Transformation of Human Urothelial Cells (UROtsa) by As 3+ and Cd 2+ Induces the Expression of Keratin 6a

This laboratory has demonstrated that both cadmium and arsenite can directly malignantly transform the UROtsa cell line (Sens et al. 2004). It was also shown that the tumor heterotransplants produced by the Cd2+- and As3+-transformed cells had histologic features consistent with human transitional cell carcinoma of the bladder. The parent UROtsa cell line was developed from a primary cell culture of human urothelium that was immortalized using the SV40 large T-antigen (Petzoldt et al. 1994, 1995). The UROtsa cells proliferate in a simple serum-containing growth medium, retain a normal cytogenetic profile, grow as a contact-inhibited monolayer, and possess an undifferentiated morphology consistent with basal epithelial cells. They are not tumorigenic as judged by their inability to form colonies in soft agar and tumors in nude mice. Our laboratory also adapted the UROtsa cells to grow in a serum-free growth medium (Rossi et al. 2001). Under serum-free conditions, the cells exhibited enhanced differentiation, as evidenced by the presence of a stratified morphology consistent with the structural features associated with the intermediate layers of the urothelium. The cells grown in serum-free medium retained the properties of immortality, contact inhibition, and nontumorigenicity. Total RNA isolated from the parental UROtsa cells and their As3+- and Cd2+-transformed sublines grown in serum-containing growth medium were subjected to microarray analysis employing the U133 Plus 2.0 Affymetric chip. One of the stronger differentially expressed signals between the parental and the As3+-transformed cells was the keratin 6a gene. Our first goal in the present study was to confirm the differential expression of keratin 6a in the parental and transformed UROtsa cell lines at both the mRNA and protein levels. The second goal was to determine the expression of the keratin 6a gene in tumor heterotransplants derived from the As3+- and Cd2+-transformed UROtsa cells. The final aim was to demonstrate that the results from this model system could potentially translate to human disease by determining if keratin 6a was overexpressed in archival specimens of human bladder cancer.