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Physicochemical and genetic evidence for specific antiestrogen binding sites
Abstract:
In rat uterus and human breast cancer MCF-7 cell cytosol, the antiestrogens tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) bind to "antiestrogen binding sites" (ABS), which do not bind estradiol (E). Demonstrated in total cytosol by binding studies with radioactive antiestrogens in the presence of a large concentration of E, ABS can be physically separated from E-binding estrogen receptor (ER) by removing the latter with an E-containing bioaffinity adsorbent or with heparin-Sepharose gel. ABS concentration is 10-20% of that of ER; the Kd for Tam and OH-Tam is 1-2 x 10(-9) M, whereas the Kd of OH-Tam binding by ER (approximately equal to 1 x 10(-10) M) is approximately equal to 1/50 that of Tam. Other triphenylethylene antiestrogens compete against Tam for binding to ABS, contrary to steroid hormones. Sucrose gradient ultracentrifugation analyses of total cytosol and of affinity gel effluents show a heterogenous pattern of ABS from 10 to 40 S, unchanged by 0.4 M KCl and limited trypsinization (which however provoke transitions of ER from 8S to 4S forms) and by 20 mM molybdate (which stabilizes the 8S form of ER and prevents large aggregates). Preliminary results suggest that ABS may be associated with particulate components of the cell. RTx6 cells of a clone selected from MCF-7 cells for resistance to the antigrowth effect of Tam have ER in the same concentration and have similar affinity for E and antiestrogens as do unselected MCF-7 cells. However, RTx6 cells have virtually no ABS detectable by binding and gradient ultracentrifugation studies. It is proposed that the double binding of Tam and OH-Tam to ER and ABS in estrogen target cells may be related to the complex double series of estrogenic and "antiestrogenic" activities displayed by nonsteroidal triphenylethylene derivatives.
Insights
Antiestrogens tamoxifen and 4-hydroxytamoxifen bind to distinct antiestrogen binding sites (ABS) in cells, separate from the estrogen receptor (ER). Loss of ABS correlates with tamoxifen resistance, suggesting ABS involvement in antiestrogen action.
Area of Science:
- Endocrinology
- Molecular Pharmacology
- Cancer Biology
Background:
- Antiestrogens like tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) exhibit complex biological activities.
- These compounds interact with both the estrogen receptor (ER) and other cellular components.
Purpose of the Study:
- To investigate the nature and function of antiestrogen binding sites (ABS).
- To determine the relationship between ABS and cellular responses to antiestrogens, particularly tamoxifen resistance.
Main Methods:
- Binding studies using radiolabeled antiestrogens in rat uterus and human breast cancer MCF-7 cell cytosol.
- Physical separation of ABS from ER using bioaffinity adsorbents and heparin-Sepharose chromatography.
- Sucrose gradient ultracentrifugation to analyze ABS heterogeneity.
- Comparison of ABS presence in tamoxifen-sensitive (MCF-7) and tamoxifen-resistant (RTx6) cell lines.
Main Results:
- ABS were identified and physically separated from ER, with distinct binding affinities for Tam and OH-Tam.
- ABS concentration was 10-20% of ER concentration, with a dissociation constant (Kd) of 1-2 x 10(-9) M for Tam and OH-Tam.
- ABS exhibited heterogeneous sedimentation patterns (10-40 S) and were unaffected by conditions altering ER.
- Tamoxifen-resistant RTx6 cells showed a significant lack of detectable ABS compared to sensitive MCF-7 cells.
Conclusions:
- ABS represent a distinct binding entity in cells, separate from the ER.
- The presence of ABS appears crucial for the antiestrogenic effects of tamoxifen and related compounds.
- The absence of ABS in resistant cells suggests their critical role in mediating tamoxifen sensitivity and action.