Functional analysis of a mutant estrogen receptor isolated from T47Dco breast cancer cells.
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.


METHODS
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.


CONCLUSIONS
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.
Leslie
K K
KK
Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver 80262.
Tasset
D M
DM
Horwitz
K B
KB
D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D011960:Receptors, Estrogen; D004247:DNA