The major emphasis of this proposal is to define the role of oncogenes, growth factors and DNA methylation in the growth and mutation of normal human urothelium and to determine how gene regulation and response to growth factors are altered during oncogenic transformation, tumor growth and metastasis. The proposal relies on the use of a serum-free medium we have developed for the growth of normal and neoplastic urothelial cells. It will be used to define the role of putative endogenous and exogenous growth factors in the division and maturation of normal urothelial cells. Studies to identify appropriate markers for maturation of urothelium in vitro will be continued and the effects of different growth conditions on the expression of the differentiated phenotype will be studied. Co-cultured experiments will investigate interactions between epithelial cells and bladder mesenchymal cells cultured from the same tissue source to determine the effect of stromal-epithelial interactions in normal maturation and growth. The next major aim is to determine how growth factor requirements and cell-cell interactions are altered in cell lines derived from papillary tumors and invasive bladder cancer cells so we may determine how these properties are modified by neoplasia, invasion and metastasis. The tissue culture studies will be complemented by a molecular analysis of genes thought to be important in epithelial differentiation and growth including the EGF receptor and the c- Ha-ras genes. The mechanisms of extracellular matrix breakdown by invasive transitional cell carcinomas will be investigated and we will continue the development of a nude mouse model we have described which allows for human bladder tumor metastasis. This model will be used to isolate metastatic cells and also to determine whether 5-azadeoxycytidine treatment can alter bladder tumor cell behaviour by alterations in DNA methylation patterns. As a complement to this study, we are isolating genes activated in osteogenic sarcoma cells by 5-azadeoxycytidine treatment and which may play a role in the tumorigenicity of these cells. Finally, we will conduct an RFLP analysis of human transitional cell carcinomas to determine the potential relevance of hemi and/or homozygosity for chromosomes 9 and 11 to bladder cancer.
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