A cell culture bioassay to analyze metabolism of phorbol diester tumor promoters.

The tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) induces ornithine decarboxylase (ODC) in confluent monolayers of transformed hamster cells and potentiates the induction of ODC in these cells by fresh medium. This observation has been used as the basis for a method to analyze metabolism of phorbol diester tumor promoters by the loss of their ODC-inducing activity. TPA-containing medium exposed to growing hamster embryo fibroblasts (HEF) for various lengths of time showed a rapid and progressive loss of the ability to potentiate ODC induction in transformed hamster cells, suggesting that the TPA had been metabolized. No loss of ODC-inducing activity occurred when TPA was exposed to cultures of growing or confluent human fibroblasts for 7 days. Mixing experiments with TPA-containing “conditioned” medium from cultures of HEF and human fibroblasts indicated that HEF treated with TPA did not elaborate an inhibitor of ODC induction. This bioassay procedure for analyzing the TPA in culture medium was directly compared with analysis of metabolism of [3H]TPA by thin-layer chromatography; the striking differences between hamster and human fibroblasts in their ability to metabolize TPA were confirmed. Cells from several other rodent species also metabolized TPA, but none of the four human cell lines tested, including SV40-transformed lines, metabolized the compound. The bioassay procedure was also used to measure metabolism of other phorbol diesters. Phorbol-12,13-didecanoate exposed to HEF did not lose its ability to induce ODC whereas phorbol-12,13-dibenzoate did. These results indicate that the metabolism of TPA renders it biologically inactive as measured by ODC-inducing activity but that not all phorbol diesters are metabolized by a given cell type or, alternatively, that their metabolism may not always lead to loss of biological activity. We conclude that a combination of the chromatographic procedure and the bioassay can be used to analyze the relationship between chemical structure and biological activity of the phorbol diester series of tumor promoters.