Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Squirrel monkeys are neotropical primates that have high circulating cortisol to compensate for expression of glucocorticoid receptors (GRs) with reduced affinity. The low binding affinity of squirrel monkey GR does not result from substitutions in the receptor, because squirrel monkey GR expressed in vitro exhibits high affinity. Rather, squirrel monkeys express a soluble factor that, in mixing studies of cytosol from squirrel monkey lymphocytes (SML) and mouse L929 cells, reduced GR binding affinity by 11-fold. In an effort to identify this factor, the cellular levels of components of the GR heterocomplex in SML and human lymphocytes (HL) were compared. The immunophilin FKBP51 was 13-fold higher in SML than in HL cytosol; FKBP52 in SML was 42% of that in HL cytosol. A role for changes in immunophilins, causing glucocorticoid resistance in neotropical primates, is supported by the following: the changes in FKBP51 and FKBP52 were observed in cells from other neotropical primates with glucocorticoid resistance; the elevated level of FKBP51 was reflected in an abundance of FKBP51 in heat shock protein 90 complexes in SML; when cytosols of SML and L929 cells were mixed, the decrease in GR binding was associated with incorporation of FKBP51 into GR heterocomplexes; the effect of SML cytosol on GR binding was reproduced with cytosol from COS cells expressing squirrel monkey FKBP51; and both the effect of SML cytosol on GR binding and the incorporation of FKBP51 into GR heterocomplexes were blocked by FK506. Regulation of GR binding by FKBP51 represents a previously unrecognized mechanism for regulating glucocorticoid sensitivity. (J Clin Endocrinol Metab 84: [663][664][665][666][667][668][669] 1999) G LUCOCORTICOIDS play an essential role in regulating many developmental and physiological processes (1). The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), a member of the steroid hormone receptor superfamily (2). The GR protein is made up of an aminoterminal transactivation domain, a central DNA-binding domain, and a carboxyterminus that includes the ligandbinding domain and sequences involved in interaction with heat shock proteins (hsps) (3). In the unliganded state, the GR resides in the cytoplasm associated with hsps and other chaperone proteins, forming a multiprotein complex (4). The ordered association of chaperone complexes with the GR is critical in establishing a receptor conformation optimally responsive to hormone (4).Our recent studies have focused on the mechanisms of glucocorticoid insensitivity in species of neotropical primates. Squirrel monkeys, cotton-top tamarins, and owl monkeys have markedly elevated plasma cortisol levels, but they show no signs of glucocorticoid excess (5, 6). They have been proposed as models for the in vivo study of glucocorticoid sensitivity (3, 5). Chrousos et al. (5) demonstrated a greater than 20-fold higher apparent dissociation constant for the GR in squirrel monkey mononuclear leukocytes than in human cells, suggesting that a dec...

The mechanisms responsible for 17␤-estradiol (E 2 )-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor ␣ (ER␣) antagonists are not fully understood. We describe a new tool for dissecting ER␣ action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor ␣ that does not compete with estrogen for binding to ER␣. TPSF noncompetitively inhibits estrogen-dependent ER␣-mediated gene expression with little inhibition of transcriptional activity by NF-B or the androgen or glucocorticoid receptor. TPSF inhibits E 2 -ER␣-mediated induction of the proteinase inhibitor 9 gene, which is activated by ER␣ binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ER␣ to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E 2 -ER␣-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ER␣-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ER␣HA cells that overexpress ER␣, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ER␣ protein levels in MCF-7 cells and several other cell lines without altering ER␣ mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ER␣ by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ER␣. TPSF represents a novel class of ER inhibitor with significant clinical potential. Estrogen receptor ␣ (ER␣)3 is a well studied member of the steroid/nuclear receptor family of transcription regulators. ER␣ acts in the nucleus to regulate gene expression by binding to estrogen response elements (EREs) and related DNA sequences (1-4) and through association with transcription factors bound at SP1 and AP-1 DNA binding sites (4 -7). In response to high affinity estrogen binding, ER␣ dimerizes, binds to ERE DNAs, and undergoes a conformational change in the ligand binding domain that facilitates the recruitment of coactivators (8). Bound coactivators promote assembly of a multiprotein complex that enables chromatin remodeling and stabilization of an active transcription complex (9 -11). In contrast, antagonist-occupied ER␣ recruits corepressors (12).At detection, growth of most human breast cancers depends on 17␤-estradiol (E 2 ) binding to ER␣ (13-16). Treatment strategies that inhibit estrogen-dependent breast cancer include selective ER modulators such as tamoxifen, which binds in the ER␣ ligand binding pocket, and aromatase inhibitors, which block estrogen production. Nearly half of patients treated with aromatase inhibitors develop resistance (17). The long-term effectiveness of tamoxifen is limited by the development of resistance in nearly all patients with metastatic breast cancer and in ϳ4...

As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 ؎ 0.16 versus 2.93 ؎ 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total ␣-tubulin levels in untreated (4.0 ؎ 0.6 versus 1.76 ؎ 0.4 ng of ␣-tubulin/mg of total protein) and nerve growth factortreated pAntisense clones (4.15 ؎ 0.4 versus 2.04 ؎ 0.5 ng of ␣-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 ؎ 8%) than control cells (116 ؎ 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 ؎ 3.0 versus 59.5 ؎ 1.2 m). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.The S100 protein family is a group of calcium-binding proteins that exhibit a high degree of conservation in amino acid sequence, secondary structure, and genomic organization (1-3). To date, there are 18 members of the S100 family, 13 of which are clustered in region q21 of human chromosome 1 (see Refs. 4 and 5). A new nomenclature that reflects the genomic organization of these proteins has recently been adopted, and S100␣ is now designated S100A1 and S100␤ is designated S100B (6). S100A1 and S100B were the original members of this family and have been implicated in a diverse group of cellular functions including cell-cell communication, cell growth, cell structure, energy metabolism, contraction, and intracellular calcium signal transduction (see Refs. 1 and 3).Because each S100 monomer contains two EF-hand high affinity calcium binding domains, they have been included in the S100-troponin-calmodulin superfamily of calcium-modulated proteins. These proteins have no known enzymatic activity and function by modulating the activity of other proteins termed target proteins. Calmodulin appears to be the "workhorse" in calcium signaling and to be responsible for generating the main events triggered by changes in intracell...

virus (EBV) transforms B-lymphocytes into continuous lines of lymphoblasts which can provide a uniform supply of donor DNA and lymphoblastoid protein (11). For example, DNA from EBV-transformed lymphocytes from humans has been used to clone and sequence mutant forms of the glucocorticoid receptor (6). EBVtransformed lymphocytes from cotton-top tamarins, on the other hand, are an abundant source of EBV (10) and have also been used for the characterization of steroid receptor inhibitory proteins which are expressed in cotton-top tamarin cells (1). Thus, transformed lymphocytes can serve as a continuous supply of biomedical products from scarce or expensive animal research models. Although the cotton-top tamarin B95-8 cell line was generated over 20 years ago (12), the transformation of B-lymphocytes from New World primates in general has not been uniformly successful (4,12), and at present, no B-lymphoblastic cell lines from owl monkeys or squirtel monkeys FIG. 1. Ultrastmcture of owl monkey I3C cells. Cell suspensions were centrifuged to form loose pellets, which were fixed in 3% glutaraldehyde (0.1 M sodium cacodylate buffer, pH 7.2). The specimens were then processed to ultrathin sections, which were stained with lead citrate and uranyl acetate and examined in a Philips CM-IO0 transmission electron microscope. (.4) The cells exhibited round and sometimes indented nuclei, prominent nucleoli, strips of rough endoplasmic reticulum, and filopodia indicative of B-cell lineage. Magnification X 5600; (B) Infrequent cells exhibited lyric changes, including breaks in the nuclear membrane and cytoplasmic swelling. These cells typically exhibited scattered viral particles approximately 95 nm in diameter which were either empty or contained a central sphere. Magnification X 27 000. 88