Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle pro gression were examined in the human breast cell line MCF10A-Neo and a deriv ative fine which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted similar to 16-24 h (IC50 similar to 0.5 nM), TPA-treated cultures produced a cytostati c conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h posttreat ment, However, cytostatic conditioned medium could be quickly regenerated b y re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supple menting the culture medium with anti-TGF beta or soluble TGF beta(II) recep tor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo prolifer ation by similar to 50%. Go-treatment with the serine protease inhibitors a protinin or plasminogen activator inhibitor-1 also suppressed the cytostati c activity of TPA similar to 50%, Conditioned medium isolated from TPA-trea ted MCF10A-Neo cultures was transiently cytostatic to MGF10A-NeoT cells. Th e proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, w as suppressed at least 72 h following TPA exposure. Conditioned medium isol ated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT prol iferation for similar to 72 h, but suppressed MCF10A-Neo proliferation for <24 h, These studies suggest that TPA quickly activates proteolytic process es in MCF10A-Neo cells leading to the activation of latent TGF beta supplie d by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not invol ving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras o ncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.