8-Alkylthio-6-thio-substituted theophylline analogues as selective noncompetitive progesterone receptor antagonists

The progesterone receptor (PR) plays a key role in reproduction and is important in cancers of the reproductive tract. Current PR antagonists usually compete for progestin binding in the PR ligand-binding pocket and often exhibit cross-binding with other members of the steroid receptor family. Using stably transfected cells expressing reporter genes, a set of ~150 theophylline analogues were screened for their ability to inhibit progesterone, estrogen, glucocorticoid and androgen signaling. The structure-activity studies presented here identify branched 8-alkylthio-6-thio-substitutions of theophylline as selective PR inhibitors. 6-thio-8-(2-ethylbutyl)thiotheophylline (51), the most extensively studied derivative, does not act by competing with progestins for binding in the ligand-binding pocket of PR. It demonstrated the ability to inhibit the mouse mammary tumor virus (MMTV)-luciferase reporter and endogenous PR-regulated alkaline phosphatase activity in T47D breast cancer cells. Compound 51 is the lead member of a novel class of PR inhibitors that act outside the PR ligand-binding pocket, thus serving as a novel probe to investigate PR action and a lead for further development

Claudin 7 expression and localization in the normal murine mammary gland and murine mammary tumors

INTRODUCTION: Claudins, membrane-associated tetraspanin proteins, are normally associated with the tight junctions of epithelial cells where they confer a variety of permeability properties to the transepithelial barrier. One member of this family, claudin 7, has been shown to be expressed in the human mammary epithelium and some breast tumors. To set the stage for functional experiments on this molecule, we examined the developmental expression and localization of claudin 7 in the murine mammary epithelium and in a selection of murine mammary tumors. METHOD: We used real-time polymerase chain reaction, in situ mRNA localization, and immunohistochemistry (IHC) to examine the expression and localization of claudin 7. Frozen sections were examined by digital confocal microscopy for colocalization with the tight-junction protein ZO1. RESULTS: Claudin 7 was expressed constitutively in the mammary epithelium at all developmental stages, and the ratio of its mRNA to that of keratin 19 was nearly constant through development. By IHC, claudin 7 was located in the basolateral part of the cell where it seemed to be localized to discrete vesicles. Scant colocalization with the tight-junction scaffolding protein ZO1 was observed. Similar results were obtained from IHC of the airway epithelium and some renal tubules; however, claudin 7 did partly colocalize with ZO1 in EPH4 cells, a normal murine mammary cell line, and in the epididymis. The molecule was localized in the cytoplasm of MMTV-neu and the transplantable murine tumor cell lines TM4, TM10, and TM40A, in which its ratio to cytokeratin was higher than in the normal mammary epithelium. CONCLUSION: Claudin 7 is expressed constitutively in the mammary epithelium at approximately equal levels throughout development as well as in the murine tumors examined. Although it is capable of localizing to tight junctions, in the epithelia of mammary gland, airway, and kidney it is mostly or entirely confined to punctate cytoplasmic structures, often near the basolateral surfaces of the cells and possibly associated with basolateral membranes. These observations suggest that claudin 7 might be involved in vesicle trafficking to the basolateral membrane, possibly stabilizing cytoplasmic vesicles or participating in cell–matrix interactions

A role for the non-conserved N-terminal domain of the TATA-binding protein in the crosstalk between cell signaling pathways and steroid receptors

Transcriptional induction by steroid receptors is coupled to cellular signal transduction pathways although, in general, the mechanisms governing these events are not well defined. Using TATA-binding protein (TBP) specificity mutants that recognize a TGTA box, we show that yeast TBP expressed in mammalian cells can support steroid-mediated gene induction to a similar degree as human TBP, however yeast TBP does not support the 8-Bromo-cAMP-mediated potentiation of glucocorticoid receptor (GR)-dependent transactivation. Chimeras between yeast and human TBP reveal that it is the non-conserved N-terminus of TBP that governs the potentiation of GR action. While the conserved core of TBP is sufficient for TATA-element binding and preinitiation complex formation, the role of the N-terminus has remained elusive. Our results suggest a role of the N-terminus of human TBP in coupling cell signaling events to steroid-mediated transcription, thereby establishing one of the few described functional roles of this polypeptide domain in a physiological process