We have previously observed that both polya-mines and an autocrine mechanism are involved in the stimulation by ovine PRL (oPRL) of growth of the N-nitrosomethylurea-induced rat mammary tumor cultured in vitro in the soft agar clonogenic assay. These experiments were designed to test the interaction between these two pathways of oPRL action in this system. In agreement with previous findings in our laboratory, conditioned medium obtained from oPRL-treated (oPRL-CM), but not untreated, tumors consistently stimulated colony formation when added to N-nitrosomethylurea mammary tumors plated in soft agar under serum-free medium conditions. Administration of α-difluoromethyl-ornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, abolished the colony-stimulating effect of oPRL-CM. The inhibitory effect of DFMO was reversed in a dose-dependent fashion by the addition of spermidine, which entirely restored the growth-promoting action of bPRL-CM. In addition, the administration of increasing amounts of spermidine potentiated the colony-stimulating effect of suboptimal concentrations of oPRL-CM. In contrast, manipulation of the polyamine environment With DFMO and/or spermidine administration did not affect the number of colonies formed when conditioned medium from untreated tumors was added instead of oPRL-CM. We conclude that the polya-mine pathway plays an essential role in the expression of auto-crine control of tumor growth by oPRL.
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