Normal Bladder Cells Research Articles

In Vitro Study of the Effect of Hyperthermia on Normal Bladder Cell Line and on Five Different Transitional Cell Carcinoma Cell Lines

Intraluminal hyperthermia is potentially useful in the management of superficial bladder cancer. The potential inhibitory effect of hyperthermia on various human bladder cancer cell lines, normal human bladder cells and the murine MBT-2 bladder cancer cell line has been studied in vitro. These cell lines were exposed for one hour to 43 ± 0.5C and compared to controls. Cell survival was assessed comparing the cell growth curve and colony formation. The human transitional cell carcinoma (TCC) cell lines vary in their sensitivity to heat. MGH-U1 was the most heat sensitive cell line. The human A-1698, CUB-2, UM-UC-3 and the murine MBT-2 lines were heat insensitive. We conclude that the cytocidal effect of hyperthermia in bladder transitional cell carcinoma is variable. Further experiments using the combination of hyperthermia and intravesical anticancer agents are in progress.

Immunohistochemical detection of acidic fibroblast growth factor in bladder transitional cell carcinoma.

Acidic fibroblast growth factor (aFGF) is a regulatory peptide which, on account of its structural homologies with the products of oncogenes, is involved in cell proliferation, differentiation, and motility. We previously reported the presence of aFGF in the urine of patients with transitional cell carcinoma (TCC). aFGF can also induce the motility of a rat-derived bladder carcinoma cell line (NBTII). This immunohistochemical study used polyclonal rabbit antibodies against acidic and basic FGF and peroxidase detection. Native NBTII nude mice xenografts and aFGF transfected NBTII (NFS14) nude mice xenografts were used as tissue controls for antibody specificity. The samples included 4 normal urothelia and 12 TCC. In addition, cytospins of 4 different tumoral cell lines of human bladder and normal bladder cells were stained. The results showed strong immunostaining in all tumoral urothelium samples using anti-aFGF and a very low amount of staining or none at all in healthy tissues. A primary analysis suggested that the strongest reaction was obtained in high-grade tumors (3 + vs + for lower-grade tumors). Using bFGF antibody, strong immunohistochemical staining was detected on basal membranes and stromal vessels and none in urothelium. These data confirm aFGF expression in the epithelial cell compartment of bladder cancer and the likely involvement of this regulatory peptide in the biology of TCC.

Photodynamic therapy of bladder cancer ?uptake and phototoxicity of photosan in vitro

The uptake of photosan and the intracellular sites of photoradiation-induced damage were investigated in vitro in bladder carcinoma cells and in normal bladder cells. Cells were examined by phase contrast, fluorescence and electron microscopy. The concentration of photosan, measured in microgram/10(6) cells, showed a good correlation to the incubation time. At all incubation times, control cells showed a lower uptake when compared with tumor cells. Following photodynamic therapy (PDT), phase-contrast microscopy revealed marked changes in tumor cells, whereas only minor effects could be detected at the cell membrane of the control cells. Following PDT, most of the investigated cells showed changes of the mitochondria and cytoplasma. These changes consisted of dissolution of the cristae, predominantly in the central part of the mitochondria. Twenty-four hours after PDT the shape of the mitochondria had changed markedly and the cristae were found to be completely destroyed. Moreover, the cystoplasma showed numerous vacuoles, and the number of mitochondria was decreased compared to non-treated cells.

Discrimination of Cell Types in Primary Transitional Cell Carcinoma by Monoclonal Anti-Cytokeratin Antibodies

The morphologic discrimination between low and high grade malignant tumor cells arising in the urinary bladder is a major diagnostic problem in cytopathology. Using immunochemical peroxidase staining of cytokeratins (CKs) of human bladder exfoliative cytology specimens, we have been able to discriminate between transitional cell carcinoma cells, atypical cells and normal bladder cells. Commercially available monoclonal antibodies used in this study were: anti-CK 13 (Sigma K8.12), anti-CK 5, 7 and 8 (Sigma K8.13), anti-CK 19 (Sigma K4.62), and anti-CK 18 (Transformation Res. 1091). When normal bladder cells are stained with these antibodies, CK 18 is specific for superficial cells; CK 13 is specific for the basal type cells and CKs 5, 7, 8 and 19 are expressed by all urothelial cell types. Four cases diagnosed by cytopathological criteria as 'atypical' and 7 diagnosed as 'positive' (various grades) were used in this study. Cytologic diagnosis was confirmed by histopathology in 7 cases. Tissue was not available for histopathology in 4 cases. Malignant cells with a 'basal' or 'immature' phenotype preferentially stained with CK 13 and were associated with increased metastatic or malignant potential. Patients with higher grade tumors had more cells positive for CK 13 than did patients with atypical or lower grade malignancies. Patients with well-differentiated, low grade tumors predominantly shed cells that expressed CK 18 and CK 19. High grade, invasive bladder lesions were characterized by many cells expressing CK 13, and fewer cells expressing CK 19. The combination of diagnosis by morphologic criteria on Papanicolaou-stained specimens with immunochemical characterization of the same cells for CKs facilitate an accurate diagnosis of bladder lesions.

Flow Cytometry Study of Cytokeratin 18 Expression According to Tumor Grade and Deoxyribonucleic Acid Content in Human Bladder Tumors

Cytokeratin 18 expression in transitional cells of 76 bladder tumors (11 grade 1, 33 grade 2 and 32 grade 3) and 10 normal biopsies was quantified by flow cytometry after immunolabeling with the anti-cytokeratin 18 monoclonal antibody RGE 53. Combined cytometric analysis of deoxyribonucleic acid content and cytokeratin 18 expression was conducted. Of 76 tumor biopsies 38 had a unimodal deoxyribonucleic acid profile with a deoxyribonucleic acid index close to 1.0, while the other 38 showed a bimodal profile: 1 peak with an index of 1.0 and a second peak with an index of greater than 1.0. The 11 grade 1 tumors belonged to the first group, whereas 10 of the 33 grade 2 and 28 of the 32 grade 3 tumors belonged to the second group. Antibody RGE 53 reacted with 4 per cent of the normal bladder cells (umbrella cells), 14 ± 3 per cent of the cells from grade 1, 33 ± 8 per cent from grade 2 and 56 ± 10 per cent from grade 3 tumors. Analysis of cytokeratin 18 expression according to deoxyribonucleic acid content at the single cell level in tumors with a bimodal deoxyribonucleic acid profile showed that cytokeratin 18 was detected frequently in cells with a high deoxyribonucleic acid index but much less so in cells with an index of 1.0. Therefore, expression of cytokeratin 18 can be recognized as a marker of aggressiveness in bladder tumors, since it increases in parallel with tumor grade and cell deoxyribonucleic acid content.

Analyses of Avidin-biotin Complexes With Lectins of Membrane Glycoproteins in the Urinary Bladder of Rats Treated With N-butyl-N-(4-hydroxybutyl)nitrosamine

The bladder epithelium was examined by staining with avidin-biotin complexes with lectins to determine the early membrane changes during bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BHBN). Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), Ricinus communis (RCA) and Arachis hypogaea (PNA) were used as probes. The cellular distributions of lectin particles were distinguished into membranous and cytoplasmic patterns. Normal bladder cells stained very slightly and showed a spotty membranous pattern. After treatment of rats with BHBN for two or three weeks, staining became stronger and its pattern changed from a membranous to a cytoplasmic type. The staining of bladder cancer cells induced by BHBN varied from area to area and with different lectins. These data indicated that changes in carbohydrates occur in the bladder cell membrane during the early phase of carcinogenesis.

Differential Growth Regulation of Human Bladder Tumor Cells (J82) in Culture by Normal Bladder Cells from Different Aged Human Donors

This study used an agarose gel culture system to study growth regulatory interactions between normal and neoplastic human bladder cells. The effects of normal human fibroblasts on tumor cell cloning efficiency in agarose was studied using 2 experimental protocols. In 1 system, the fibroblasts proliferated as anchored cells beneath the agarose, and in the other the fibroblasts were nongrowing cells embedded within the agarose as nonanchored cells. In contrast to the largely inhibitory effects of anchored fetal fibroblasts on tumor cell growth, their nonanchored counterparts were exclusively stimulatory. Similar experiments with normal fibroblasts derived from the bladders of increasingly older donors showed a progressive decline in their capacity to inhibit tumor cell growth in agarose. Further experiments modulating the growth rate of neonatal fibroblasts with fetal bovine serum have indicated that the diverse tumor responses elicited by anchored versus nonanchored cells, as well as those observed for the aged bladder fibroblasts, were a function of fibroblast growth rate. We concluded that there was an inverse regulatory relationship between the growth of normal and tumor cells; rapidly growing fibroblasts were inhibitory whereas nongrowing fibroblasts stimulated tumor cell growth in agarose culture.

Quantitative fluorescence measurements of AO-stained normal and malignant bladder cells.

AbstractQuantitative, corrected fluorescence emission spectra from the nuclei of acridine orange‐stained bladder epithelial cells were obtained with a corrected spectrum microspectrofluorophotometer (MSF)5 which is calibrated with a phosphor particle fluorescence standard. The spectra demonstrated that the green fluorescence band (λmax≅530 nm) is stoichiometrically related to the amount of nucleic acids (NA) present in a given nucleus, provided this spectroscopic species has reached saturation. The shape of this spectral band is constant. On the basis of this information, we used a simple microfluorometer, (SFM), calibrated with the phosphor particle standard, to routinely measure the amount of green fluorescence emission from the nuclei of AO‐stained bladder epithelial cells. The amount of green fluorescence per normal nucleus never exceeded 0.5 phosphor particle units (ppu). In contrast, in every sample containing malignant or cancerrelated cells, one or more cells contained nuclei that exhibited more than 0.5 ppu total green nuclear fluorescence. Results obtained with the SFM on 794 AO‐stained cells from 80 samples taken from 40 patients are reported here. Samples consisted of urine specimens, bladder washings, and biopsies. One or more malignant cells from every grade 3 or 4 tumor and 17 of 17 grade 1 or 2 tumors showed markedly elevated levels of total (integrated) green nuclear fluorescence indicating the presence of DNA aneuploidy, strongly, suggestive of the presence of malignant or potentially malignant cells. Comparison with diagnoses based on H and E‐stained biopsy material and Papanicolaou‐stained cytological preparations show excellent correlation. The quantitative fluorescence measurements demonstrate a clear difference between negatives and positives with linear ordering up through Grade 4 and CIS. Grades 1 and 2 were clearly different from 3 and 4 (p<0.001). The results indicate that the quantitative fluorescence methodology reveals the presence and extent of DNA aneuploidy which may prove to be a more sensitive cytologic method for detection of low grade tumors and may also provide an objective biochemical basis for tumor grade classification. Detecting the presence of DNA aneuploidy may even serve to provide earlier detection than is possible with conventional means, but this remains to be confirmed by longitudinal studies.

Suppression of granulocyte-macrophage colony formation in vitro by conditioned medium derived from human bladder cancer cells.

Media conditioned with 8 cell lines were assayed for stimulating and inhibiting activities on granulocyte-macrophage colony formation from bone marrow cells in soft agar (CFUc). Conditioned media from 3 human bladder cancer cell lines (EJ, T24 and KK47) and a rat bladder cancer cell line (BC50-TC) showed marked inhibition of mouse CFUc. Conditioned medium from normal human bladder cells showed a weak colony-stimulating activity, and only a slight inhibition when added to CFUc culture. Conditioned media from mouse or rat fibroblast cell lines (BLP and 3Y1) showed no colony-inhibiting activity, and the conditioned media from human lymphoblastoid cell line (Raji) and human epithelia-like cell line from amniotic membrane (FL) showed moderate inhibitory activity. No colony-stimulating activity was detected in any of the cell lines tested. EJ-conditioned medium inhibited human CFUc, but not erythroid progenitors. Colony-inhibiting activity in EJ-conditioned medium appears not to be prostaglandins; it needs more than 3 hr to inhibit CFUc, is stable at 56 degrees for 30 min but not at 75 degrees for 20 min, and is susceptible to proteolytic enzymes.

Collection and evaluation of normal exfoliated urinary bladder cells in man using scanning electron microscopy.

Normal exfoliated urinary bladder cells were collected from midstream urine samples from 21 male patients. Procedures for urine filtration using Millipore filters of Nucleopore filters, cell fixation and preparation for scanning electron microscopy investigation were developed. Correlations were made between the surface morphology of exfoliated cells and that of biopsies from different anatomical regions of the urinary bladder. Scanning electron microsocopy (SEM) of exfoliated normal urinary bladder cells proved to be a more sensitive method for discovering cellular surface details than light microscopy. This information forms the basis for further SEM studies of cell surface alterations due to urothelial diseases.

Cellular microcytotoxicity in a human bladder cancer system: Analysis of in vitro lymphocyte-mediated cytotoxicity against cultured target cells

The microcytotoxicity test (MCT) has been used to determine the cytotoxic effects of purified peripheral blood lymphocytes from patients with carcinoma of the urinary bladder (BT), tumor control patients (TC) (tested after therapy), and healthy donors (HD) against cultured bladder tumor cells, melanoma cells, and normal bladder cells. Lymphocytes from all three donor groups were tested in parallel. Disease-specific cytotoxicity (CTX) is defined as statistically significant and selective destruction of disease-related tumor target cells by the test lymphocytes in comparison with the baseline controls. Nonspecific CTX is defined as statistically significant destruction of a proportion (selective) or all (nonselective) disease unrelated target cells by the effector cells.