Multistage transformation of cultured rat urothelium: the effects of

Rat bladder epithelial cell transformation was found to in-volve at least three distinct phenotypic stages in vitro. The effects on this process of N-methyl-N-nitrosourea (MNU), sodium saccharin, sodium cyclamate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) alone or in combination were investigated. MNU was a potent inducer of proliferating pre-neoplastk epithelial foci. Sodium saccharin alone induced a transient hyperpiastic response of the cultured urothetium but did not induce significantly more foci than in controls, though a late toxic effect of saccharin may have masked focus induc-tion. In combination with a single dose of 250 /ig/ml MNU, sodium saccharin did not increase focus incidence compared with MNU treatment alone but following a single treatment with 25 /ig/ml MNU which alone induced no foci, saccharin induced a significant number of foci indicating a promoting effect under these conditions. Sodium cyclamate alone induced a marked and prolonged epithelial hyperplasia and a signifi-cant increase in focus incidence above controls and above MNU-treated cultures. Following a single treatment with 250 pg/ml MNU, cyclamate increased focus incidence still fur-ther. TPA, the potent skin tumour promoting agent, alone induced significantly more foci than in controls. Rapidly-proliferating cell lines were established from nine foci induc-ed by treatment with MNU alone or MNU + saccharin. Four of these were tumorigenic. These results indicate that MNU is a potent inducer of preneoplastic foci in rat urothelial cultures. Sodium saccharin and sodium cyclamate can act as promoting agents in this in vitro system as in the in vivo in-duction of tumours and sodium cyclamate appears to be active as a complete inducer under these conditions