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.

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.

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.

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.

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.

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.

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.

Urothelial cell transplantation using biodegradable synthetic scaffolds.

Reconstruction of the urinary bladder with bowel to restore storage capacity is associated with significant complications arising from substituting an absorptive, mucus-producing intestinal epithelium for the barrier urothelium of the bladder. To overcome these problems, we are developing a "composite enterocystoplasty" procedure to replace the epithelium of the bowel with autologous in vitro-propagated normal urothelial cells. The aims of this study were to evaluate synthetic biomaterials as delivery vehicles for the cultured urothelial cells and provide support during transfer and cell adherence to the de-epithelialized bowel wall. The surgical compliance of 12 biomaterials was evaluated, along with their ability to support urothelial cell attachment. Transfer of urothelial cells onto biomaterials as single cell suspensions or intact cell sheets was investigated. Seeding of a single cell suspension on to non-woven mesh resulted in poor cell attachment. Seeding onto woven mesh was more efficient, but the most effective transfer method involved producing an intact cell sheet that could be combined with woven, knitted and non-woven biomaterials. Transfer of the cell sheet : mesh complexes onto a de-epithelialized bladder stroma produced a stratified epithelium incorporating the strands of the mesh and expressing urothelial-associated antigens after 48 h in organ culture. Thus, we have developed and evaluated a suitable transfer method for in vitro propagated urothelial cells to be used in "composite enterocystoplasty".

N;-3 and n;-6 polyunsaturated fatty acids induce cytostasis in human urothelial cells independent of p53 gene function.

The role of long-chain polyunsaturated fatty acids (PUFA) in the etiopathology and treatment of cancer is poorly understood. We have studied the effects of n;-3 and n;-6 PUFA on the proliferation and survival of normal human uroepithelial (NHU) cells, cells with disabled p53 function after stable transfection with the human papillomavirus 16 (HPV16) E6 gene (HU-E6), and p53-disabled cells that had passed through crisis and acquired karyotypic abnormalities (HU-E6P). The n;-3 and n;-6 PUFA had distinct reversible antiproliferative and irreversible cytostatic effects according to concentration and exposure time. The reversible antiproliferative effect was partly due to the production of lipoxygenase metabolites. NHU and HU-E6 cells were equally sensitive to n;-3 and n;-6 PUFA, but HU-E6P cells were more resistant to both the antiproliferative and cytostatic effects. Cytostatic concentrations of n;-3 and n;-6 PUFA did not induce apoptosis, but caused permanent growth arrest ("interphase" or "reproductive" cell death) and mRNA levels for genes involved in cell cycle control (p21, p16, p27, cdk1, cdk2, and cdk4) were not altered. Neither n;-3 nor n;-6 PUFA promoted acquisition of karyotypic abnormalities in HU-E6 cells, suggesting that n;-3 and n;-6 PUFA do not cause genotoxic damage. In conclusion, our studies show that the antiproliferative and cytostatic effects of n;-3 and n;-6 PUFA are not dependent on p53 function and, further, that transformation results in a loss of sensitivity to n;-3 and n;-6 PUFA-mediated growth inhibition.

Effects of long-chain fatty acids on human urothelial cells in organ culture.

It has been suggested that tumour-derived cells are differentially sensitive to the anti-proliferative and cytotoxic effects of long chain n-3 and n-6 polyunsaturated fatty acids (PuFAs). We have previously shown that PuFAs are also growth suppressive to highly proliferative normal human urinary bladder uro-epithelial (NHU) cells grown in monolayer culture. To determine if the effects on NHU cells are directly related to the proliferative index, we have studied the effects of long chain fatty acids in a bladder organ culture system, where proliferation and differentiation of the urothelium is under homeostatic control. A 50 microM concentration of fatty acids was chosen as this concentration of PuFA was profoundly growth inhibitory to NHU cells in monolayer culture. In organ culture, 50 microM PuFAs had no detectable effect on the proliferation or on the preservation of urothelial differentiated histioarchitecture, as assessed using a panel of phenotypic markers. These results suggest that the effects of PuFA may be modulated by the tissue microenvironment.

Subcutaneous administration of recombinant glycosylated interleukin 6 in patients with cancer: pharmacokinetics, pharmacodynamics and immunomodulatory effects.

This is the first report of the serum profile of a glycosylated recombinant form of human IL-6 (rhIL-6) administered subcutaneously (1-10 microg/kg/day) in a phase I/II trial as a thrombopoietic agent in patients with advanced cancer. The pharmacodynamic effects of IL-6 were also examined. Detailed pharmacokinetic measurements were made in four patients. Peak concentrations at 5-8 h and a median t0.5 of ca. 5 h were similar to those previously reported for non-glycosylated IL-6. However, higher peak concentrations and apparent differences in effective dose levels to those previously reported with the non-glycosylated form were seen. Indications of an apparent attenuation in circulating IL-6 concentrations with continuing injections were seen in eight of 10 patients examined but anti-IL-6 antibody generation was seen in only two patients. Soluble interleukin 6 receptor concentrations generally decreased. No major changes in T cell subsets were seen but expression of CD25 and CD54 by T lymphocytes significantly increased, accompanied by marked increases in soluble CD25 (sIL-2R) and CD54 (sICAM-1). No consistent change in B cells, monocytes or NK cells were seen. No evidence for induction of TNF-alpha was found. This study demonstrates similar biological effects of glycosylated rhIL-6 to those reported for the non-glycosylated form but illustrates several apparent differences which are discussed further.

Inhibition of T cell activation by normal human biliary epithelial cells.

BACKGROUND/AIMS: Human intrahepatic biliary epithelial cells can express immune recognition elements and are targets for immune attack in several liver pathologies. The aim of this study was to investigate the ability of biliary epithelial cells to act as accessory cells for T cell activation in normal and inflammatory conditions. METHODS: Normal biliary epithelial cells were cocultured with allogeneic unstimulated and mitogen- or antigen-stimulated peripheral blood lymphocytes. T cell responses were assessed by flow cytometry. RESULTS: Biliary epithelial cells did not induce allostimulation in resting T cells and inhibited T cell activation in response to either phytohaemagglutinin, mitogenic anti-CD3 antibody or recall antigen, irrespective of the presence of accessory cells. Biliary epithelial cells did not affect T cell viability, promote or inhibit activation-induced apoptosis nor modulate expression of CD95/Fas. In presence of biliary epithelial cells, stimulated T cells failed to develop an antigen-committed (CD45R0hi) phenotype and were unresponsive to subsequent CD3 ligation. However, T cells underwent normal activation in the presence of biliary epithelial cells which had been pre-treated with Interferon gamma or TGFbeta, cytokines implicated in liver disease. CONCLUSIONS: In normal liver, biliary epithelial cells inhibit rather than promote T cell activation, but their anergising effects may be overcome in response to trauma.

Application of a marker of ciliated epithelial cells to gynaecological pathology.

BACKGROUND: The assessment of neoplastic disease in gynaecological histopathology can be complicated by the high incidence of metaplasia seen in tissues of the female genital tract. There is a need to identify specific tissue markers which can be applied in routine histopathological practice. AIM: To examine the clinical potential of a monoclonal antibody, LhS28, which reacts with basal bodies of ciliated epithelial cells. METHODS: A panel of normal and pathological gynaecological tissues was processed and labelled with LhS28. RESULTS: LhS28 immunoreactivity was found in the normal Fallopian tube where it was confined to ciliated rather than secretory epithelial cells. In the remaining specimens, LhS28 was associated exclusively with ciliated cells in tubal metaplasias of the cervix and endometrium and in benign serous lined inclusion cysts. CONCLUSIONS: LhS28 may be a valuable marker for identifying metaplasia of tubal type and may find application in distinguishing tubal metaplasia from low grade cervical glandular intraepithelial neoplasia.

Expression of an antigen associated with basal bodies of human ciliated epithelial cells.

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000-45,000 M(r) protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9 + 0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.

Expression of activation and costimulatory elements by human intestinal intraepithelial lymphocytes.

It is unclear whether human intestinal intraepithelial T lymphocytes (iIEL) are resting or activated cells. To address this question, an improved isolation procedure was developed for small bowel iIEL, which were analysed by two-colour flow cytometry and compared with resting and mitogen-activated peripheral blood lymphocytes. iIEL expression of CD44 isoforms, Bcl-2 and Ki67 antigen was also determined in tissue sections. iIEL expressed CD69 at levels comparable with 48-72 h phytohaemagglutinin blasts, but did not express CD25 or CD95. iIEL were Bcl-2+ but not Ki67+. alphaEbeta7 and alpha4B7 expression was relatively high, whereas alphaLbeta2, CD5 and CD28 were expressed at low density. Isolated iIEL expressed CD44 (core epitopes) at lower levels than peripheral blood lymphocytes, although almost all CD44 contained splice variant 6 (CD44v6). Peripheral blood lymphocytes expressed CD44 at very high density, but little CD44v6, even after activation. However, in tissue sections, iIEL showed differential labelling with CD44 core epitope antibodies and no detectable CD44v6, implying CD44 receptor occupancy or epitope masking in situ. Thus, normal iIEL express a quasi-activated phenotype with unusual patterns of adhesion receptors, which may act as costimulatory elements. These may permit iIEL to assume effector functions, with absence of CD25 preventing entry into the cell cycle, thereby maintaining an apoptosis-resistant phenotype.

Cytokeratin expression patterns in normal and malignant urothelium: a review of the biological and diagnostic implications.

The cytokeratins are the intermediate filament proteins characteristic of epithelial cells. In human cells, some 20 different cytokeratin isotypes have been identified. Epithelial cells express between two and ten cytokeratin isotypes and the consequent profile which reflects both epithelial type and differentiation status may be useful in tumour diagnosis. The transitional epithelium or urothelium of the urinary tract shows alterations in the expression and configuration of cytokeratin isotypes related to stratification and differentiation. In transitional cell carcinoma, changes in cytokeratin profile may provide information of potential diagnostic and prognostic significance. The intensification of immunolabelling with some CK8 and CK18 antibodies may underly an active role in tumour invasion and foci of CK17-positive cells may represent proliferating populations. Loss of CK13 is a marker of grade and stage and de novo expression of CK14 is indicative of squamous differentiation and an unfavourable prognosis. However, perhaps the most important recent finding is the demonstration that a normal CK20 expression pattern is predictive of tumour non-recurrence and can be used to make an objective differential diagnosis between transitional cell papilloma and carcinoma. This review will consider cytokeratin expression in urothelium and discuss the application of cytokeratin typing to the diagnosis and prognosis of patients with TCC.