The immortalized UROtsa cell line as a potential cell culture model of human urothelium.

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

Heat shock protein expression in testis and bladder cancer cell lines exhibiting differential sensitivity to heat.

Testis cancer cells are more sensitive than bladder and most other cancer cells to chemotherapeutic drugs both in the clinic and in vitro. In this study we show that they are also more sensitive than bladder cancer cells to heat. Since heat and drug sensitivity may be related to the ability of a cell to mount a stress response, constitutive and induced levels of heat shock proteins (HSPs) in three testis and three bladder human cancer cell lines were measured using Western blotting and scanning densitometry. No correlation between constitutive levels of HSP 90 or HSP 73/72 and cellular heat sensitivity was found. However, HSP 27 levels were much lower in the testis tumour cells, suggesting that low HSP 27 expression might contribute to heat sensitivity. Protein synthesis studies using [35S]methionine indicated that, for the same heat shocks, the kinetics of synthesis and decay of HSP 90 and HSP 73/72 in 833K (the most heat sensitive testis cells) was similar to or greater than that in HT1376 (the most heat-resistant bladder cells). Both 833K and HT1376 developed thermotolerance, and this followed an increase in synthesis of HSPs. These results indicate that, although there are differences in the constitutive levels of HSPs between testis and bladder cancer cells, both cell types are capable of mounting an induced heat shock response and can develop a similar degree of thermotolerance.

Factors influencing the sensitivity of two human bladder carcinoma cell lines to cis-diamminedichloroplatinum(II).

A two-fold difference in sensitivity to cis-diamminedichloroplatinum(II) (cisplatin), as judged by colony forming assays, has been demonstrated in two human bladder carcinoma continuous cell lines. Approximately twice as many DNA-DNA interstrand cross-links (ISL) and a 2-fold greater inhibition of DNA synthesis occurred in the more sensitive T24 cell line than in the RT112 cell line after exposure to the same concentrations of cisplatin. Equitoxic concentrations of cisplatin resulted in similar extents of ISL and inhibition of DNA synthesis in both cell lines. Although drug uptake was identical, twice as much cisplatin was bound to the DNA of T24 cells than RT112 cells. However after equitoxic concentrations of cisplatin the DNA from both cell lines was platinated to a similar extent. In addition, levels of glutathione (GSH), glutathione reductase (GR) and total glutathione-S-transferases (GST) were higher in the less sensitive RT112 cell line.

Comparison of the cytotoxic activities of chemotherapeutic drugs using a human bladder cancer cell line.

Many chemotherapeutic drugs have been used to treat patients with advanced bladder cancer, but few of these have been evaluated adequately in phase II clinical trials. Continuous cell lines provide one means for comparing the in vitro cytotoxicities of anticancer agents. In this study, a continuous cell line derived from a transitional cell cancer of the human bladder, which still produces tumours histologically similar to the tumour of origin on xenotransplantation, was used to measure the in vitro cytotoxicities of twelve chemotherapeutic drugs by clonogenic assay. The most cytotoxic agents tested were methotrexate, mitoxantrone, adriamycin, mitomycin C and cisplatin. These in vitro findings are compatible with the activity of these drugs given systemically as single agents in phase II clinical trials in patients with advanced bladder cancer.