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Biomedical subjects

G C O'Sullivan

Publications and source records attributed to G C O'Sullivan.

At least 19 recordsLinked to original sources
PubMed2001

Constitutive ERK1/2 activation in esophagogastric rib bone marrow micrometastatic cells is MEK-independent.

In this study, we examined the mitogen-activated protein kinase (MAPK) cascade in micrometastatic cell lines generated from rib bone marrow (RBM) of patients undergoing resection of esophagogastric malignancies. The molecular mechanism(s) involved in esophagogastric MAPK activation have not previously been investigated. Constitutive activation of both ERK1 and -2 isoforms was evident in each of the five RBM cell lines. Elk-1, a transcription factor activated by the ERK1/2 pathway was also found to be constitutively activated. Cell lines generated from metastases of involved lymph nodes (OC2) and ascites (OC1) of patients with esophageal cancer do not display, however, hyperphosphorylation of ERK1/2. Constitutive RBM ERK1/2 activation is protein kinase C and phosphatidylinositol 3-kinase dependent. Surprisingly, constitutive ERK1/2 activation is MEK-independent. Pharmacological inhibition of MEK with two specific inhibitors, PD 98059 and U0126, were both ineffective in blocking ERK activation. Similarly, the use of a dominant negative MEK mutant was without effect. Interestingly, experiments overexpressing two different dominant negative Pak1 mutants significantly reduced RBM ERK1/2 activation, albeit not to the same extent for all cell lines. We also examined the role of three different phosphatases, PAC1, MKP-1, and -2. While RBM ERK1/2 activation was found to be PAC1- and MKP-2-independent, surprisingly, MKP-1 was down-regulated in all five RBM cell lines. In conclusion, we provide evidence for the first time for a MEK-independent constitutive ERK1/2 activation pathway in esophagogastric RBM cell lines. These findings have important implications for drug treatment strategies which currently target MEK in other forms of cancer.

Base Sequence↗
PubMed2001

Immune privilege or inflammation? Insights into the Fas ligand enigma.

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.

Fas Ligand Protein↗
PubMed2001

Probiotics.

Explore the source record for details and available documents.

Animals↗
PubMed2001

In vitro selection criteria for probiotic bacteria of human origin: correlation with in vivo findings.

The enteric flora comprises approximately 95% of the total number of cells in the human body and can elicit immune responses while protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract may also be implicated in the pathogenesis of diseases such as inflammatory bowel disease (ulcerative colitis and Crohn disease). The objectives of the Probiotic Research Group based at University College Cork were to isolate and identify lactic acid bacteria exhibiting beneficial probiotic traits, such as bile tolerance in the absence of deconjugation activity, acid resistance, adherence to host epithelial tissue, and in vitro antagonism of pathogenic microorganisms or those suspected of promoting inflammation. To isolate potentially effective probiotic bacteria, we screened the microbial population adhering to surgically resected segments of the gastrointestinal tract (the environment in which they may subsequently be reintroduced and required to function). In total, 1500 bacterial strains from resected human terminal ilea were assessed. From among these organisms, Lactobacillus salivarius subsp. salivarius strain UCC118 was selected for further study. In mouse feeding trials, milk-borne L. salivarius strain UCC118 could successfully colonize the murine gastrointestinal tract. A human feeding study conducted in 80 healthy volunteers showed that yogurt can be used as a vehicle for delivery of strain UCC118 to the human gastrointestinal tract with considerable efficacy in influencing gut flora and colonization. In summary, we developed criteria for in vitro selection of probiotic bacteria that may reflect certain in vivo effects on the host such as modulation of gastrointestinal tract microflora.

Animals↗
PubMed2001

Fas ligand upregulation is an early event in colonic carcinogenesis.

BACKGROUND/AIMS: Fas ligand (FasL) is a mediator of apoptosis via the Fas receptor (Fas/CD95/APO-1). Normal colonic epithelium expresses Fas, and appears to be relatively sensitive to Fas mediated apoptosis. Colonic adenocarcinomas coexpress FasL and Fas without undergoing widespread apoptosis. This study investigates the expression of FasL in colonic carcinogenesis from the earliest stages of the adenoma-carcinoma sequence. METHODS: FasL expression was determined in colonic adenomas (n = 38) of varying degrees of dysplasia and histological type by immunohistochemistry. Adenomas that contained areas of carcinomatous change were included (n = 12 of 38). Normal colonic epithelium (n = 10), hyperplastic polyps (n = 8), and serrated adenomas (n = 3) from patients without colonic adenocarcinomas were used for comparison. Cell death was detected in situ in adenomas using TUNEL (terminal transferase mediated dUTP nick end labelling). RESULTS: In normal colonic epithelium and hyperplastic polyps, FasL expression was restricted to the luminal surface of the crypts, where Fas-FasL coexpression was coincident with a high frequency of TUNEL positive epithelial cells. All adenomas (n = 38) had an altered distribution of positive FasL staining; FasL expression was found in most cells (> 70% of neoplastic cells). Expression of Fas was also detected throughout the adenomas, but coexpression of FasL and Fas was not associated with TUNEL positivity in most cells. CONCLUSIONS: FasL upregulation occurs early in the adenoma-carcinoma sequence of colon carcinogenesis, and is evident at the level of mild dysplasia. The lack of pronounced apoptosis in areas of adenomas coexpressing Fas and FasL suggests that colonocytes acquire resistance to Fas mediated apoptosis early in the transformation process.

Adenoma↗
PubMed2001

High-resolution detection of loss of heterozygosity of dinucleotide microsatellite markers.

Dinucleotide microsatellite markers are frequently investigated to study inheritance, genetic stability, and allele frequency distribution in a wide variety of genetic disorders. Previous studies have encountered significant problems regarding resolution and detection of dinucleotide, microsatellites. In this study, a useful method to investigate loss of heterozygosity (LOH) of dinucleotide microsatellite markers is described that involves the use of nondenaturing (Spreadex) submerged gel electrophoresis and SYBR Green I nucleic acid staining. This method omits the gel casting step and the use of hazardous radioactive materials frequently used in many microsatellite studies that employ polyacrylamide gel nucleic acid denaturation analysis. Using this method, 62 patients' paired tumor and normal samples were investigated to detect allele deletions in a region of chromosome 7q31.1, which is believed to harbor a tumor suppressor gene. Interpretable results were obtained in all cases. These results were compared to those attained using ABI Prism Genetic Analyzer 310 and Gene-Scan. There were no discrepancies in results obtained between the two assays. The Spreadex system is cheap, does not require larger equipment costs, and may prove to be a useful system for high-throughput investigation of microsatellites. It may have diagnostic significance and also prove useful if applied to population-based genomic screening and linkage analysis.

Dinucleotide Repeats↗
PubMed2000

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell's sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

Antineoplastic Agents↗
PubMed2000

Rapid activation of basolateral potassium transport in human colon by oestradiol.

1. We investigated the effect of oestradiol on basolateral potassium channels in human colonic epithelium. 2. Ion transport was quantified using short circuit current (I:(sc)) measurements of samples mounted in Ussing chambers. Serosal K transport was studied using nystatin permeabilization of the apical membrane. Intracellular pH changes were quantified using spectroflouresence techniques. 3. Experiments were performed with either 10 nM or 1 microM Ca(2+) in the apical bathing solution. With 10 nM Ca(2+) in the apical bathing solution addition of oestradiol (1 nM) to the basolateral bath produced a rapid increase in current (delta I(K)=11.2+/-1.2 microA.cm(-2), n=6). This response was prevented by treatment of the serosal membrane with tolbutamide (1 microM). With 1 microM Ca(2+) in the apical bathing solution addition of oestradiol produced a rapid fall in current (delta I(K)=-12.8+/-1.4 microA.cm(-2)), this response was prevented by treatment of the basolateral membrane with tetra-pentyl-ammonium (TPeA). These responses were rapid and occurred independently of protein synthesis. 4. Inhibition of basolateral Na(+)/H(+) exchange with either amiloride or a low sodium bathing solution prevented this response. These responses were prevented by inhibition of protein kinase C (PKC) with bis-indolyl-maleimide. 5. Oestradiol (1 nM) produced a rapid intracellular alkanization (mean increase=0.11 pH units; n=6; P<0.01). 6. These results suggest that oestradiol rapidly modulates serosal K transport in human colon. These effects depend upon intact Na(+)/H(+) exchange and protein kinase C. We propose a non-classical, possibly membrane linked, mechanism for oestradiol action in human colonic epithelium.

Calcium↗
PubMed2000

Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon.

BACKGROUND: Oestrogens are important mitogens in epithelial cancers, particularly where tumours express complementary receptors. While the traditional model of oestrogen action involves gene-directed (genomic) protein synthesis, it has been established that more rapid, non-genomic steroid hormone actions exist. This study investigated the hypothesis that oestrogen rapidly alters cell membrane activity, intracellular pH and nuclear kinetics in a mitogenic fashion. METHODS: Crypts isolated from human distal colon and colorectal cancer cell lines were used as robust models. DNA replication and intracellular pH were measured by radiolabelled thymidine incorporation (12 h) and spectrofluorescence imaging respectively. Genomic protein synthesis, sodium-hydrogen exchanger (NHE) and protein kinase C (PKC) activity were inhibited with cycloheximide, ethylisopropylamiloride and chelerythrine chloride respectively. RESULTS: Oestrogen induced a rapid (less than 5 min) cellular alkalinization of crypts and cancer cells that was sensitive to NHE blockade (P < 0.01) or PKC inhibition (P < 0.01). Oestrogen increased thymidine incorporation by 44 per cent in crypts and by up to 38 per cent in cancer cells (P < 0.01), and this was similarly reduced by inhibiting the NHE (P < 0.01) or PKC (P < 0.05). CONCLUSION: Oestrogen rapidly activates cell membrane and nuclear kinetics by a non-genomic mechanism mediated by PKC but not gene-directed protein synthesis.

Cell Membrane↗
PubMed2000

Biological behaviour and clinical implications of micrometastases.

BACKGROUND: The most important prognostic determinant in cancer is the identification of disseminated tumour burden (metastases). Micrometastases are microscopic (smaller than 2 mm) deposits of malignant cells that are segregated spatially from the primary tumour and depend on neovascular formation (angiogenesis) to propagate. METHODS: The electronic literature (1966 to present) on micrometastases and their implications in malignant melanoma and epithelial cancers was reviewed. RESULTS: Immunohistochemical techniques combined with serial sectioning offer the best accuracy for detection of nodal micrometastases. Molecular techniques should be reserved for blood samples or bone marrow aspirates. Detection of micrometastases in regional lymph nodes and/or bone marrow confers a poor prognosis in epithelial cancers. The concept of sentinel node biopsy combined with serial sectioning and dedicated screening for micrometastases may improve staging procedures. Strategies against angiogenesis may provide novel therapies to induce and maintain micrometastatic dormancy. CONCLUSION: The concept of micrometastases has resulted in a paradigm shift in the staging of epithelial tumours and our overall understanding of malignant processes.

Biopsy, Needle↗
PubMed2000

Bone-marrow micrometastases in patients with brain metastases from epithelial cell tumours.

Carcinoma that has metastasized to the central nervous system (CNS) poses a particular clinical problem regarding confirmation of the diagnosis and subsequent management. Prior to excision, thorough evaluation for coexisting systemic disease is essential, but current imaging techniques are limited by their spatial resolution and under-stage many patients. We evaluated the potential utility of bone-marrow evaluation for micrometastatic cells in patients with CNS metastasis. Bone-marrow aspirates were examined for cytokeratin-positive cells in 12 consecutive patients who presented with symptomatic space-occupying lesions of the CNS. These patients had previously undergone surgical excision of either gastrointestinal or breast cancers. All twelve had micrometastases in their bone marrow at the time of presentation with the CNS disease and all had a fatal outcome within 13 months. In nine of the 12 patients, bone-marrow micrometastases were the only evidence for systemic spread. Three patients had elevated serum tumour markers and two of these had radiologically detectable recurrence elsewhere. Bone-marrow micrometastases indicate concurrent systemic involvement and a poor prognosis. The results suggest that bone-marrow evaluation for systemic spread is a useful diagnostic adjunct and should be performed before considering diagnostic biopsy or excision.

Adenocarcinoma↗