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Expression of novel ERE‐binding proteins in human breast & uterine cells
Author(s) -
Kruer Traci L,
Smolenkova Irina A,
Wittliff James L
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a967-a
Subject(s) - estrogen receptor , estrogen , cytosol , chemistry , myometrium , microbiology and biotechnology , biology , uterus , endocrinology , medicine , biochemistry , breast cancer , cancer , enzyme
While investigating ERE‐binding properties of estrogen receptor‐α (hERα) in human breast cancer cytosols, lower molecular weight ERE‐binding proteins (ERE‐BP) were observed. Recognition properties of ERE‐BP were evaluated by electrophoretic mobility shift (EMSA) and super‐shift assays with ERE sequences of estrogen responsive genes. Cytosols, prepared from human breast cancer and uterine reference samples in 50 mM K 2 HPO 4 /KH 2 PO 4 buffer, pH 7.4 with 1.5 mM EDTA, 10 mM Na 2 MoO 4 , 1 mM PMSF & 10 μM monothioglycerol, were incubated 16 hr, 4° C with [ 32 P]ERE sequences (VitA2, CathD, pS2, jun, h‐fos and prolactin) and separated by EMSA. Four ERE‐BP expression profiles were detected exhibiting different ERE recognition and affinity. No ERE‐BP super‐shifted when incubated with ERα monoclonal antibodies AER 314, 320, 611, 1D5 and 5D11 (NeoMarkers®) or a polyclonal antibody (PanVera®/Invitrogen). Cytosols from breast cancer, uterus and fibroids, but not from myometrium exhibited ERE‐BP. Neither rat nor calf uterine cytosol exhibited ERE‐BP, but ververt monkey uterine cytosol expressed two distinct ERE‐BP. To ascertain if ERE‐BP influenced ERE recognition by ERα, competition experiments, performed with increasing [cytosol] and constant [hERα] and [ERE], revealed breast cancer and uterine cytosols inhibited hERα binding in a concentration dependent manner with simultaneous ERE‐BP appearance. The biological significance and distribution of these unique proteins are being evaluated in a wider selection of estrogen and non‐estrogen responsive tissues. Supported in part by grants from the NIH/NCI 1 R43 CA106059‐01A1& Phi Beta Psi Sorority Res. Fund.