Karen Gray

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action

Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rho-guanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound specifically to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogen-responsive reporter revealed that Brx augmented gene activation by the ER in an element-specific and ligand-dependent manner. Moreover, activation of ER by Brx could be specifically inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

Detection of adrenomedullin, a hypotensive peptide, in amniotic fluid and fetal membranes.

OBJECTIVE Our purpose was to determine whether adrenomedullin, a multifunctional regulatory peptide involved in blood flow regulation and growth stimulation and with antimicrobial activity, was a component of amniotic fluid from second-trimester human fetus and to determine the source of this peptide. STUDY DESIGN A prospective descriptive study was performed on 134 patients undergoing amniocentesis after genetic counseling, ultrasonography, and informed consent. Adrenomedullin expression was determined by immunocytochemical analysis, Western blot analysis, reverse transcriptase-polymerase chain reaction, and in situ reverse transcriptase-polymerase chain reaction in fetal membranes and with radioimmunoassay in amniotic fluids. RESULTS Radioimmunoassay of the 134 amniotic fluid specimens revealed adrenomedullin-like immunoreactivity in all of them, ranging in concentration from 10 to 300 fmol/25 microliters (170 +/- 62 fmol/25 microliters). Immunocytochemical analysis, Western blot analysis, reverse transcriptase-polymerase chain reaction, and in situ reverse transcriptase-polymerase chain reaction further established the expression of adrenomedullin protein and messenger ribonucleic acid in fetal amniotic membranes, suggesting that this organ is the source of amniotic adrenomedullin. CONCLUSIONS Our results clearly demonstrate the presence of adrenomedullin in second-trimester human amniotic fluid and adrenomedullin messenger ribonucleic acid and protein in amniotic membranes, suggesting that adrenomedullin is a hormone involved in the maintenance of normal pregnancy. Further studies with these molecular tools are in progress to determine the precise role of this hormone and whether adrenomedullin plays a role in the pathogenesis of various disorders of pregnancy.

Modulation of endometrial steroid receptors and growth regulatory genes by tamoxifen.

Objective We investigated tamoxifens effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation. Methods Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-α, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques. Results Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P < .003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-α, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens. Conclusion These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.

Neoplastic transformation of the endocervix associated with downregulation of lactoferrin expression

The incidence of cervical adenocarcinomas in young women over the last two decades has increased. Even with increasing knowledge of the role of human papillomavirus in the etiology of adenocarcinoma of the cervix, there is a paucity of data concerning the genetic and epigenetic factors that contribute to the histologic features and biologic behaviors of these tumors. Lactoferrin is a basic glycoprotein found in human milk, secondary granules of neutrophils, and many body secretions, and it has been associated with carcinogenesis of the endometrium, breast, and lymphoid systems. In this study, we examined the expression of lactoferrin in normal human endocervical epithelium and in cervical adenocarcinomas in relation to proliferative index, steroid receptor status, p53 protein expression, and apoptosis. Immunohistochemical and in situ studies demonstrated that lactoferrin protein and mRNA were strikingly downregulated upon neoplastic transformation of the endocervix as early as in adenocarcinoma in situ when compared with the prominent expression exhibited by the normal cervical epithelium. Furthermore, neoplastic transformation of endocervical epithelial cells was accompanied by a pronounced stimulation of proliferation and a substantial reduction in the expression of the estrogen and progesterone receptors and p53 but little or no change in the number of apoptotic cells. In conclusion, we identified lactoferrin as a novel cancer‐specific marker of endocervical adenocarcinomas that may be useful in the early detection of the disease, prediction of prognosis, and the development of new therapeutic modalities. Mol. Carcinog. 20:240–250, 1997.

Expression of brx proto-oncogene in normal ovary and in epithelial ovarian neoplasms☆☆☆★

OBJECTIVE We previously identified a protein, Brx, that interacted with estrogen receptor alpha. Sequence analysis determined that Brx is a novel member of the Dbl family of oncoproteins involved in signaling pathways that regulate cell growth. Because the Brx protein was found to be highly expressed in hormoneresponsive breast epithelium, the objective of this study was to determine whether Brx was expressed in both normal and neoplastic ovarian tissues. STUDY DESIGN A polyclonal antiserum directed against the Brx protein was used to perform immunolocalization on sections from 5 normal ovaries and 20 ovarian neoplasms. Chromosomal localization of the brx gene was accomplished by means of fluorescence in situ hybridization. Northern and Western blot analyses were performed on extracts prepared from human ovaries. RESULTS Brx protein was localized to the cytoplasm of granulosa cells from mature graafian follicles, the corpus luteum, and islands of hilar cells in normal ovaries. In tumors with low malignant potential and ovarian carcinomas the neoplastic epithelium stained strongly for Brx protein. Northern and Western blot analyses, respectively, confirmed expression of Brx messenger ribonucleic acid and protein in normal ovary. Finally, the brx gene was localized to 15q25. CONCLUSION The proto-oncogene brx is expressed in specific normal human ovarian tissues and is also present in ovarian epithelial neoplasms.

Human Pulmonary Acinar Aplasia: Reduction of Transforming Growth Factor-|[beta]| Ligands and Receptors

Pulmonary hypoplasia has been found in the human neonatal autopsy population and has been attributed to an alteration in epithelial-mesenchymal interactions during development of the lung. Pulmonary acinar aplasia is a very rare and severe form of pulmonary hypoplasia. The transforming growth factor-betas (TGF-β) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells and are expressed in developing organs including the lung; their tissue distribution patterns have possible significance for signaling roles in many epithelial-mesenchymal interactions. Here, we report our examination of TGF-β in the lungs of a term female infant diagnosed with pulmonary acinar aplasia whose autopsy revealed extremely hypoplastic lungs with complete absence of alveolar ducts and alveoli. Immunohistochemical and in situ hybridization analyses were used to localize and measure the proteins and mRNA, respectively, for TGF-β1, TGF-β2, TGF-β3, and TGF-β type I and type II receptors (TGF-β RI and RII) in formalin-fixed and paraffin-embedded sections of these hypoplastic lungs and normal lungs. Immunostaining for TGF-β1, TGF-β2, and TGF-β RI and RII was significantly lower in the bronchial epithelium and muscle of the hypoplastic lungs than in normal lungs, whereas no difference was detected in staining for other proteins including Clara cell 10-kD protein, adrenomedullin, hepatocyte growth factor/scatter factor, and hepatocyte growth factor receptor/Met in the hypoplastic and normal lungs or in the liver and kidneys of this infant compared with normal liver and kidney. In addition, in situ hybridization showed that TGF-β1 and TGF-β RI transcripts were considerably reduced in the bronchial epithelium of the hypoplastic lung compared with normal lung. These results show that there is a selective reduction of TGF-β in pulmonary acinar aplasia and suggest that the signaling action of TGF-β in epithelial-mesenchymal interactions in the lungs of this developmental condition may be compromised.