Binding analysis of the estrogen receptor to its specific DNA target site in human breast cancer.
The estrogen receptor (ER) is a nuclear protein with a hormone- and a DNA-binding domain. We examined the DNA binding of ER in MCF-7 cells and 79 primary breast cancers by gel mobility shift assay using as a probe the estrogen response element (ERE). The mobility shift assay showed saturable, specific binding of ER to ERE in crude, high molar extracts containing greater than or equal to 4 mg/ml protein. Nonspecific binding was reduced by increasing concentrations of poly(deoxyinosidylate.deoxycytidylate) and shortening of the ERE probe from 35 to 15 base pairs. In the presence of Mg2+ the ER-ERE complex formation was hormone dependent at 22 degrees C but not at 37 degrees C. In the absence of Mg2+ estradiol was not necessary for ER-ERE complex formation. Correlation of the mobility shift assay with the hormone-binding (E2) assay showed agreement in 55 of the 79 tumors. Both assays were positive (E2 +/ERE+) in 35 cases and both were negative (E2-/ERE-) in 20 cases. In 11 tumors the hormone-binding assay was positive and the mobility shift assay negative (E2+/ERE-), suggesting an alteration of the DNA-binding domain. In 13 cancers the hormone-binding assay was negative and the mobility shift assay positive (E2-/ERE+) suggesting an alteration of the hormone-binding domain. By performing both hormone- and DNA-binding assays of ER and the hormone-binding assay of progesterone receptor (PR), we found the following subgroups of breast cancer: (a) E2+/ERE+/PR+, (b) E2+/ERE+/PR-, (c) E2+/ERE-/PR+, (d) E2+/ERE-/PR-, (e) E2-/ERE+/PR+, (f) E2-/ERE+/PR-, (g) E2-/ERE-/PR-. The simultaneous determination of 17 beta-estradiol and ERE binding may provide a better definition of the ER status of individual tumors and prove useful in refining endocrine therapy of patients with breast cancer.
Foster
B D
BD
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37232.
Cavener
D R
DR
Parl
F F
FF
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011960:Receptors, Estrogen; D004958:Estradiol; D008274:Magnesium