Nonhomologous end-joining ligation transfers DNA regulatory elements between cointroduced plasmids.

Cointroduction of plasmids into mammalian cells is commonly used to investigate transcription factor regulation of reporter genes or to normalize transfection efficiency. We report here that cotransfected DNA molecules commonly transfer enhancer elements from one plasmid to another. Using separate Renilla or Firefly luciferase reporters, we found that an estrogen response element (ERE) originally linked to one of the reporters stimulated expression of the non-ERE-containing reporter.

Expression of estrogen receptor beta in prostate carcinoma cells inhibits invasion and proliferation and triggers apoptosis.

The involvement of estrogen receptor beta (ERbeta) in prostate carcinogenesis has been hypothesized. Several reports have shown that ERbeta expression was decreased when prostate cells undergo neoplastic transformation, suggesting that it could play a tumor-suppressor role. By restoring ERbeta expression in prostatic carcinoma cells by adenoviral delivery, we aimed to test this hypothesis.

Estrogenic effects of resveratrol in breast cancer cells expressing mutant and wild-type estrogen receptors: role of AF-1 and AF-2.

Resveratrol, a hydroxystilbene found in grapes and wine, has previously been shown to be a non-flavonoid phytoestrogen, and to act as an estrogen receptor (ER) superagonist in MCF-7 cells transiently transfected with estrogen-responsive reporter constructs. Several additional hydroxystilbenes, including diethylstilbestrol (DES) and piceatannol, were tested, and all showed ER agonism or partial agonism, but superagonism was specific to resveratrol. Moreover, superagonism was observed in cells carrying a stably integrated reporter gene, indicating that this phenomenon is not a result of transient transfection.

Functional characterization of pendrin in a polarized cell system. Evidence for pendrin-mediated apical iodide efflux.

Pendred's syndrome is an autosomal recessive disorder characterized by sensorineural deafness, goiter, and impaired iodide organification. It is caused by mutations in the PDS/SLC26A4 gene that encodes pendrin. Functionally, pendrin is a transporter of chloride and iodide in Xenopus oocytes and heterologous mammalian cells and a chloride/base exchanger in beta-intercalated cells of the renal cortical collecting duct.

An estrogen receptor (ER)alpha deoxyribonucleic acid-binding domain knock-in mutation provides evidence for nonclassical ER pathway signaling in vivo.

We created a nonclassical estrogen receptor (ER) knock-in mouse model by introducing a mutation that selectively eliminates classical ER signaling through estrogen response elements, while preserving the nonclassical ER pathway. Heterozygous nonclassical ER knock-in (NERKI) females are infertile. Their ovaries contain no corpora lutea, reflecting a defect in ovulation, and the stromal cells contain lipid droplets, suggesting altered steroidogenesis.

Activation of phosphatidylinositol 3-kinase contributes to insulin-like growth factor I-mediated inhibition of pancreatic beta-cell death.

To begin to determine whether IGF-I treatment represents a potential means of enhancing the survival of islet cell grafts after transplantation, the present studies established a model of beta-cell death secondary to loss of trophic support and examined the ability of IGF-I to prevent cell death. The studies were performed using the rat pancreatic beta-cell line, INS-1. Incubating INS-1 cells in RPMI 1640 and 0.

Estrogen regulates a tissue-specific calpain in the anterior pituitary.

A PCR-based cDNA subtraction hybridization was performed to identify the genes stimulated by estrogen in the pituitary. A novel tissue-specific calpain (nCL-2'), previously shown to be expressed mainly in the stomach, was markedly induced in the pituitary after estrogen treatment. The 5'-flanking region of the calpain nCL-2' gene was analyzed to assess the molecular mechanism of estrogen regulation.

Cell-specific Cre-mediated activation of the diphtheria toxin gene in pituitary tumor cells: potential for cytotoxic gene therapy.

Diphtheria toxin has been suggested for the treatment of malignant cancer. In this paper, we describe a strategy for targeting the expression of the diphtheria toxin gene to growth hormone (GH)-producing pituitary tumor cells using adenoviral vectors. We generated adenoviral vectors in which a stuffer DNA fragment, flanked by two loxP sequences, was placed between the GH or cytomegalovirus (CMV) promoters and the diphtheria toxin gene (GH-loxP-DT, and CMV-loxP-DT) or the beta-Gal gene (GH-loxP-Gal, and CMV-loxP-Gal).