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J Southgate

Publications and source records attributed to J Southgate.

At least 19 recordsLinked to original sources
PubMed2006

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation.

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.

Apoptosis↗
PubMed2006

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells.

Comparing normal human urothelial (NHU) cells to a panel of six representative urothelial cell carcinoma (UCC)-derived cell lines, we showed that while TRAIL receptor expression patterns were similar, susceptibility to soluble recombinant crosslinked TRAIL fell into three categories. 4/6 carcinoma lines were sensitive, undergoing rapid and extensive death; NHU and 253J cells were partially resistant and HT1376 cells, like normal fibroblasts, were refractory. Both normal and malignant urothelial cells underwent apoptosis via the same caspase-8/9-mediated mechanism. Rapid receptor downregulation was a mechanism for evasion by some UCC cells. TRAIL resistance in malignant urothelial cells was partially dependent on FLIP(L) and was differentially mediated by p38(MAPK), whereas in normal cells, resistance was mediated by NF-kappaB. Importantly, extensive killing of UCC cells could be induced using noncrosslinked TRAIL after prolonged exposure, with no damage to their homologous, normal urothelial cell counterparts.

Apoptosis↗
PubMed2006

Novel urothelium specific gene expression identified by differential display reverse transcriptase-polymerase chain reaction.

PURPOSE: Understanding the molecular basis of differential gene expression among different tissues at various developmental stages and in neoplastic transformation is an important biological goal. The potential clinical applications of this improved understanding are more precise diagnosis of disease, prediction of prognosis, novel targeted therapies and prediction of response to therapy. MATERIALS AND METHODS: Differential display reverse transcriptase-polymerase chain reaction was used to compare gene expression in bovine urothelium to that in autologous lung, esophagus, liver and spleen. Products that appeared to have urothelial specific expression were sequenced and assessed for homology with known sequences. Ribonuclease protection assays were used to further confirm the expression pattern. RESULTS: A total of 32 discrete cDNAs were identified, including 3 products from genes known to be urothelium specific in their expression, 16 with significant homology to bovine, human or mouse expressed sequence tags and 5 with no sequence homology to any currently available sequence. Urothelium specific mRNA expression was confirmed for 3 genes by ribonuclease protection assays and one (Udd06) was further characterized as a urea transporter. CONCLUSIONS: The use of differential display reverse transcriptase-polymerase chain reaction and other complementary techniques for parallel gene expression analysis will permit the complete characterization of the urothelial transcriptome and help identify potential molecular targets for rationally targeted therapy.

Animals↗
PubMed2005

Experimental models of human bladder carcinogenesis.

Bladder cancer is the fifth most common cancer in the UK, yet human bladder carcinogenesis remains poorly understood and the response of bladder tumours to radio- and chemo-therapy is unpredictable. The aims of this article are to review human bladder carcinogenesis and appraise the different in vitro and in vivo approaches that have been developed to study the process. The review considers how in vitro models based on normal human urothelial (NHU) cells can be applied to human bladder cancer research. We conclude that recent advances in NHU cell culture offer novel approaches for defining urothelial tissue-specific responses to genotoxic and non-genotoxic carcinogens and elucidating the role of specific genes involved in the mechanisms of bladder carcinogenesis and malignant progression.

Animals↗
PubMed2005

Transcriptional control of the human urothelial-specific gene, uroplakin Ia.

The transcriptional control elements of tissue-specific genes may be exploited in the design of therapeutic constructs for use in human gene therapy. The uroplakins are a family of four proteins which form the asymmetric unit membrane of the urothelium. We have cloned the human uroplakin Ia gene and defined its genomic structure and transcriptional start site. Using quantitative RT-PCR in an extended panel of normal tissues, we have demonstrated highly urothelial-specific expression of this gene. A Dual-Luciferase assay was used to assess the transcriptional activity of a variety of promoter fragments of the human uroplakin Ia gene. A highly specific promoter fragment (consisting of 2147 bp of 5'-flanking sequence, intron 1 and the 5' UTR) was identified which regulated urothelial-specific expression in vitro. The human uroplakin Ia promoter identified has potential use in future gene therapy strategies to restrict transgene expression to the urothelium.

5' Flanking Region↗
PubMed2005

A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function.

The urinary bladder and associated tract is lined by the urothelium. Once considered as just an impermeable epithelium, it is becoming evident that the urothelium not only functions as a volume-accommodating urinary barrier but has additional roles, including sensory signaling. Lack of access to normal human urothelium has hampered physiological investigation, and although cell culture systems have been developed, there has been a failure to demonstrate that normal human urothelial (NHU) cells grown in vitro retain the capacity to form a functional differentiated urothelium. The aim of this study was to develop a biomimetic human urothelium from NHU cell cultures. Urothelial cells isolated from normal human urothelium and serially propagated as monolayers in serum-free culture were homogeneous and adopted a proliferative, nondifferentiated phenotype. In the presence of serum and physiological concentrations of calcium, these cells could be reproducibly induced to form stratified urothelia consisting of basal, intermediate, and superficial cells, with differential expression of cytokeratins and superficial tight junctions. Functionally, the neotissues showed characteristics of native urothelium, including high transepithelial electrical resistance of >3,000 Omega.cm(2), apical membrane-restricted amiloride-sensitive sodium ion channels, basal expression of Na(+)-K(+)-ATPase, and low diffusive permeability to urea, water, and dextran. This model represents major progress in developing a biomimetic human urothelial culture model to explore molecular and functional relationships in normal and dysfunctional bladder physiology.

Adult↗
PubMed2005

Modelling cell signalling and differentiation in the urothelium.

Urothelial cells line the bladder. If the urothelium is damaged, it is vital that it repairs itself quickly. Experimental results shedding light on how this repair process works are presented, revealing in particular the dependence of the response on the length of time for which the drug Troglitazone (TZ) is applied. A simple mathematical model for the basic mechanism (comprising ordinary differential equations) is then developed and analysed, seeking specifically to clarify and quantify the mechanisms governing the dependence of the cell differentiation response on the TZ administration time, rather than providing a comprehensive model of differentiation. Through biologically justified simplifications, analysis reveals that the model gives results in accord with the experimental observations, and suggests new experiments that may aid further understanding. Directions in which this preliminary modelling of the PPAR gamma (peroxisome proliferator activated receptor gamma) pathway could be usefully extended are also indicated.

Cell Differentiation↗
PubMed2004

Agent-based computational modeling of wounded epithelial cell monolayers.

Computational modeling of biological systems, or in silico biology, is an emerging tool for understanding structure and order in biological tissues. Computational models of the behavior of epithelial cells in monolayer cell culture have been developed and used to predict the healing characteristics of scratch wounds made to urothelial cell cultures maintained in low- and physiological [Ca2+] environments. Both computational models and in vitro experiments demonstrated that in low exogenous [Ca2+], the closure of 500-microm scratch wounds was achieved primarily by cell migration into the denuded area. The wound healing rate in low (0.09 mM) [Ca2+] was approximately twice as rapid as in physiological (2 mM) [Ca2+]. Computational modeling predicted that in cell cultures that are actively proliferating, no increase in the fraction of cells in the S-phase would be expected, and this conclusion was supported experimentally in vitro by bromodeoxyuridine incorporation assay. We have demonstrated that a simple rule-based model of cell behavior, incorporating rules relating to contact inhibition of proliferation and migration, is sufficient to qualitatively predict the calcium-dependent pattern of wound closure observed in vitro. Differences between the in vitro and in silico models suggest a role for wound-induced signaling events in urothelial cell cultures.

Algorithms↗
PubMed2004

A surgical model of composite cystoplasty with cultured urothelial cells: a controlled study of gross outcome and urothelial phenotype.

OBJECTIVES: To study the outcome of composite cystoplasty using cultured urothelial cells combined with de-epithelialized colon or uterus in a porcine surgical model, using appropriate controls, and to characterize the neo-epithelium created by composite cystoplasty. MATERIALS AND METHODS: Urothelial cells were isolated and propagated in vitro from open bladder biopsies taken from nine female minipigs. Cohesive sheets of confluent urothelial cells were transferred to polyglactin carrier meshes and sutured to de-epithelialized autologous colon in four animals and de-epithelialized autologous uterus in five. These composite segments were then used for augmentation cystoplasty. Conventional colocystoplasty, de-epithelialized colocystoplasty and sham operations were carried out in six control animals. After killing the animals at approximately 90 days the bladders were removed for examination and immunohistochemical analysis, using a panel of antibodies against cytokeratins and urothelial differentiation-associated antigens. RESULTS: Macroscopically, the bladders augmented with composite segments derived from uterine muscle had no evidence of shrinkage or contracture. Histological analysis showed that in four of five composite uterocystoplasties, the neo-urothelium was stratified and had a transitional morphology, although in some areas coverage was incomplete. Immunohistochemical analysis showed evidence of squamous differentiation in both native and augmented segments. All composite and de-epithelialized colonic segments showed significant contraction with poor urothelial coverage, reflecting the unsuitability of the thin-walled porcine colon for de-epithelialization. CONCLUSIONS: The functional and macroscopic outcome of bladder augmentation with a composite derived from cultured urothelium and de-epithelialized smooth muscle of uterine origin endorses the feasibility of composite cystoplasty.

Animals↗
PubMed2003

Bladder reconstruction--from cells to materials.

Surgical reconstruction of the urinary bladder is performed on patients of all ages for a diverse range of conditions, including congenital abnormalities, bladder dysfunction, trauma and cancer. The most common material utilized to augment or replace the bladder during these procedures is a segment of the patient's own intestine. However, this procedure ('enterocytoplasty') is associated with significant clinical complications that arise due to the exposure of the epithelial lining of the intestine to urine. A number of alternative approaches are being actively developed to find a practical and functional substitute for native bladder tissue. These range from 'composite enterocystoplasty', where the de-epithelialized intestine wall is lined with bladder epithelial cells that have been propagated in vitro, to augmenting the urinary system with natural or synthetic biomaterials that may incorporate in vitro-propagated cells. However, if tissue-engineered products are to have therapeutic application in bladder reconstruction, a number of issues remain to be addressed; these issues are discussed briefly below.

Bioartificial Organs↗
PubMed2002

Regulation of apoptosis in the bovine blastocyst by insulin and the insulin-like growth factor (IGF) superfamily.

Insulin and the insulin-like growth factors, IGF-I and IGF-II, have been reported to exert a mitogenic effect on the preimplantation mammalian embryo. Furthermore, it has been proposed that loss of imprinting of the insulin-like growth factor II receptor gene and the consequent over-production of IGF-II may be involved in the aetiology of the Enlarged Offspring Syndrome, which occurs as an artefact of in vitro embryo production. We have previously shown that apoptosis occurs in the preimplantation bovine embryo and is influenced by in vitro culture conditions. We have therefore sought to establish the effects of insulin, IGF-I and IGF-II on apoptosis and cell proliferation in bovine blastocysts in vitro. Zygotes, obtained by in vitro maturation and fertilization of follicular oocytes, were cultured to blastocysts, with or without exogenous growth factors. Embryos were stained with propidium iodide to label all nuclei and by TUNEL to label apoptotic nuclei and analyzed by epifluorescent and confocal microscopy. IGF-I and IGF-II, but not insulin, were found to increase the proportion of embryos which formed blastocysts. Insulin decreased the incidence of apoptosis without affecting blastocyst cell number. IGF-I acted to decrease apoptosis and increase total cell number and IGF-II increased cell number alone. These data suggest roles for insulin and the IGFs as mitogens and/or apoptotic survival factors during early bovine development. Perturbation of IGF-II regulated growth may be involved in fetal oversize.

Animals↗
PubMed2002

Inter-individual variation in urothelial DNA repair gene expression in vitro.

DNA repair efficiency may play a significant role in individual susceptibility to bladder cancer, the third most common cancer in Europe. Bladder cancer arises from the urothelial cell layer which lines the urinary tract. As DNA repair gene expression levels should reflect DNA repair capacity, we investigated the expression of genes from the base excision, nucleotide excision and mismatch repair pathways in normal human urothelial (NHU) cells in vitro. RNA was extracted from six independent NHU cell lines and expression of 26 DNA repair genes was determined by ribonuclease protection assay. The results show that all the genes analysed were detected in NHU cells in vitro with a similar expression pattern in most cell lines. However, there was some variation between cell lines, with one expressing base excision repair genes very strongly, but another having weak expression of mismatch repair genes. These results suggest that DNA repair genes are constitutively expressed by NHU cells and that there is some inter-individual variation. Prospective studies are required to determine whether these differences in gene expression may play a role in susceptibility to bladder cancer.

Cell Line↗
PubMed2002

Effects of insulin-like growth factors I and II on tumour-necrosis-factor-alpha-induced apoptosis in early murine embryos.

The proposition that members of the insulin-like growth factor superfamily act as rescue factors from apoptosis in murine preimplantation embryos was tested. The cytokine tumour necrosis factor alpha (TNFalpha) was used to induce apoptosis. Zygotes were cultured for 5 days to the blastocyst stage in the presence or absence of TNFalpha and in the presence or absence of the insulin-like growth factors, IGF-I or IGF-II. Tumour necrosis factor alpha significantly increased the percentage of apoptotic cells and reduced the total cell count in Day 5 blastocysts. When IGF-I or IGF-II were added to the culture medium in the presence of TNFalpha, the cell number and apoptotic dead cell index (DCI) were restored to control values. Insulin-like growth factor-I alone had a greater effect on total cell number than IGF-II alone, but did not significantly decrease the apoptotic DCI. In contrast, IGF-II significantly reduced the number of apoptotic cells. This study shows that IGFs may play a role as apoptotic survival factors in the early mouse embryo.

Animals↗
PubMed2001

Role of p53 in the responses of human urothelial cells to genotoxic damage.

Loss of p53 function is a feature of many types of malignancy, including transitional-cell carcinoma (TCC), where it is associated with high-grade lesions and the development of muscle-invasive disease. Genotoxic agents used as part of the treatment strategy may contribute to tumour progression by inducing further non-lethal DNA damage in surviving cells. To determine the role of p53 in cellular responses to genotoxic agents, we used cultured normal human urothelial (NHU) cells and NHU cells with disabled p53 function. Mitomycin C and gamma-radiation caused normal cells to undergo an extended period of cell-cycle arrest, followed by complete recovery of proliferative potential. In contrast, cells with disabled p53 function, whether karyotypically normal (HU-E6 cells) or post-crisis with karyotypic abnormalities (HU-E6P cells), underwent extensive apoptosis. Overall survival was dose-dependent, and surviving HU-E6 cells from low-dose treatments showed clonal karyotypic abnormalities. These findings demonstrate that p53 status is a crucial factor in determining the ability of urothelial cells to survive DNA damage and suggest caution in the use of genotoxic treatments for low-grade tumours as our data imply that malignancies that have not yet lost p53 function will show the same "repair-and-recovery" response as normal cells.

Alkylating Agents↗
PubMed2001

Identification of genes involved in human urothelial cell-matrix interactions: implications for the progression pathways of malignant urothelium.

Interactions between epithelial cells and the extracellular matrix are central to tissue homeostasis and have a dynamic role in tissue remodeling and repair. Regulation of these pathways is balanced by positive and negative feedback elements, many of which have been implicated in the pathways of malignant progression. We have used differential display to identify genes that are up-regulated in normal human urothelial cells in response to exposure to extracellular matrix proteins (Matrigel) in vitro. This approach has identified genes that have key roles in cell-cell and cell-matrix interactions and that have been implicated in the progression of carcinomas of urothelial or other epithelial cell origins. One confirmed but unknown differentially expressed sequence was used to isolate a full-length gene, MIG-C4, from a human urothelial cDNA library. This gene was found to encode a novel urokinase plasminogen-activator receptor-like member of the Ly-6 family of glycosyl-phosphatidylinositol-anchored glycoproteins, and was identified as the human homologue of the rat metastasis-associated C4.4A gene. By in situ hybridization, MIG-C4 was expressed variably in normal urothelium and intensely in the tumor component of some noninvasive superficial lesions and in invasive and metastatic urothelial cancers. Thus, our approach has identified previously nonimplicated gene products involved in normal urothelium-matrix interactions that could be tumor-invasion or suppressor-gene targets in the development of invasive and metastatic tumor phenotypes.

Amino Acid Sequence↗
PubMed2001

Transforming growth factor-beta promotes 'death by neglect' in post-activated human T cells.

Transforming growth factor-beta (TGF-beta) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound-healing, we studied the effects of TGF-beta on mitogen-stimulated peripheral blood cells. TGF-beta added at the initiation of mitogenesis did not significantly alter T-cell activation, proliferation, CD45 isoform switching, or activation-induced cell death. By contrast, TGF-beta added 72 hr post-activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF-beta had no effect on mitogen-induced up-regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas-sensitive Jurkat cell line by activated T cells. Furthermore, TGF-beta had no direct effect on levels of mRNA for members of the bcl family (bcl-X, bfl-1, bik, bak, bax, bcl-2 and mcl-1). These findings suggest that TGF-beta does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin-2, which can 'rescue' lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro-apoptotic effects of TGF-beta on post-activated T cells, thus demonstrating that TGF-beta increases the cytokine-dependence of T cells for survival. We propose a novel role for TGF-beta in the suppression of inflammation by promoting the elimination of post-activated T cells once the initiating stimulus has been resolved.

Apoptosis↗