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S Dooley

Publications and source records attributed to S Dooley.

At least 19 recordsLinked to original sources
PubMed2006

Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Animals↗
PubMed2006

TGF-beta/Smad signaling in the injured liver.

TGF-beta, acting both directly and indirectly, represents a central mediator of fibrogenic remodeling processes in the liver. Besides hepatic stellate cells (HSCs), which are induced by TGF-beta to transdifferentiate to myofibroblasts and to produce extracellular matrix, hepatocytes are also strongly responsive for this cytokine, which induces apoptosis during fibrogenesis and provides growth control in regeneration processes. Based on this, TGF-beta-mediated hepatic responses to injury are the result of a complex interplay between the different liver cell types. In this review we summarize the knowledge about TGF-beta signal transduction in HSCs with special impact on Smad pathways. We further describe a molecular cross-talk between profibrogenic TGF-beta and antifibrogenic IFN-gamma signaling in liver cells. Finally, we introduce hepatocyte plasticity and epithelial-to-mesenchymal transition in the liver, which is well established in tumorigenesis, as a potential feature of fibrogenesis and highlight possible action points of TGF-beta in these contexts.

Animals↗
PubMed2005

Thrombospondin 1 acts as a strong promoter of transforming growth factor beta effects via two distinct mechanisms in hepatic stellate cells.

BACKGROUND AND AIMS: Thrombospondin 1 (TSP-1) is an important activator of latent transforming growth factor beta (TGF-beta) but little is known of the expression patterns and functions of TSP-1 in liver cells. We therefore analysed if and how TSP-1 acts on TGF-beta during fibrogenesis. METHODS AND RESULTS: Using reverse transcription-polymerase chain reaction, we demonstrated that hepatocytes from normal liver expressed no TSP-1 mRNA whereas Kupffer cells and sinusoidal endothelial cells did. TSP-1 mRNA and protein were detected in quiescent and activated cultured hepatic stellate cells (HSC) and TSP-1 expression was highly inducible by platelet derived growth factor BB (PDGF-BB) and, to a lesser extent, by tumour necrosis factor alpha in activated HSC. Furthermore, addition of PDGF-BB directly led to enhanced TGF-beta mRNA expression and a TSP-1 dependent increase in TGF-beta/Smad signalling. Using either a peptide specifically blocking the interaction of TSP-1 with latent TGF-beta or antibodies against TSP-1 not only abrogated activation of latent TGF-beta but also reduced the effects of the active dimer itself. CONCLUSIONS: Our data suggest that TSP-1 expression is important for TGF-beta effects and that it is regulated by the profibrogenic mediator PDGF-BB in HSC. Furthermore, the presence of TSP-1 seems to be a prerequisite for effective signal transduction by active TGF-beta not only in rat HSC but also in other cell types such as human dermal fibroblasts.

Animals↗
PubMed2001

Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells.

To current knowledge, transforming growth factor beta (TGFbeta) signaling is mandatory to establish liver fibrosis and various molecular interventions designed to affect the TGFbeta system were successfully used to inhibit fibrogenesis. Activated hepatic stellate cells (HSC), which are one important source of TGFbeta, are the major producers of extracellular matrix proteins in liver injury. We have previously shown that the TGFbeta response of this cell type is modulated during the transdifferentiation process. This work delineates the activation of TGFbeta downstream mediators, the Smads, in quiescent HSC and transdifferentiated myofibroblasts (MFB). The expression level of all Smads remained largely unchanged during this process. The response of HSC to TGFbeta, leading to, e.g., induction of alpha2 (I) collagen expression, is mediated by phosphorylation of Smad2 and Smad3 and subsequent nuclear translocation of a Smad containing complex. Neither TGFbeta-dependent nor endogenously phosphorylated Smad2/3 was detectable in comparable amounts in transdifferentiated MFB, indicating loss of TGFbeta sensitivity. Ectopic expression of Smad7 in HSC led to inhibition of Smad2 phosphorylation and abrogated TGFbeta response. In transdifferentiated MFB, expression of a constitutively active TGFbeta receptor I, but not treatment with TGFbeta1, resulted in transcriptional activation of a TGFbeta responsive promoter, thereby demonstrating completely restored TGFbeta signal transduction. Our data indicate that in contrast to a postulated mechanism of enduring autocrine TGFbeta signal transduction, early and late stages of HSC activation have to be distinguished, which is of importance for antifibrotic therapies.

3T3 Cells↗
PubMed2001

Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts.

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.

Alternative Splicing↗
PubMed2001

Oral fluid collection by post for viral antibody testing.

BACKGROUND: The objective of this study was to estimate the prevalence of hepatitis B virus exposure (HBV) in the population of the Republic of Ireland, by using oral fluid (saliva) collection by post for hepatitis B anti-core antibody (anti-HBc). This paper discusses the methodological approach used and the strategies that were adopted to improve response rates. METHODS: The sampling frame used was the Register of Electors for Irish parliamentary elections. A multistage stratified cluster sample was taken, and a total of 962 households were selected nationally. A four-letter approach was employed for sample collection. Households received an initial letter outlining the purpose of the study. This was followed by a letter containing six swabs for oral fluid collection, along with easy-to-follow instructions. Non-respondents received two reminder letters, and were also telephoned where possible. A telephone helpline was provided. All testing was anonymous and unlinked. RESULTS: The study achieved a good household response rate (60.4%), and more than 98% of the 1738 specimens received were suitable for testing. The prevalence of anti-HBc in the Irish population was estimated to be 0.51%. The observed design effect was 1.29. DISCUSSION: From a review of the literature, this is the first study where a representative sample of a national population was asked to self-collect oral fluid samples and return these by post for serological testing. The technique may have many future applications in epidemiological research.

Adult↗
PubMed2001

Antenatal hepatitis B screening - is there a need for a national policy?

Routine antenatal testing for hepatitis B carriage with maternal consent was introduced at the Rotunda in January 1998. The uptake of testing has been excellent; 99.98% of women presenting for antenatal care accepted hepatitis B (HBV) screening in the 30-months from January 1998 through June 2000. The prevalence of HBV carriage was 0.35% (58 pregnancies of 16,222 tested) increasing from 0.25% in 1998 (16 of 6227) to 0.45% in the first six months of 2000 (16 of 3484). Fifty-five women had 58 pregnancies (three women had two pregnancies). Two of these were e-antigen positive. HBV carrier status was previously unknown in 48 (87%). Two additional women had acute HBV infection in pregnancy. Forty-five infants have been born to mothers included in this screening programme. Audit of infant outcome reveals excellent compliance with immunisation and follow-up: 29 (64%) have completed the 3 dose HBV vaccination schedule to date. Thirteen infants (31%) are still attending; three are lost to follow-up including one whose family has emigrated. Routine antenatal screening for hepatitis B carriage is cost-effective and should be considered a standard of care in maternity practice.

Carrier State↗
PubMed2000

Participation of Smad2, Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. THE TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.

Smad7 has recently been identified as a player that antagonizes transforming growth factor beta (TGF-beta) signals by acting downstream of TGF-beta receptors. TGF-beta rapidly induces expression of Smad7 mRNA in a variety of cell types, suggesting participation in a negative feedback loop to control TGF-beta responses. We have previously described the genomic locus of rat Smad7 including the promoter region. Here we report polymerase chain reaction cloning of the corresponding promoter regions of human and murine Smad7 genes and functional characterization of the rat Smad7 promoter. Using transient transfection experiments of HepG2 cells, we identified the TGF-beta response element within a strongly conserved region, containing a perfect Smad binding element (SBE; GTCTAGAC). Performing electrophoretic mobility shift assay and cotransfection experiments, we were able to delineate DNA-binding complexes and identified Smad3, Smad4, and Smad2. Mutation of the SBE completely abolished TGF-beta inducibility of Smad7 in HepG2 cells, indicating that this sequence is necessary for TGF-beta-induced transcription. Furthermore, a 3-base pair adjacent E-box is additionally essential for TGF-beta-dependent promoter activation and an overlapping AP1 site is also involved. We conclude that regulation of Smad7 transcription by TGF-beta is mediated via a specific constellation of recognition motifs localized around the SBE, which is conserved in human, rat, and murine genes.

Animals↗
PubMed2000

Genomic locus and promoter region of rat Smad7, an important antagonist of TGFbeta signaling.

SMAD proteins are essential components of the intracellular signaling pathways utilized by members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. Certain SMAD proteins (Smad1, 2, 3, and 5) can act as regulated transcriptional activators. This process involves phosphorylation of these proteins by activated TGFbeta receptors. Recently, Smad6 and Smad7 were identified; they antagonize TGFbeta signaling by preventing the activation of signal-transducing SMAD complexes. TGFbeta rapidly induces the expression of Smad7 mRNA, suggesting participation of Smad7 in a negative feedback loop to control TGFbeta responses. Similarly, epidermal growth factor (EGF) and interferon gamma (IFN-gamma) have been reported to induce Smad7 expression. In a rat model system of liver fibrosis, TGFbeta inducibility of Smad7 is abrogated during transformation of hepatic stellate cells (HSC), indicating an important switch in transcriptional regulation of the gene. With the detailed characterization of the rat Smad7 genomic organization including the promoter region, we present the first identified Smad7 gene so far. The gene is composed of four exons separated by three introns covering a DNA region of about 30 kilobases (kb) in total. The major transcription start site is conserved between rat and mouse, and two polyadenylation signals were detected. In the promoter region, a potential CAGA box, a signal transducer and activator of transcription (STAT) factor-related recognition site, and different AP1 sites were identified, which could be the targets of TGFbeta, IFN-gamma, and EGF-dependent Smad7 transcription initiation.

Animals↗
PubMed2000

Prevalence of hepatitis B anti-core antibody in the Republic of Ireland.

The aim of this study was to estimate the prevalence of hepatitis B exposure in the population of the Republic of Ireland, by measuring the prevalence of hepatitis B anti-core antibody in oral fluid collected by postal survey. A random multi-stage stratified sample of Irish households was obtained, using the Irish electoral register as the sampling frame. A total of 962 households were selected, and a household response rate of 60.4% was achieved. Oral fluid specimens totalling 1714 were tested for antibody to hepatitis B core antigen (anti-HBc), using an Immune Capture Enzyme Immuno-Assay. Five specimens (0.29%) were found to contain anti-HBc. Adjusting for study design, the estimated anti-HBc prevalence in the Republic of Ireland is 0.51%. This study demonstrates that self-collection of oral fluid samples is acceptable to the public, and based upon the data generated, that the Republic of Ireland has a low prevalence of hepatitis B infection.

Adolescent↗
PubMed2000

Modulation of transforming growth factor beta response and signaling during transdifferentiation of rat hepatic stellate cells to myofibroblasts.

Activation of hepatic stellate cells (HSCs) is the key step in liver fibrogenesis. Increased transforming growth factor beta (TGF-beta) expression and extracellular matrix production in patients with hepatic fibrosis and experimental models of liver fibrogenesis support implication of TGF-beta in the pathogenesis of this disease. However, a causative role for TGF-beta during transdifferentiation of HSCs has not been delineated in molecular detail. Using a rat cell culture model of HSC transdifferentiation, we analyzed TGF-beta signal transduction and identified changes between stellate cells and their transdifferentiated phenotype. Fully transdifferentiated myofibroblasts, opposed to HSCs, were not inhibited in proliferation activity on treatment with TGF-beta1. Furthermore, stimulation of alpha2 (I) collagen and Smad7 messenger RNA (mRNA) expression by TGF-beta1 was achieved in stellate cells but not in myofibroblasts. Northern and Western blot analyses indicated significant expression of TGF-beta receptors I and II in both cell types. In contrast, [(125)I]-TGF-beta1 receptor affinity labeling displayed strongly reduced types I, II, and III receptor presentation at the cell surface of myofibroblasts. Moreover, myofibroblasts did not display DNA-binding SMAD proteins in electrophoretic mobility shift assays with a CAGA box. These data indicate that stellate cells are responsive to TGF-beta1 treatment and transduce a signal that may play an important role in liver fibrogenesis. Myofibroblasts display decreased availability of surface receptors for TGF-beta, which could be based on autocrine stimulation. However, lack of activated SMAD complexes with DNA-binding activity and absence of alpha2 (I) collagen transcription inhibition by latency-associated peptide (LAP)/anti-TGF-beta antibody raise the possibility of TGF-beta signaling independent receptor down-regulation in myofibroblasts.

Activin Receptors, Type I↗
PubMed1998

In situ hybridization analysis of genes coding collagen IV alpha1 chain, laminin beta1 chain, and S-laminin in prostate tissue and prostate cancer: increased basement membrane gene expression in high-grade and metastatic lesions.

BACKGROUND: Recent immunohistochemical data have shown that invasive prostate cancer cells are separated from the host tissue by basement membranes (BM), and express associated adhesive molecules that bind to these de novo synthesized extracellular matrices. METHODS: In the present study, we used in situ hybridization techniques to determine steady-state levels of genes coding BM components (alpha1 chain of collagen IV, laminin beta1 chain, and S-laminin) in prostate tissue obtained from 15 radical prostatectomy specimens and 5 lymph node metastases of common prostatic adenocarcinomas. RESULTS: In benign prostate tissue, transcripts of these genes were detected predominantly in the basal cell layer, indicating that components of epithelial BMs are synthesized by basal cells and not by stromal cells. The cancerous lesions investigated revealed increasing collagen IV, laminin beta1 chain, and S-laminin mRNA levels when compared with benign prostate tissue. The highest steady-state levels were found in high grade (primary Gleason grade 4 and 5) carcinoma and lymph node metastases, and were predominantly localized in epithelial compartments of the cancerous tissue. CONCLUSIONS: These findings indicate that neoplastic BM in prostatic adenocarcinoma derive from tumor cells and not from the host tissue. Increasing transcriptional activities of genes coding BM components detected in poorly differentiated and metastatic lesions may accelerate the BM-forming process, which probably contributes to the ability of tumor cells to penetrate the extracellular matrix during the process of stromal invasion and metastasis.

Adenocarcinoma↗
PubMed1997

Identification of human semaphorin E gene expression in rheumatoid synovial cells by mRNA differential display.

Rheumatoid arthritis (RA) is characterised by chronic inflammation of synovial tissue with aggressive proliferation of synovial cells causing destruction of cartilage and bone. Immunopathological mechanisms, infectious causes and genetic factors have been discussed, but the etiology of the disease has not been understood until now. Especially, the mechanisms driving tumourlike growth and invasive behaviour of fibroblastoid synovial cells have not been identified yet. Our aim is to find cellular factors which are mediators for such pathways. One possibility to approach this, is searching for disease-relevant genes. We applied the mRNA-differential display technique to compare mRNA expression patterns of normal and rheumatic synovial fibroblasts. We identified an upregulation of the human semaphorin E gene in rheumatoid synovial fibroblastoid cells. Interestingly semaphorin E is a member of a protein-family described to play an immunosuppressive role via inhibition cytokines. A relevance of this finding towards the pathogenesis of RA is discussed.

Arthritis, Rheumatoid↗
PubMed1997

Expression of the candidate tumor suppressor gene nm23 in the bronchial system of patients with squamous cell lung cancer.

OBJECTIVE: A number of oncogenes and tumor suppressor genes participating in tumorigenesis have been identified, one of them being nm23. The expression of the candidate tumor suppressor gene nm23 depends on the cell type of tumors. Both, reduced expression as well as overexpression of nm23 is associated with a high potential of malignancy. In a variety of tumor cell lines secretion of the nm23 protein can be detected. In an earlier investigation we showed, that the nm23 expression in squamous cell lung carcinoma is considerably elevated. In order to establish the potential diagnostic value of this finding we investigated the nm23 expression in healthy and diseased lungs in patients with squamous cell lung cancers. METHODS: We examined bronchial lavage samples of 20 patients with bronchogenic squamous cell carcinoma. The lavage was separately performed in the bronchus of the tumor bearing lobe and in the corresponding bronchus of the unaffected contralateral lung. RESULTS: Using Western blot analysis we found 2-7 fold elevated amount of nm23 protein in bronchial lavage of the tumor bearing lung in comparison to the healthy side. This finding was neither related to tumor stage nor to tumor location. Thus we have a strong hint that the nm23 protein is secreted by the bronchogenic squamous cell carcinoma. CONCLUSIONS: With respect to these results the proof of nm23 protein in bronchial lavage fluid might be of relevance to establish the diagnosis when pulmonary nodules of unknown etiology are found.

Biomarkers, Tumor↗
PubMed1996

Specific in vitro binding of p53 to the promoter region of the human mismatch repair gene hMSH2.

MSH2 is one of the genes involved in DNA-mismatch repair. Mutations in the coding region of the human gene (hMSH2) have been shown to be directly involved in microsatellite instability in hereditary nonpolyposis colorectal tumors. Examination of the promoter region of hMHS2 revealed a site with homology to the p53 consensus binding sequence. Using gel mobility shift experiments we were able to show that purified p53 has at least in vitro the potential to specifically bind the hMSH2-p53 motif. This binding activity was even stronger than the binding activity measured with the p53-consensus site. These data identify the hMSH2 gene as a possible novel p53-regulated target gene and indicate a direct involvement of p53 in repair mechanisms via DNA binding of a mismatch repair gene.

Animals↗
PubMed1996

Isolation and characterization of the human mismatch repair gene hMSH2 promoter region.

Hereditary nonpolyposis colorectal cancer (HN-PCC) is one of man's commonest hereditary diseases. Several studies have identified four responsible genes that are involved in a process known as DNA mismatch repair; hMSH2 is the most important of these four genes. In addition to mutational analysis of these genes, investigations of transcriptional regulatory mechanisms are important. Therefore, our purpose has been to isolate the hMSH2 promoter region. Using direct sequencing of P1 recombinant DNA we have characterized 1100 bp of the hMSH2 promoter.

Base Sequence↗
PubMed1996

A second trefoil protein, ITF/hP1.B, is transcribed in human breast cancer.

Trefoil proteins form a specific group of stable secreted polypeptides. They are expressed in a lot of human cancers and during inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B, was isolated. Until now no studies of the activity of this gene in human solid tumors exist. In our examination we show for the first time that this gene is transcribed in human breast cancer. In contrast to another trefoil protein, pS2, the expression of ITF/hP1.B is not under control of estrogen in the human breast cancer cell line MCF-7. We suggest that the gene activity of ITF/hP1.B in addition to pS2 expression may be an improved prognostic marker in human breast cancer.

Biomarkers, Tumor↗