Utilization of site-specific recombination for generating therapeutic protein producing cell lines.

The AttSite Recombinase Technology from Intrexon, Blacksburg, VA, utilizes specific DNA sequences and proprietary recombinase enzymes to catalyze the insertion of a gene of interest at a specific location in the host cell genome. Using this technology, we have developed Chinese Hamster Ovary (CHO) cell lines that have incorporated attB recombination sites at highly transcriptionally active loci or 'hot spots' within the cell genome. Subsequently, these attB site containing host cell lines could then be used for the expression of future Centocor products.

Structure-function relationships in estrogen receptors and the characterization of novel selective estrogen receptor modulators with unique pharmacological profiles.

This article summarizes recent research on the development of estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta) subtype-selective ligands based on our understanding of structure-activity relationships in these two estrogen receptors and differences in their ligand binding domains and activation function domains. The use of these ligands should enable greater understanding of the unique biologies mediated by ER alpha versus ER beta and may, as well, provide selective estrogen receptor modulators having unique biological and pharmacological profiles optimal for prevention and treatment of breast cancer, for menopausal hormone replacement, for prevention of osteoporosis, and for potential cardiovascular benefit.

Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members.

Ligands for the estrogen receptor (ER) that have the capacity to selectively bind to or activate the ER subtypes ERalpha or ERbeta would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. In this study, we examine three compounds of novel structure that act as ER subtype-selective ligands. These are a propyl pyrazole triol (PPT), which is a potent agonist on ERalpha but is inactive on ERbeta, and a pair of substituted tetrahydrochrysenes (THC), one enantiomer of which (S,S-THC) is an agonist on both ERalpha and ERbeta, the other (R,R-THC) being an agonist on ERalpha but an antagonist on ERbeta.

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor.

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A).

Analysis of estrogen receptor interaction with a repressor of estrogen receptor activity (REA) and the regulation of estrogen receptor transcriptional activity by REA.

The transcriptional activity of nuclear hormone receptors is known to be modulated by coregulator proteins. We found that the repressor of estrogen receptor activity (REA), a protein recruited to the hormone-occupied estrogen receptor (ER), decreased the transcriptional activity of ER, both when ER was acting directly through DNA response elements as well as when it was tethered to other transcription factors. Administration of antisense REA resulted in a 2-4-fold increase in ER transactivation, implying that endogenous REA normally dampens the stimulatory response to estradiol.

Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity.

We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells.

Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor.

The vitamin D receptor (VDR) contains an alpha-helical, ligand-inducible activation function (AF-2) at the COOH-terminus of the ligand-binding domain (LBD). In this study, a second distinct activation domain was identified in the VDR LBD. Using a yeast-based system to screen a random mutant library of GAL4-VDR (93-427), a mutant GAL4-VDR fusion protein with constitutive transcriptional activity was isolated.

The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction.

A ligand-inducible transactivation function (AF-2) exists in the extreme carboxyl terminus of the vitamin D receptor (VDR) that is essential for 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-activated transcription and p160 coactivator interaction. Crystallographic data of related nuclear receptors suggest that binding of 1, 25-(OH)2D3 by VDR induces conformational changes in the ligand-binding domain (LBD), the most striking of which is a packing of the AF-2 helix onto the LBD adjacent to helices H3 and H4. In this study, a panel of VDR helix H3 mutants was generated, and residues in helix H3 that are important for ligand-activated transcription by the full-length VDR were identified.

Transcriptional activation through the vitamin D receptor in osteoblasts.

Osteoblasts are bone-forming cells that play an essential role in the development and maintenance of a mineralized bone extracellular matrix and they are target cells for vitamin D. Osteoblasts express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates the expression of osteoblastic-specific genes such as osteocalcin and osteopontin. VDR is a ligand-inducible transcription factor which heterodimerizes with retinoid X receptor (RXR) and binds as a heterodimer to vitamin D-responsive elements (VDREs) in the promoter region of vitamin-D responsive genes, ultimately leading to their increased transcription.

Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.