PURPOSE: We investigated the inducible response of transcription factor Nuclear Factor Kappa B(NFkB) in response to lipopolysaccharide(LPS), interferons(INFs), and tumor necrosis factor(TNF) on the normal urothelial cells derived from the ureter and the bladder. MATERIALS AND METHODS: Urothelial cells were harvested from the ureter and the bladder, cultured, passaged and expanded in serum free medium. Immunostaining of the urothelial cells with reacting anti-cytokeratin antibodies was done to identify a stable epithelial phenotype. Cultured human urothelial cells were either untreated and treated with LPS, INFgamma, INFalpha and TNFalpha. Cell extracts were prepared and used for electrophoretic mobility shift assay(EMSA) using PRD II-kB as probes for NFkB respectively. RESULTS: We cultured urothelial cells successfully confirmed by immunohistochemical staining. In both urothelial cell types, NFkB bindings with their respective probe were induced by treatment with LPS, INFgamma and TNFalpha. NFkB was weakly induced by INFalpha. For the NFkB complex, a distinctly different migrating pattern of the NFkB was noted between the diffrent urothelial cells. CONCLUSIONS: Many urothelial diseases are unique to specific areas of the urinary collecting system. The characterization of different inducible responses of transcription factors involved in cytoplasmic and nuclear signaling of genes encoding immunologically relevant proteins provide a unique opportunity for understanding disease presentation and designing specific treatment interventions.
Antibodies ; Cell Culture Techniques ; Cell Extracts ; Cytoplasm ; Humans* ; Necrosis ; Phenotype ; Transcription Factors ; Tumor Necrosis Factor-alpha ; Ureter ; Urinary Bladder ; Urothelium

PURPOSE: While the exact inflammatory and immunological mechanisms involved in the induction of immunotherapy for superficial bladder carcinoma are mostly unknown, many of these mechanisms are mediated through the activation of transcription factors involved in signal transduction pathways Nuclear Factor Kappa B(NFkappaB) signal transduction pathways induce transcription factors that activate genes encoding immunological proteins such as cytokines and cell Surface maskers. This study investigates the activation pattern of NFkappaB by lipopolysaccharide(LPS), interferon gamma(INFgamma), and tissue necrosis factor alpha(TNFalpha) in low(RT4) and high(T24) grade transitional carcinoma cell lines. MATERIALS AND METHODS: Low and high grade transitional carcinoma cell lines were cultured with Eagle's minimum essential medium. Using electrophoretic mobility shift assays(EMSA) of whole cell extracts from cell cultures of RT4 and T24 after exposure to LPS, INFgamma and TNFalpha, activation of NFkappaB complex has been demonstrated. Degradation of IkappaB has been demonstrated by Western blot analysis using IkappaBalpha /MAD-3. RESULTS: NFkappaB complex was induced by TNFalpha in both RT4 and T24 cells as determined by EMSA. NFalphaB complex was also Induced by INFgamma in the RT4 cells but not in the T24 cells. However in LPS treatment, the NFkappaB complex was strongly induced in T24 cells and the induction was very weak in RT4 cells. Inhibitor of NFkappaB (IkappaBalpha) was degraded rapidly after LPS treatment in the T24 cells as determined by Western blot analysis with IkappaBalpha specific antibody. However, the level of IkappaBalpha protein was same in the RT4 cells before and after LPS treatment. CONCLUSIONS: Identification of differences in low and high grade tumor cell lines in the inducibility of transcription factors by LPS and INFgamma provide an opportunity for understanding the observed differences in the mechanisms within the signal transduction pathways will ultimately create disease specific as well as patient specific treatments for these malignant urothelial disorders.
Blotting, Western ; Cell Culture Techniques ; Cell Extracts ; Cell Line* ; Cell Line, Tumor ; Cytokines ; Humans ; Immunotherapy ; Interferons ; Necrosis ; Signal Transduction ; Transcription Factors ; Tumor Necrosis Factor-alpha ; Urinary Bladder ; Urinary Bladder Neoplasms