Normal Human Urothelium Research Articles

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.

MUCIN GENE EXPRESSION IN HUMAN UROTHELIUM AND IN INTESTINAL SEGMENTS TRANSPOSED INTO THE URINARY TRACT

The repertoire of mucin (MUC) gene expression in the normal human urothelium is poorly defined and the alterations in MUC gene expression following transposition of intestinal segments into the urinary tract has not previously been studied. The aims of this study were to define MUC gene expression in the normal human urothelium; and in transposed intestinal segments. Non-isotopic in-situ hybridization was carried out using eight digoxigenin labeled oligonucleotide mucin gene probes (MUC 1 - 7). Immunohistochemistry using NCL-MUC1 and NCL-MUC2 monoclonal antibodies was performed on sections of paraffin-embedded tissues. Twenty-seven patients were investigated (normal human urothelium, n = 6; transposed ileal segments, n = 14 and normal ileal controls, n = 7). MUC1 and MUC4 were the predominant mucin genes expressed in the normal urothelium with MUC3 being expressed in a third of cases studied; MUC2, 5AC, 5B, 6 and 7 were not expressed. Despite the morphological changes seen in transposed ileal segments, MUC2 and MUC3 continued to be expressed in these segments albeit in a disorganised fashion. Both MUC1 and MUC4 were up-regulated in transposed ileal segments, genes expressed by the normal human urothelium. All eight mucin genes were expressed in an area of pyloric-type metaplasia found in one transposed ileal segment. In patients with clam enterocystoplasty there was evidence of increasing up-regulation of MUC2, 3, 4 and 5AC expression in the urothelium toward the anastomotic site. Transposition of ileal segments into the urinary tract results in up-regulation of MUC1 and MUC4, the predominant MUC genes expressed in the human bladder. The clinical implication of the up-regulation of some MUC genes toward the anastomotic site in patients with an enteroplasty and the aberrant expression of MUC5AC - MUC7 by transposed segments is at present unclear.

Mesenchymal reprogramming of adult human epithelial differentiation

The objective of this study was to determine whether neonatal rat seminal vesicle mesenchyme (rSVM) can reprogram epithelial differentiation in a fully differentiated adult human bladder epithelium. For this purpose neonatal rSVM was isolated from newborn (0-day) Sprague-Dawley rats, and normal adult human bladder epithelium (hBLE) was isolated from radical cystoprostatectomy specimens to prepare rSVM+hBLE tissue recombinants in vitro. After overnight culture the tissue recombinants were grafted beneath the renal capsule of male athymic rodent hosts and allowed to grow in vivo for 6 months. As controls, rSVM and hBLE were grafted separately and allowed to grow for the same period. Tissue recombinants and control tissue grafts were harvested, and secretions were collected for biochemical studies. Tissues were fixed both for histologic as well as immunohistochemical staining. Neonatal rSVM induced normal adult human bladder urothelium to form glandular structures resembling prostate. The induced prostatic acini were filled with secretions that expressed human prostate-specific secretory proteins. These findings demonstrate that adult human urothelial cells retain a responsiveness to neonatal prostatic mesenchymal inductors. Change in urothelial histodifferentiation was associated with change in functional activity. The ability of the neonatal rat mesenchymal tissues to induce morphologic as well as biochemical changes in normal adult human urothelium provides a basis for human tissue engineering and organ reconstruction.

Expression of transforming growth factor β1 and its receptors in normal human urothelium and human transitional cell carcinomas

Previous studies indicated that transforming growth factor β 1 (TGFβ 1) is expressed by normal urothelial cells and exerts regulatory autocrine functions in urothelial maintenance and wound healing. However, little is known about the expression patterns of TGFβ 1 and its receptors in bladder tumors. Therefore, we studied the protein and mRNA localization of TGFβ 1 and TGFβ receptor types I and II (TGFβRI and TGFβRII) in normal human urothelium and transitional cell carcinomas (TCCs) of different grades and stages. Expression of TGFβ 1 and its receptors was examined by immunocytochemistry and mRNA in situ hybridization in normal urothelium and TCCs using a semiquantitative method. By immunocytochemistry, the expression of TGFβ 1 and TGFβRII was higher in superficial and basal cell layers of normal urothelium than in the intermediate layer. A similar localization was seen in superficial TCCs. TGFβRI was mainly present in basal and intermediate cell layers of normal urothelium and superficial TCCs. In contrast, in muscle invasive TCCs, all tumor cells stained intensely for all three proteins. No correlation was found between immunostaining and TCC grade. In situ hybridization pointed out that all cell layers in normal urothelium exhibit similar TGFβ 1 mRNA levels. Elevated TGFβ 1 mRNA levels were noted in TCCs irrespective of grade or stage. In conclusion, these data indicate that in normal urothelium TGFβ 1, TGFβRI, and TGFβRII expression depend on maturation and differentiation. This pattern is particularly lost in muscle invasive TCCs, in which the expression of the three proteins is enhanced. These data suggest autocrine TGFβ 1 mechanisms in human TCC cells that may be more pronounced in muscle invasive TCC cells.

Cloning of the human uroplakin 1B cDNA and analysis of its expression in urothelial-tumor cell lines and bladder-carcinoma tissue.

The human uroplakin 1B (UPK1B) gene codes for a structural protein which is a terminal differentiation component of the asymmetric unit membrane on the apical surface of the mammalian bladder. UPK1B is a member of the tetraspan family of proteins, many of which have de-regulated patterns of expression in cancer. Using polymerase-chain-reaction techniques, we have cloned a partial human UPK1B cDNA which codes for the putative full open reading frame for the UPK1B protein. The deduced human UPK1B protein sequence has 92% and 93% amino-acid homology with bovine UPK1b and mink TI1 proteins respectively. Using Northern analysis, we show that the human UPK1B gene is highly expressed in normal human urothelium. However, expression of UPK1B mRNA was undetectable or markedly reduced in 11 out of 16 samples of transitional-cell-bladder-carcinoma tissue and in all 5 bladder-carcinoma cell lines when compared with normal urothelial tissue. The molecular mechanism of down-regulation of RNA expression does not appear to involve gross gene rearrangements or allelic loss.

Uroplakin Gene Expression by Normal and Neoplastic Human Urothelium

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

Interleukin-6 functions as an autocrine growth factor in human bladder carcinoma cell lines in vitro.

Interleukin (IL)-6 is reported to function as a growth factor for renal and prostatic carcinomas. We conducted the present study to define the role of IL-6 in the growth of normal and neoplastic urothelial cells. Human bladder carcinoma cell lines (253J, RT4 and T24) and primary cultured human urothelial cells derived from normal ureters were used. Recombinant human IL-6 stimulated the growth of bladder carcinoma cell lines far better than that of normal urothelial cells (p < 0.001). All carcinoma cell lines tested produced and released IL-6, whereas normal urothelial cells did so only at marginal levels. Furthermore, treatment with lipopolysaccharide derived from Escherichia coli, tumor necrosis factor-alpha or IL-1 increased IL-6 secretion by bladder carcinoma cell lines but not by normal urothelial cells. Growth of bladder carcinoma cells was significantly inhibited by anti-IL-6 neutralizing antibody or the anti-sense oligonucleotide for IL-6 cDNA. We conclude that IL-6 functions as an autocrine growth factor for bladder carcinoma cells but not for normal urothelial cells and that it may be a factor accounting for the marked enhancement of inflammation-associated bladder carcinogenesis and tumor growth.

Induction of multiple PCR-SSCPE mobility shifts in exons in cultures of normal human urothelium exposed to low-dose gamma radiation

We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma -radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma -radiation from a 60Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene postirradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53 -related genomic instability.

Apoptosis and other effects of radiation in normal human urothelial cells.

In this paper, an attempt is made to identify endpoints that might be of potential use in the quantification of radiation effects in human tissues. Irradiated cultures of cells that are not selected for clonogenic survival but are left in situ to grow after irradiation show a wide variety of morphological and biochemical abnormalities. These include nuclear fragmentation and other evidence of programmed cell death, but they also include a considerable amount of lysis, necrosis, and persistent abnormal growth and function, which are expressed in the progeny of irradiated cells. Induction of proteins associated with stress or shock responses, growth and cell cycle control, and control of apoptosis are also seen and may persist. The dose dependence of these various responses is documented, because it probably determines to a large extent the outcome of radiation exposure in terms of whether a cell dies, divides normally, or develops genomic instability, mutation, and ultimate carcinogenic progression of the progeny. Clearly, a cell that dies presents no further threat to the organism, nor does a fully repaired cell. Therefore, a major challenge facing radiation protection research is to define the population at risk of surviving with damage. The results show that there is a variation in response to radiation between different patient cultures that is detectable in an explant culture system of primary normal human urothelium. The growth pattern and protein expression postirradiation is consistent with apoptosis being a major determinant of low dose response to radiation. This form of death appears to be suppressed at higher doses and, in the majority of subjects, results in the presence of a highly abnormal population of cells, even though the population size is the same whether their progenitors were irradiated or not.

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.

Individual variation in the expression of proteins involved in the regulation of growth and apoptosis post‐DNA damage in normal human urothelium

AbstractThe changes in expression of five oncoproteins involved in the regulation of repair of DNA damage and postdamage proliferation of cells were measured in cultures of normal urothelium from over 60 patients. The results for oncoprotein expression were correlated with apoptotic index and growth rate postexposure. Both radiation and a metabolite of nitrosamine found in urine led to increased levels of p53 in cultured urothelium. There was significant interpatient variation. PCR‐SSCPE (single strand conformational polymorphism electrophoresis) analysis of foci occurring in the exposed cultures showed multiple mobility shifts in exons 5–8 of the p53 gene. These were present in irradiated cultures at high frequency, but they were only present in two of six cultures exposed to nitrosamine. Cells surrounding foci, although they were p53+ by immunocytochemistry, were mainly negative for mutations using SSCPE analysis. Expression of bcl‐2 was significantly increased following radiation exposure, but it was unchanged following nitrosamine treatment. EGFr and p21ras were uniformly increased in all carcinogen‐exposed cultures The results suggest that an early breakdown in control mechanisms of growth arrest and apoptosis may occur in urothelium following carcinogen exposure. The problem of interindividual variation in response must be considered in studies of this sort. © 1995 Wiley‐Liss, Inc.

Cellular heterogeneity in human transitional cell carcinoma: an analysis of optical properties and lectin binding.

Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Ward et al., 1987). This difference was most marked with Maclura pomifera agglutinin (MPA), Ricinus communis agglutinin (RCA) and Ulex europeus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.

Plasminogen Activator Regulation by Transforming Growth Factor-β in Normal and Neoplastic Human Urothelium

Plasminogen activators (PA) are thought to participate in the invasive and metastatic processes of malignancies and are known to be modulated by certain growth factors (Danø, K; Andreasen, P.A.; Grondahl-Hansen, J.; Kristensen, P.; Nielsen, L.S.; Skriver, L. Adv. Cancer Res. 44:139-266; 1985 and Laiho, M.; Keski-Oja, J. Cancer Res. 49:2533-2553; 1989). This report describes the effect of TGF-beta on the regulation of secreted PA activity produced by human fetal urothelium and neoplastic urothelial cell lines. Epidermal growth factor was previously shown to induce substantially different effects on PA production by normal versus neoplastic urothelial (Dubeau, L.; Jones, P.A.; Rideout, W.M.; Laug, W.E. Cancer Res. 48:5552-5556; 1988). This report demonstrates that log phase normal urothelium, but not transformed cells, responded to TGF-beta (1-10 ng/mL) by diminishing the total secreted PA activity. Northern and western analyses showed that the reduction in protease activity resulted from an increased level of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. Additionally, northern analysis of total mRNA levels at varying cell densities demonstrated modulation of tPA, PAI-1, and TGF-beta transcripts in normal urothelial cells as a function of growth in vitro, suggesting the presence of an intact regulatory pathway to control extracellular proteolysis.

Antigen common to several species, recognized by a rat monoclonal antibody raised against syngeneic rat bladder tumor

A rat monoclonal antibody (MAb) termed RS-II (Ig M) was obtained by syngeneic immunization with rat bladder tumor cells induced by N-butyl, N-hydroxybutylnitrosamine (BBN). Immunocytochemical analysis showed that RS-II is also reactive with mouse, dog and human bladder tumor-cell lines and some other human tumor-cell lines but not myeloma or leukemia cells. Immunohistochemical examination of paraffin-embedded tissues has shown that RS-II is reactive with mouse, rat, dog and human bladder tumors (5/5, 5/5, 1/1, 31/49) and some other tumors, but not with normal human urothelium or normal rat tissues. The antigen is expressed on the majority of low-grade or well-differentiated tumors, but less on advanced, invasive tumors. The immunofluorescence staining pattern of cultured cells was cytoplasmic and filamentous. Immunoblotting revealed that the antigen is a Mr 54,000 to Mr 56,000 protein which was extracted with difficulty from the cultured cells with Triton X-100. The antigen was also detected in the culture supernatant by means of ELISA. Our results suggest that the epitope is expressed on cytoskeletons common to a wide variety of mammalian cells at a certain stage of differentiation.

Cellular heterogeneity in normal and neoplastic human urothelium: a study using murine monoclonal antibodies.

To assist in the description of the cellular heterogeneity present in normal and neoplastic urothelium, a panel of monoclonal antibodies (MoAbs) was raised against human transitional cell carcinoma (TCC) of the urinary bladder. All immunizations were carried out using whole cells and membrane preparations from well differentiated human TCCs. Two fusions produced 145 hybridomas. Following primary screening by ELISA and secondary screening with immunohistochemistry, three useful antibodies were identified. MoAb 35.48 binds to all cell layers of the normal urothelium and well differentiated tumours, but not to the majority of poorly differentiated tumours. MoAb 21.48 binds preferentially to the basal cell layer of normal urothelium and to some well differentiated papillary TCCs, but poorly differentiated tumours exhibit diffusely positive staining. MoAb 21.48 also shows cross-reactivity with basal cell layers of other epithelia. MoAb 5.48 binds preferentially to the superficial cell layers of normal urothelium and well differentiated TCCs, but exhibits less binding in poorly differentiated tumours with loss of the preferential superficial staining. Quantitative flow cytometric studies indicate that MoAb 5.48 binds to a cell-surface antigen which is present on significantly fewer cells of poorly differentiated tumours than on either normal urothelium (P less than 0.05), or well differentiated tumours (P = 0.05).

Cellular heterogeneity in normal human urothelium: quantitative studies of lectin binding.

A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II+) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.

X-ray microanalysis--a method for determination of elements in bladder biopsies.

Chemical elements in biopsies of normal human urothelium and transitional cell carcinoma (TCC) are determined by X-ray microanalysis in combination with scanning electron microscopy. The methodology presented is intended to be used in subsequent studies to compare data of normal urothelium and TCC of different stage and grade.

Cellular heterogeneity in normal human urothelium: an analysis of optical properties and lectin binding.

Flow cytometry was used to study the optical properties of normal urothelial cells in suspension. Narrow-angle light scatter, which is a function of cell size, defined one major and one minor cell population, and 90 degrees light scatter, a function of intracellular structure, showed three distinct cell populations. These properties were displayed as a 2-dimensional dot plot or "fingerprint" which proved to be characteristic and reproducible from one specimen of urothelium to the next. Cell sorting on the basis of these two parameters demonstrated that the small cells of the basal layer occupy the low narrow angle, low 90 degrees light-scatter region; the giant cells of the superficial layer lie in the high narrow angle, high 90 degrees scatter region; and the pyramidal cells of the intermediate layer lie in an intermediate zone. Studies of tissue sections using the galactose-specific, FITC-conjugated Maclura Pomifera lectin (MPA) demonstrated preferential binding to the superficial layers of intact urothelium. In order to quantify the apparent differences in lectin binding between the superficial and basal layers, urothelial cell suspensions were labeled with FITC-conjugated MPA and studied by flow cytometry. The resolution obtained on the basis of light scatter made it possible to quantify the difference in lectin binding to the three morphologically recognized cell types present in normal urothelium.

Immunohistochemical identification of lymphocyte subsets and macrophages in normal human urothelium using monoclonal antibodies.

Monoclonal antibodies were used to examine the pattern of distribution of lymphocyte subpopulations and macrophages in normal human urothelium. T lymphocytes (T11-positive cells) were demonstrated within the epithelium and within the lamina propria of both the ureter and the urinary bladder. Suppressor/cytotoxic T cells (T8-positive cells) predominated, with the average ratio of suppressor/cytotoxic T cells to the helper/inducer T cells being approximately 8.9 in the epithelium and 1.5 in the lamina propria. Leu M3+ cells (monocytes/macrophages) were detected in both epithelium and lamina propria. The character and distribution of these immunocompetent cells within the urothelium suggest a protective function, especially against infection and in tumour surveillance.

Growth of a Malignant Human Urothelial Cell Line (J82) in a Serum-Free Medium (HMRI-1) Developed Previously for Normal Human Urothelium

A malignant human urothelial cell line (J82) has been cultured in a serum-free medium (HMRI-1) developed specifically for normal human urothelium. Unlike the normal human urothelium which grew as an attached monolayer, the J82 cells proliferated as free floating cellular aggregates. Comparative growth kinetic studies have shown that the J82 cells, unlike normal cultured urothelial cells, did not require epidermal growth factor, bovine pituitary extract or transferrin as single additives in the HMRI-1. However, deletion of certain combinations of these factors markedly reduced growth. It was concluded that the J82 cells had a reduced dependency on the normal urothelial cell growth factors suggesting that the J82 cells had altered nutritional requirements which might possibly be associated with the production of autocrine growth factors.