Regulation of Antitrypsin and Antichymotrypsin Synthesis by MCF-7 Breast Cancer Cell Sublines*

Abstract

We have examined the synthesis of the protease inhibitors .alpha.1-antitrypsin (.alpha.1-AT) and .alpha.1-antichymotrypsin (.alpha.1-ACHY) by variants of the MCF-7 human breast cancer cell line. Spent medium from MCF-7 203P cells, grown in the absence of serum, was found to contain immunoreactive .alpha.1-AT and .alpha.1-ACHY by Western blotting. In the presence of 10-8 M estradiol, levels of both inhibitors were increased 3- to 6-fold. Incubation of spent medium with [125I]trypsin or [125I]chymotrypsin resulted in the formation of stable 75- and 90-kDa complexes identical to the complexes formed between these proteases and the protease inhibitors in plasma, showing the release of active protease inhibitors by MCF-7 cells in culture. Immunoprecipitation of 35S-labeled proteins from the medium of cells grown in the presence of [35S]methionine yielded comparable results, confirming hormonally sensitive synthesis of both protease inhibitors. Northern blot analysis suggests that stimulation of estradiol occurs at the level of transcription. Tetradecanoyl phorbol acetate (50 ng/ml) also stimulated .alpha.1-AT and .alpha.1-ACHY synthesis 2- to 4-fold, suggesting the involvement of protein kinase-C. Comparison studies with MCF-7 cell sublines ML, BK, 203P, and 300P (a variant spontaneously appearing after 100 passages of 203P) show a wide variation in synthesis of .alpha.1-AT and .alpha.1-ACHY proteins; sublines 203P and 300P synthesize both inhibitors, the ML subline synthesizes detectable amounts only of .alpha.1-ACHY, while no detectable synthesis of either inhibitor was seen in the BK subline. Similar results were obtained for protease inhibitor mRNA transcription by Northern blotting, although low levels of .alpha.1-AT mRNA transcription by the ML subline and of .alpha.1-AT and .alpha.1-ACHY mRNA transcription by the BK subline could be detected.