GENERATION OF METASTATIC VARIANTS BY TRANSFECTION OF A RAT NONMETASTATIC EPITHELIAL-CELL LINE WITH GENOMIC DNA FROM RAT PROSTATIC-CARCINOMACELLS

Prostate cancer is the second leading cause of male death from maligna nt disease in Europe and in the USA. Failure to prevent or eliminate m etastatic dissemination is a fundamental problem underlying the curren t inadequate treatment of prostate cancer, and novel therapeutic strat egies are required if this disease is to be successfully managed. No i ndependent markers are yet available to predict the behaviour of any i ndividual prostate cancer, particularly its potential to metastasize, and there is now an urgent prerequisite to identify and characterize g enes specifically involved in determining the metastatic phenotype of prostate cancer cells before any biologically appropriate treatment mo dality can be devised. To identify DNA sequences that trophically prom ote the metastatic phenotype, we have established a new transfection a ssay with which to monitor activity of prostate cancer genomic DNA. Ra t prostatic G and AT6.1 cell lines derived from the same original Dunn ing R3327 rat prostatic carcinoma exhibit, respectively, low- and high -metastatic phenotypes when grown in syngeneic Copenhagen rats. Rat ma mmary epithelial cell line 'Rama 37' derived originally from Wistar-Fu rth rats yields benign non-metastasizing adenomas when inoculated subc utaneously into syngeneic animals. In this report, the Rama 37 cell li ne is successfully used as the recipient cell-line for transfected DNA fragments extracted from rat prostatic carcinoma G and AT6.1 cells. N ew metastatic Variants of Rama 37 cells have been generated. Enzymatic ally fragmented genomic DNA from rat metastatic prostate carcinoma cel l 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 identificati on of metastasis-promoting DNA sequences, the fragmented genomic DNA s equences were covalently attached to specifically engineered linker DN A molecules to flank the genomic DNA before transfection. Thereafter, the resulting transfectants were pooled and inoculated into mammary fa t pads of female Wistar-Furth rats. Metastases produced by the transfe ctant cells in vivo were reestablished from secondary tumours and prob ed for the presence of the specific synthetic oligonucleotide sequence s that flanked, and hence identified the presence of the transfected D NA. These new metastatic cells are shown to provide a sensitive assay system with which to detect DNA sequences responsible for conveying th e metastatic phenotype of prostate cancer when inoculated into syngene ic rats.