Guo M, Reiners J J
Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201, USA.
Carcinogenesis. 2000 Jul;21(7):1303-12.
The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.
研究了12 - O -十四烷酰佛波醇-13 -乙酸酯（TPA）对人乳腺细胞系MCF10A - Neo及其表达Ha - ras癌基因的衍生细胞系（MCF10A - NeoT细胞）细胞周期进程的影响。将MCF10A - Neo培养物暴露于TPA会诱导G(1)期阻滞，持续约16 - 24小时（半数抑制浓度（IC(50)）约为0.5 nM）。经TPA处理的培养物产生一种细胞生长抑制条件培养基。细胞生长抑制活性在TPA处理后1小时内即可检测到，3 - 7小时后达到峰值，并在处理后16至24小时消失。然而，通过用新鲜培养基重新培养先前处理过的培养物，可迅速再生细胞生长抑制条件培养基。从培养基中去除潜在的转化生长因子β（TGFβ）、用抗TGFβ或可溶性TGFβ（II）受体补充培养基，或用抗TGFβ预吸附条件培养基，均使TPA或条件培养基对MCF10A - Neo增殖的细胞生长抑制作用降低约50%。与丝氨酸蛋白酶抑制剂抑肽酶或纤溶酶原激活物抑制剂-1共同处理也使TPA的细胞生长抑制活性降低约50%。从经TPA处理的MCF10A - Neo培养物中分离出的条件培养基对MCF10A - NeoT细胞具有短暂的细胞生长抑制作用。与MCF10A - Neo细胞相反，MCF10A - NeoT培养物的增殖在TPA暴露后至少72小时受到抑制。从经TPA处理的MCF10A - NeoT培养物中分离出的条件培养基也抑制MCF10A - NeoT增殖约72小时，但抑制MCF10A - Neo增殖不到24小时。这些研究表明，TPA迅速激活MCF10A - Neo细胞中的蛋白水解过程，导致培养基中血清提供的潜在TGFβ活化。TPA还刺激产生一种额外的细胞生长抑制因子，其通过不涉及TGFβ（II）受体的机制发出信号。最后，活化的Ha - ras癌基因的表达改变了TPA处理后产生的细胞生长抑制因子的类型以及对这些因子的反应性。
