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Two types of antiprogestins identified by their differential action in transcriptionally active extracts from T47D
L Klein-Hitpass1, A C Cato, D Henderson
1Institut für Zellbiologie, Universitätsklinikum Essen, FRG.
Abstract:
Transcriptionally active nuclear extracts from human breast carcinoma cells (T47D) were used to compare the action of progestins and several antiprogestins of the 11 beta-aryl substituted steroid series on the DNA-binding properties and the trans-activating potential of progesterone receptor (PR) in vitro. Using the gel-shift assay we identified a novel type of antiprogestin (ZK98299, type I), which in contrast to type II antiprogestins, including RU486, does not induce binding of PR to progesterone response elements (PREs). In competition experiments excess of type I antiprogestin inhibits induction of DNA binding of PR by progestins and type II antiprogestins suggesting that its binding to PR interferes with the formation of stable receptor dimers. Moreover, we demonstrate that the antagonistic action of ZK98299 can be fully mimicked in vitro by using cell-free nuclear extracts from T47D cells and a 'simple' test promoter. In contrast, type II antiprogestins known to induce certain promoters in vivo exert strong agonistic effects on in vitro transcription of the test template used.
Insights
A novel antiprogestin, ZK98299, blocks progesterone receptor (PR) binding to DNA, unlike RU486. This type I antiprogestin prevents PR dimerization, inhibiting transcription in breast cancer cells.
Area of Science:
- Endocrinology
- Molecular Biology
- Cancer Research
Background:
- The progesterone receptor (PR) plays a crucial role in various physiological processes and is a target for hormone therapies in conditions like breast cancer.
- Antiprogestins are drugs that block the action of progesterone, with different mechanisms influencing PR activity.
Purpose of the Study:
- To investigate the in vitro effects of novel 11 beta-aryl substituted steroid antiprogestins on progesterone receptor (PR) DNA-binding and trans-activation.
- To compare the activity of a new type of antiprogestin (ZK98299) with existing type II antiprogestins like RU486.
Main Methods:
- Utilized transcriptionally active nuclear extracts from human breast carcinoma cells (T47D).
- Employed gel-shift assays to assess PR DNA-binding properties in the presence of progestins and antiprogestins.
- Used cell-free nuclear extracts and a test promoter for in vitro transcription studies.
Main Results:
- Identified ZK98299 as a novel type I antiprogestin that does not induce PR binding to progesterone response elements (PREs).
- Demonstrated that ZK98299 inhibits PR-DNA binding by interfering with stable receptor dimer formation.
- Showed that type II antiprogestins (e.g., RU486) exhibit agonistic effects on in vitro transcription, unlike their in vivo activity.
Conclusions:
- ZK98299 represents a distinct class of antiprogestins that antagonize PR function by preventing DNA binding.
- The in vitro transcription system effectively models the antagonistic actions of ZK98299.
- Understanding these distinct mechanisms is crucial for developing targeted therapies for hormone-dependent diseases.