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J Southgate

Publications and source records attributed to J Southgate.

At least 73 records · Page 4Linked to original sources
PubMed1995

Effect of cell density on the expression of adhesion molecules and modulation by cytokines.

Little is known about the regulation of integrin expression. Using flow cytometry, we have characterized the expression of integrin subunits and other adhesion molecules in bladder and colonic cell lines and studied their modulation by the cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). We show that although TNF-alpha and IFN-gamma may modulate integrin expression, these effects are, in the case of some integrin subunits, secondary to cytokine-induced changes in cell density. Cell density-dependent changes in the expression of alpha 2-, alpha 3-, alpha 6-, beta 1-, and beta 4-integrin subunits were seen. IFN-gamma produced density-independent decreases in expression of alpha 2- and beta 1-integrins and increases in expression of intercellular adhesion molecule-1 (ICAM-1), alpha 3-, alpha 6-, and beta 4-integrins. TNF-alpha produced increases in beta 1-integrin and ICAM-1 expression which were also density-independent. The expression of some integrin subunits is clearly density-dependent and the alterations seen are dependent on the cell line studied. This study illustrates the importance of including appropriate density-related controls in all investigations examining adhesion molecule expression.

Carcinoma, Transitional Cell↗
PubMed1995

Patterns of splice variant CD44 expression by normal human urothelium in situ and in vitro and by bladder-carcinoma cell lines.

CD44 core and splice variant exon expression was investigated in the stratified transitional epithelium of the urinary tract and in normal and malignant bladder epithelial cell lines in vitro. Antibodies against core CD44 epitopes and splice exon variants v3, v4/5, v5 and v6 showed an intense reaction in the basal and lower intermediate urothelial cell layers, which was consistently lost from the upper intermediate and superficial cell layers. Seven independent cell lines established from normal human urothelium expressed complex multiple-spliced CD44 mRNA transcripts when tested by RT-PCR and were positive with antibodies against CD44 core epitopes and splice variants v3, v4/5, v5 and v6. Of the 13 bladder-carcinoma cell lines, all were positive for CD44 core. The more differentiated cell lines had retained some splice-variant antibody reactivity and showed multiple but less complex CD44 mRNA transcript patterns, compared with normal cells. Anaplastic cell lines did not react with variant antibodies and did not contain multiple-spliced CD44 transcripts. These data suggest that loss of alternatively-spliced CD44 may reflect a selection pressure in the evolution of anaplastic bladder cancers.

Antigens, Neoplasm↗
PubMed1995

Loss of cyclin-dependent kinase inhibitor genes and chromosome 9 karyotypic abnormalities in human bladder cancer cell lines.

Loss of cell cycle control through the structural or functional aberration of checkpoint genes and their products is a potentially important process in carcinogenesis. In this study, a panel of well-characterised established human bladder cancer cell lines was screened by the polymerase chain reaction for homozygous loss of the cyclin-dependent kinase inhibitor genes p15, p16 and p27. The results demonstrate that, whereas there was no genetic loss of p27, homozygous deletion of both p15 and p16 genes occurred in seven of 13 (54%) independent bladder cell lines tested. Differential loss of either the p15 or p16 gene was not seen. The p15 and p16 genes are known to be juxtaposed on chromosome 9p21 at the locus of a putative tumour-suppressor gene involved in the initiation of bladder cancer. Cytogenetic analysis of the cell lines revealed karyotypes ranging from near diploid to near pentaploid with complex rearrangements of some chromosomes and a high prevalence of chromosome 9p rearrangements, although all cell lines contained at least one cytogenetically normal 9p21 region. These observations support a role for p15/p16 gene inactivation in bladder carcinogenesis and/or the promotion of cell growth in vitro and lend support to the hypothesis that homozygous deletion centred on 9p21 is a mechanism by which both p15 and p16 genes are co-inactivated.

Base Sequence↗
PubMed1995

Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction.

Circulating cancer cells in the blood play a central role in the metastatic process. Their number can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumour cells in haematological cancer in which abnormalities in DNA are sufficiently consistent to make this possible. For most solid tumours this not yet feasible. However, we have found that reverse transcriptase (RT)-PRC for tissue-specific gene expression is a useful technique for identifying small numbers of circulating cells in melanoma and neuroblastoma patients. In this report we describe detection of colon carcinoma cells by RT-PCR using CK 20 mRNA as a marker. Unlike other cytokeratin genes examined (CK 8 and CK 19), CK 20 was not transcribed in normal haematopoietic cells. This suggests a role for RT-PCR in the detection of colon carcinoma metastasis in blood and bone marrow, using CK 20 as the target gene. Future analysis of clinical material will determine the clinical significance of this technique.

Adenocarcinoma↗
PubMed1995

Cytokeratin 20 expression by non-invasive transitional cell carcinomas: potential for distinguishing recurrent from non-recurrent disease.

Although approximately 50% of patients with non-invasive (Ta) papillary transitional cell carcinoma show no recurrence of their disease, current histopathological approaches cannot distinguish this sub-group from those patients in whom the disease will recur. In this 5 year retrospective study, we have shown that cytokeratin 20 (CK20) was expressed in 19 of 29 (65.5%) of non-invasive papillary tumours of grades 1 or 2. CK20 expression patterns were predictive of disease non-recurrence in a sub-group of eight patients, representing 51.7% of patients with non-recurrent disease. In normal bladder mucosa, CK20 expression was restricted to the terminally-differentiated superficial cell. In eight CK20-positive tumours which showed no recurrence at 5 years, CK20 expression was either restricted to, or most intense in, the luminal cells of the papillae. This pattern of expression was not seen in any of the 15 tumours from the recurrent group. Disruption of normal CK20 expression was highly significantly correlated with recurrent tumours. These results suggest that changes in the expression of differentiation-associated antigens, such as CK20, may be useful in predicting benign versus malignant behaviour and may, therefore, be useful in defining treatment strategies.

Adult↗
PubMed1995

Expression and in vitro regulation of integrins by normal human urothelial cells.

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Adolescent↗
PubMed1994

Normal human urothelial cells in vitro: proliferation and induction of stratification.

BACKGROUND: The purpose of the work was to establish urothelium as an in vitro model for the study of proliferation, stratification, and differentiation in "complex" epithelia. EXPERIMENTAL DESIGN: Normal human urothelial cells were cultured in a serum-free medium. The effects of epidermal growth factor (EGF), cholera toxin (CT), extracellular calcium and 13-cis-retinoic acid on cell growth, morphology, phenotype, and cytodifferentiation were studied using phase-contrast microscopy and indirect immunofluorescence. Stratification-related changes were additionally analyzed by transmission electron microscopy. RESULTS: Under optimized conditions, long-term cultures were successful in 44 (74.5%) out of 59 specimens. Bacterial infection was the most common cause of failure (9 cases). Primary urothelial cells required an initial plating density of > or = 10(4) cells/cm2 for survival; passaged cells survived much lower plating densities (> or = 2.5 x 10(2) cells/cm2). CT significantly improved cell attachment, but neither CT nor EGF were essential for growth. By contrast, cells failed to proliferate without bovine pituitary extract. In media containing bovine pituitary extract, CT, and EGF, cultures had a mean population doubling time of 14.7 +/- 1.8 hours, maintained a nonstratified phenotype, and expressed the cytokeratin (CK) profile of basal/intermediate urothelium: CK7, CK8, CK17, CK18 and CK19, with variable expression of CK13. CK20 was not expressed in vitro. CK14 and CK16 were also expressed, suggestive of squamous metaplasia in culture, which could be inhibited with 13-cis-retinoic acid. Increasing extracellular calcium from 0.09 to 0.9-4.0 mM slowed cell proliferation, induced stratification and desmosome formation, and increased expression of E-cadherin. High calcium, EGF, CT, and retinoic acid did not induce markers of late/terminal urothelial cytodifferentiation. CONCLUSIONS: We describe a simplified technique for the isolation and long-term culture of human urothelial cells. Urothelial cells in vitro are capable of rapid proliferation and can be induced to form integrated stratifying cell layers in high calcium medium. Stratification-related changes are not necessarily accompanied by urothelial cell maturation and differentiation.

Adolescent↗
PubMed1993

Urothelial tissue culture for bladder reconstruction: an experimental study.

An in vitro system for the growth of normal human urothelial cells has been developed. Urothelial cells were isolated from tissue samples in 38 patients and cultured in a defined serum-free medium. Confluent cell monolayers of 25 cm.2 were produced after 7 days in 32 cases. Subsequent subcultures at a 1:20 split ratio achieved confluency within another 7 days and a consistently high rate of growth was sustained for at least 7 passages. Characterization by immunofluorescence with a panel of antibodies demonstrated that the cultured cells were exclusively epithelial and retained the characteristic antigenic profile of normal urothelium, even after extended periods in culture. The only consistent cause of failure (6 of 38 cases) was bacterial contamination secondary to an underlying urinary tract infection in these patients.

Adolescent↗
PubMed1993

Use of primary cultures of rabbit oviduct epithelial cells to study the ionic basis of tubal fluid formation.

A pure population of rabbit oviduct epithelial cells was isolated and grown as a polarized monolayer on collagen-impregnated filters in primary culture. The cells were shown to be epithelial by immunocytochemical staining. The cells were mounted in a modified Ussing chamber which enabled ion transport across the cells to be studied. There was a net flux of Cl- in a basal to apical direction which was reversed by 1 mmol dibutyryl cyclic AMP l-1 (cAMP). A small but consistent transepithelial electrical potential difference (p.d.) of 0.86 mV was recorded with the apical side of the cells negative with respect to the basal. Adrenaline added to the basal side of the cells induced large transient increases in p.d. across the monolayer, involving both alpha and beta receptors. Adrenaline also induced a small increase in basal to apical Cl- transport across the cells. It is proposed that adrenergic agonists and cAMP modulate rabbit oviduct fluid formation in part via an effect on transepithelial chloride transport.

Animals↗
PubMed1991

Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction.

Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.

Amino Acid Sequence↗
PubMed1991

p68 RNA helicase: identification of a nucleolar form and cloning of related genes containing a conserved intron in yeasts.

The human p68 protein is an RNA-dependent ATPase and RNA helicase which was first identified because of its immunological cross-reaction with a viral RNA helicase, simian virus 40 large T antigen. It belongs to a recently discovered family of proteins (DEAD box proteins) that share extensive regions of amino acid sequence homology, are ubiquitous in living organisms, and are involved in many aspects of RNA metabolism, including splicing, translation, and ribosome assembly. We have shown by immunofluorescent microscopy that mammalian p68, which is excluded from the nucleoli during interphase, translocates to prenucleolar bodies during telophase. We have cloned 55% identical genes from both Schizosaccharomyces pombe and Saccharomyces cerevisiae and shown that they are essential in both yeasts. The human and yeast genes contain a large intron whose position has been precisely conserved. In S. cerevisiae, the intron is unusual both because of its size and because of its location near the 3' end of the gene. We discuss possible functional roles for such an unusual intron in an RNA helicase gene.

Amino Acid Sequence↗
PubMed1990

Identification of a putative RNA helicase in E.coli.

The human p68 protein, an SV40 large T related antigen, is an RNA dependent ATPase and RNA helicase. It belongs to a new large and highly conserved gene family, the DEAD box proteins, whose members are involved in a variety of processes requiring manipulation of RNA secondary structure such as translation and splicing. Multiple DEAD box genes are present in S.cerevisiae, but only one has previously been described in E.coli. Low stringency screening of an E.coli genomic library with a p68 cDNA probe led to the identification of dbpA, a new E.coli DEAD box gene located at 29.6 minutes on the W3110 chromosome. We report here the nucleotide and deduced amino acid sequences of the gene. We have overexpressed dbpA from its own promoter on a high copy number plasmid and identified the gene product as a approximately 50 kD protein by immunoblotting with an anti-DEAD antibody.

Amino Acid Sequence↗
PubMed1989

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ.

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.

Animals↗
PubMed1987

Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics.

Epithelial cell cultures were initiated from explants of normal human oral mucosa. Growth parameters, cell type, and degree of maturation/cytodifferentiation were assessed by morphological and surface topographical criteria (light and scanning electron microscopy) together with immunofluorescence studies with a panel of antibodies to cytokeratins and extracellular matrix components. The effects of different media formulations were compared. Whereas stromal cell over-growth soon became apparent in media containing 10% serum, in low serum (0.5%) media containing insulin, hydrocortisone, epidermal growth factor (EGF), and/or cholera toxin (CT), epithelial growth was maintained with minimal or absent stromal cell contamination. Cell proliferation, maturation, and differentiation were modulated by EGF and CT: cultures maintained on EGF showed optimal growth but cells typically displayed only limited differentiation. By contrast, CT promoted considerably more cytodifferentiation but at the expense of proliferative capacity. Both factors together were complementary, resulting in maintenance of cells of a more mature phenotype of high proliferative capacity. Cytokeratins of normal oral epithelium in situ demonstrated characteristic changes in patterns of expression associated with differentiation. In culture, proliferative epithelial cells expressed keratins typical of the basal layer, whereas the most differentiated cells were identified by their strong reactivity with antibodies to epidermal keratins. Less mature cells showed expression of keratins associated with nonstratified epithelia. In cultures maintained with CT but no EGF, there was a tendency for weaker expression of basal type keratins, further suggesting that these cells were maintaining a more differentiated phenotype. Extracellular matrix components (fibronectin, laminin, collagen type IV) were not expressed by any epithelial cells in culture. Irrespective of medium composition, cultures did not survive beyond 100 days (5 or 6 subcultivations) before undergoing an irreversible 'crisis' of growth arrest and onset of degenerative changes.

Adolescent↗
PubMed1987

Colloidal gold--a powerful tool in scanning electron microscope immunocytochemistry: an overview of bioapplications.

Colloidal gold may be conjugated to a wide variety of macromolecules, provides a versatile system for immunocytochemical studies by various types of microscopy (light and fluorescent microscopy, scanning (SEM) and transmission (TEM) electron microscopy), and is significantly contributing to the development of SEM immunocytochemistry as a routine analytical procedure. A comprehensive overview has been compiled of the literature on SEM bioapplications of colloidal gold. This is illustrated through a selected series of studies focussing on a) cell surface receptor-ligand interactions; b) expression of cell surface lectin-binding sites; c) surface distribution of extracellular matrix components; and d) visualization of gold-labelled cytoskeletal elements with emphasis on the use of backscattered electron imaging as a powerful analytical adjunct in the development of SEM immunocytochemistry.

Animals↗