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Prostate cancer is the second leading cause of male death from malignant disease in Europe and in the USA. Failure to prevent or eliminate metastatic dissemination is a fundamental problem underlying the current inadequate treatment of prostate cancer, and novel therapeutic strategies are required if this disease is to be successfully managed. No independent markers are yet available to predict the behaviour of any individual prostate cancer, particularly its potential to metastasize, and there is now an urgent prerequisite to identify and characterize genes specifically involved in determining the metastatic phenotype of prostate cancer cells before any biologically appropriate treatment modality can be devised. To identify DNA sequences that trophically promote the metastatic phenotype, we have established a new transfection assay with which to monitor activity of prostate cancer genomic DNA. Rat prostatic G and AT6.1 cell lines derived from the same original Dunning R3327 rat prostatic carcinoma exhibit, respectively, low- and high-metastatic phenotypes when grown in syngeneic Copenhagen rats. Rat mammary epithelial cell line 'Rama 37' derived originally from Wistar-Furth rats yields benign non-metastasizing adenomas when inoculated subcutaneously into syngeneic animals. In this report, the Rama 37 cell line is successfully used as the recipient cell-line for transfected DNA fragments extracted from rat prostatic carcinoma G and AT6.1 cells. New metastatic variants of Rama 37 cells have been generated. Enzymatically fragmented genomic DNA from rat metastatic prostate carcinoma cell lines was co-transfected together with plasmid pSV2neo into parental Rama 37 cells, followed by culture in the presence of Geneticin-G418 to select for the transfected cells. To enable subsequent identification of metastasis-promoting DNA sequences, the fragmented genomic DNA sequences were covalently attached to specifically engineered linker DNA molecules to flank the genomic DNA before transfection. Thereafter, the resulting transfectants were pooled and inoculated into mammary fat pads of female Wistar-Furth rats. Metastases produced by the transfectant cells in vivo were reestablished from secondary tumours and probed for the presence of the specific synthetic oligonucleotide sequences that flanked, and hence identified, the presence of the transfected DNA. These new metastatic cells are shown to provide a sensitive assay system with which to detect DNA sequences responsible for conveying the metastatic phenotype of prostate cancer when inoculated into syngeneic rats.

Generation of metastatic variants by transfection of a rat non-metastatic epithelial cell line with genomic DNA from rat prostatic carcinoma cells