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Functional analysis of a mutant estrogen receptor isolated from T47Dco breast cancer cells
K K Leslie1, D M Tasset, K B Horwitz
1Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver 80262.
Objective:
Estrogen receptor-positive cancers that initially respond to hormone therapy often progress to a resistant state. The breast cancer cell line T47Dco is a model for such resistance. It is a polymorphic line, composed of multiple cell populations that demonstrate the presence of mutant estrogen receptors by cloning and sequencing techniques. Our objective was to isolate and analyze the structural and functional characteristics of the T47Dco mutant estrogen receptor complementary deoxyribonucleic acid clones.
Study Design:
We constructed two independent T47Dco complementary deoxyribonucleic acid libraries. We isolated and sequenced T47Dco estrogen receptors and have identified a mutant receptor that is truncated near the end of the deoxyribonucleic acid binding domain. This mutant has now been recreated with site-directed mutagenesis and tested for its ability to bind to deoxyribonucleic acid, dimerize with other estrogen receptors, and activate gene transcription by the chloramphenicol acetyltransferase assay and by gel shift assays.
Results:
The chloramphenicol acetyltransferase assays reveal that in the absence of estradiol low levels of conversion of chloramphenicol to acetylated products occur when the mutant estrogen receptor is used to activate chloramphenicol acetyltransferase gene transcription, supporting that it has some constitutive function. Also, gel mobility shift assays demonstrate low levels of deoxyribonucleic acid binding with the mutant protein.
Conclusion:
This mutant estrogen receptor may contribute to the estrogen receptor-positive, hormone-resistant phenotype of T47Dco cells by constitutively binding to and activating genes that were previously estradiol dependent.
Insights
Researchers identified a mutant estrogen receptor in hormone-resistant breast cancer cells. This altered receptor shows constitutive activity, potentially driving tumor growth independent of estradiol.
Area of Science:
- Molecular Biology
- Cancer Research
- Endocrinology
Background:
- Estrogen receptor-positive (ER+) breast cancers often develop resistance to hormone therapy.
- The T47Dco cell line serves as a model for acquired hormone resistance.
- Polymorphic cell lines like T47Dco harbor mutant estrogen receptors.
Purpose of the Study:
- To isolate and characterize mutant estrogen receptor (ER) complementary DNA (cDNA) clones from T47Dco cells.
- To analyze the structural and functional properties of these mutant ERs.
Main Methods:
- Construction of two independent T47Dco cDNA libraries.
- Isolation and sequencing of ERs to identify mutations.
- Site-directed mutagenesis to recreate a truncated ER mutant.
- Functional assays including DNA binding (gel shift) and gene transcription (chloramphenicol acetyltransferase assay).
Main Results:
- A mutant ER was identified, truncated within the DNA binding domain.
- The mutant ER exhibited low-level constitutive transcriptional activity in the absence of estradiol.
- The mutant protein showed reduced DNA binding affinity.
Conclusions:
- The identified mutant estrogen receptor may contribute to the ER-positive, hormone-resistant phenotype in T47Dco cells.
- Constitutive activation of previously estradiol-dependent genes by the mutant ER is a potential mechanism for resistance.