Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.     

Centrifugal separation of carcinoma or atypical cells in voided urine

A simple density gradient method was used to separate atypical and cancer cells from non-cancer cells in voided urine from patients with transitional cell atypia (moderate and grave atypia) and bladder cancer (squamous cell carcinoma and transitional cell carcinoma). Prior to cell separation, the Saccomanno preserved cells were dispersed by homogenization. After cell separation (5 min x 1400 rpm), atypical and cancer cells were enriched up to 20-fold. Also, most of the leucocytes (68-98%) and squamous cells (47-82%) were absent from density gradient specimen fractions containing the largest percentages of atypical and cancer cells. Peak purity ranges of atypical or cancer cells from different sample classes showed a large degree of overlap. This permitted the pooling of density gradient fractions enriched for atypical or cancer cells, thus increasing the efficiency of the method. Also, following centrifugation, the Papanicolaou-stained specimen fractions showed less background staining than the unprocessed controls, and the cells retained diagnostic morphologic features. We infer that this method may be a useful, low-cost approach for the morphologic study of developing cancers, not only from the urinary bladder, but also from the respiratory tract.     

Ras oncogene mutations in urine sediments of patients with bladder cancer

Early detection of bladder cancer is particularly important since it dramatically affects the survival rates. However, neither urinary cytology nor tumor markers that are currently used are sensitive enough for the early detection of bladder cancer or recurrent disease. The ras genes are frequently mutated in cancer. In this study, we investigated the diagnostic potential of ras mutation analysis in urinary sediments of patients with bladder cancer using a single-strand conformation polymorphism analysis and polymerase chain reaction. Mutation in codon 12 of the H-ras gene was observed in 39% of the patients. Our results indicate that this approach may significantly improve diagnostic sensitivity in detecting bladder tumors.     

Alterations in microvilli of uterine epithelial cells after colchicine treatment

Colchicine has been injected intra-peritoneally to pregnant rats to determine the effect of this drug on the microvilli of uterine luminal epithelial cells. Microvilli in control animals (saline injected) were 0.51 +/- 0.07 micron tall, whereas in colchicine treated they were only 0.24 +/- 0.02 micron tall. We conclude that colchicine reduces microvillar height in these cells via interaction with microtubules, and suggest that modulations of microtubules probably contribute to the alterations in the microvilli of these cells seen during normal pregnancy.     

Cytologic evaluation of low grade transitional cell carcinoma and instrument artifact in bladder washings

OBJECTIVE: To identify key cytologic features for the separation of low grade transitional cell carcinomas (TCCs) from nonneoplastic lesions in bladder washings. STUDY DESIGN: The cytomorphologic features of 95 bladder washing specimens showing papillary fragments, which included 50 low grade TCCs and 45 nonneoplastic lesions, were reviewed retrospectively. RESULTS: Bladder washings from low grade TCCs showed papillary and irregular groups of cells with ragged borders, cytoplasmic homogeneity and subtle nuclear changes, such as increased nuclear/cytoplasmic ratio and irregular nuclear border. Bladder washings after instrumentation from nonneoplastic lesions of the bladder showed cellular specimens with cohesive, ball-shaped and papillary clusters with smooth borders lined with a denser-staining cytoplasmic collar. Reactive urothelial cells often displayed loose aggregates with irregular borders but no cytoplasmic collar. CONCLUSION: In bladder washing cytology, nuclear changes and cytoplasmic homogeneity play a major role in the diagnosis of carcinoma.     

Abundant dendritic cells express HLA-DR in pleomorphic adenomas

Most pleomorphic adenomas were found to contain abundant dendritic cells (DC) with major histocompatibility complex (MHC) class II (HLA-DR) expression. Their immunohistochemical staining features were suggestive of dendritic histiocytic cells. Extensive phenotypic characterization by two-colour immunofluorescence staining for various cell markers was performed. The DC expressed both HLA class I and II determinants, vimentin, S-100 protein, and various monocyte-related markers (10G11, 3D10, 7G5 or CD11a, 8C2) but were negative for leucocyte common antigen (CD45), Leu-6 (CD1), and the myelomonocytic L1 antigen. Characterization of HLA-DR positive DC isolated by an immunomagnetic bead method confirmed the immunohistochemical staining pattern that corresponds to the phenotype of interdigitating cells. Morphological and immunological implications of the abundant presence of these cells in pleomorphic adenomas are discussed.     

Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new view of mechanotransduction and strain amplification in cells with microvilli and cell processes

In this paper we shall describe new mechanical models for the deformation of the actin filament bundles in kidney microvilli and osteocytic cell processes to see whether these cellular extensions, like the stereocilia on hair cells in the inner ear, can function as mechanotransducers when subject to physiological flow. In the case of kidney microvilli we show that the hydrodynamic drag forces at the microvilli tip are <0.01 pN, but there is a 38-fold force amplification on the actin filaments at the base of the microvilli due to the resisting moment in its terminal web. This leads to forces that are more than sufficient to deform the terminal web complex of the microvillus where ezrin has been shown to couple the actin cytoskeleton to the Na(+)/H(+) exchanger. In the case of bone cell processes we show that the actin filament bundles have an effective Young's modulus that is 200 times > the measured modulus for the actin gel in the cell body. It is, therefore, unlikely that bone cell processes respond in vivo to fluid shear stress, as proposed in [59]. However, we show that the fluid drag forces on the pericellular matrix which tethers the cell processes to the canalicular wall can produce a 20-100 fold amplification of bone tissue strains in the actin filament bundle of the cell process.     

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples

OBJECTIVES: Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine. METHODS: Flow cytometry was utilized to detect protoporphyrin IX in an artificial model consisting of normal urinary bladder transitional epithelial cells (NBECs) from healthy volunteers' urine and an established human urinary bladder carcinoma cell line, TCCSUP, after incubation with hexaminolevulinate (HAL). In addition, urine samples from 19 patients with histopathologically confirmed superficial bladder cancer were examined. RESULTS: Incubation of NBECs or TCCSUP cells with HAL for 1 hour resulted in production of protoporphyrin IX only in the TCCSUP cells. Incubation of a mixture of NBECs and TCCSUP cells with HAL gave rise to a separated subpopulation of cells with protoporphyrin IX fluorescence. After cell sorting by flow cytometry the protoporphyrin IX-containing subpopulation of cells was confirmed as TCCSUP cells on cytological examination. It was possible to detect 5% TCCSUP cells in the mixture of NBECs/TCCSUP cells. To test the feasibility of the method in clinica diagnosis, urine samples from patients with bladder cancer were also measured with comparable, although preliminary and limited, results to those of cytological examination. CONCLUSIONS: The preliminary results show that the technique may be feasible for the detection of bladder cancer cells in urine with possible advantages of simplicity, reliability and objectivity.     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.