing radiation (IR), which damages DNA. Both treatments induced G2 delay in normal human fibroblasts. Ataxia telangiectasia fibroblasts with defective G2 checkpoint response to IR displayed normal G2 delay after treatment with ICRF-193, demonstrating that ATM kinase was not required for signaling when chromatid decatenation was blocked. The G2 delay induced by ICRF-193 was reversed by caffeine, indicating that active checkpoint signaling was involved. ICRF-193-induced G2 delay also was independent of p53 function, being evident in cells expressing HPV16E6 to inactivate p53. However, as fibroblasts expressing HPV16E6 aged in culture, they lost the ability to delay entry to mitosis, both after DNA damage and when decatenation was blocked. This age-related loss of G2 delay in response to ICRF-193 and IR in E6-expressing cells was blocked by induction of telomerase. Expression of telomerase also prevented chromosomal destabilization in aging E6-expressing cells. These observations lead to a new model of genetic instability, in which attenuation of G2 decatenatory checkpoint function permits cells to enter mitosis with insufficiently decatenated chromatids, leading to aneuploidy and polyploidy.

Ataxia Telangiectasia Mutated Proteins↗