Endocrinology, Vol 120, 1140-1151, Copyright © 1987 by Endocrine Society

ARTICLES

Antiestrogen stimulation of the production of a 37,000 molecular weight secreted protein and estrogen stimulation of the production of a 32,000 molecular weight secreted protein in MCF-7 human breast cancer cells

YY Sheen and BS Katzenellenbogen

Antiestrogens (AE), such as tamoxifen, selectively increase the production of a secreted protein of 37,000 mol wt (Mr) in estrogen receptor-containing human breast cancer cells (MCF-7, but not in estrogen receptor-negative MDA-MB-231 cells), and the production of this protein by AE is inhibited by concomitant estradiol (E) treatment. Likewise, E increases the production of a 32,000 Mr secreted protein whose synthesis is inhibited by AE. Proteins were detected by [35S]methionine and [35S]cysteine labeling of cells and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gels and fluorography. Enhanced production of the 37,000 Mr protein is observed within 6 h of AE treatment, with maximal synthesis seen at 1-2 days when this protein represents about 6% of the total radiolabeled secreted proteins. This protein is stimulated maximally (approximately 4-fold) by 10(-8) M trans-hydroxytamoxifen or LY117018 or 10(-6) M tamoxifen, and its AE specificity is seen by the fact that trans- tamoxifen increases this protein, whereas cis-tamoxifen, an estrogen, does not. In addition to stimulating the synthesis of previously identified 160,000 and 52,000 Mr secreted proteins, E increases the production of a 32,000 Mr secreted protein. When cells are grown in estrogen-free conditions, i.e. in charcoal-dextran-treated serum in medium lacking the estrogen phenol red, the basal level of the 32,000 Mr protein is extremely low, and E stimulation results in a 10-fold increase in the production of this protein, with increases observable by 6 h and maximal stimulation at 2 days. Interestingly, the basal level of synthesis of the 37,000 Mr protein is high in the absence of E and is then stimulated only minimally by the addition of AE, suggesting that this protein is clearly produced as an estrogen-antagonistic protein. Amino acid incorporation conducted in the presence of tunicamycin and endoglycosidase H indicates that both of these proteins are glycoproteins. These proteins should serve as useful markers for AE and E action and may be involved in AE and E modulation of cell proliferation and/or cell function.

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