Barbara M. Iwasiow

The prolactin-inducible protein (PIP/GCDFP-15) gene: cloning, structure and regulation.

The androgen and prolactin responsive prolactin-inducible protein (PIP)/gross cystic disease fluid protein (GCDFP-15) is expressed in benign and malignant human breast tumors and in such normal exocrine organs as sweat, salivary and lacrimal glands. In this paper we report the cloning and structure of the human gene, and describe potential mechanisms involved in its regulation by hormones. The entire PIP gene, 7 kb long, was found in a single recombinant phage clone. The gene has 4 exons ranging from 106 bp to 223 bp in length. Nuclear run-on experiments utilizing PIP genomic fragments to detect nascent PIP transcripts revealed that both androgen and prolactin increased transcription of the PIP gene. Neither hormone had any effect on the stability of PIP precursor RNA or mature mRNA. Therefore the PIP gene is an excellent model by which to study the molecular events associated with the actions of prolactin and androgen in the regulation of gene expression in mammalian cells.

C-myc gene expression alone is sufficient to confer resistance to antiestrogen in human breast cancer cells

C‐myc is implicated in the initiation, progression and estrogen response of breast cancer. To further investigate the role of c‐myc in breast cancer, we have developed clonal MCF‐7 human breast cancer cell lines harboring a stably‐transfected human c‐myc gene, whose expression was stringently controlled by the bacterial reverse tetracycline transcription activator protein. The expression of the endogenous genomic c‐myc gene in MCF‐7 cells was abolished by the potent pure estrogen antagonist, ICI 182,780. Functional c‐Myc protein was identified by both Western immunoblotting and by its ability to transactivate a chimeric plasmid consisting of E‐box sequences upstream of the luciferase reporter gene. One MCF‐7 clone, 35im, was chosen for further characterization. C‐myc induction by doxycycline was rapid and dose dependent; c‐myc mRNA appeared as early as 30 min after doxycycline addition and stimulation of c‐myc expression required as little as 50 ng/ml doxycycline, with c‐myc mRNA levels reaching a plateau at 2.5 μg/ml doxycycline. ICI 182,780 or doxycycline (a tetracycline analog) treatment did not alter the mRNA levels of Max, the c‐myc binding partner. As in wildtype MCF‐7 cells, the growth of clone 35im was inhibited by 1 μM or less of ICI 182,780 and stimulated by 10 nM to 1 μM 17β‐estradiol. When maintained in a complete medium containing 5% normal fetal bovine serum (FBS) and ICI 182,780, doxycycline induced cell growth by 400% in an 8‐day assay. A similar level of growth was achieved with doxycycline treatment in cells that were arrested by the use of charcoal‐stripped FBS. Doxycycline had no effect on the growth of a control MCF‐7 clone (18c). Apoptosis, assessed by caspase‐dependent cleavage of poly(ADP‐ribose) polymerase, was unchanged in clone 35im cells after treatments with doxycycline or ICI 182,780. The present study demonstrates that c‐myc alone is sufficient to confer antiestrogen resistance in human breast cancer. Our novel c‐myc‐inducible MCF‐7 cell model offers a unique opportunity to study the diverse actions of the c‐myc proto‐oncogene in human breast cancer.

Biological actions of prolactin in human breast cancer.

Publisher Summary In birds, prolactin promotes nesting behavior and the development of the crop sac. In mammals, the most widely studied effects of prolactin are those related to the reproductive organs. In the male, prolactin affects the prostate and the testis; in the female, prolactin is both luteotropic and luteolytic for the ovary. The best known effect of prolactin in mammals is on the mammary gland: prolactin promotes both growth and differentiation in this organ and is the principal hormone that influences the production of milk. Apart from its ability to stimulate lipid synthesis and alter growth behavior, prolactin also stimulates the synthesis of unique proteins in the T-47D HBC cells. This chapter discusses an experiment in which [ 35 S]methionine-labeled proteins secreted by T-47D cells under the influence of various hormone combinations were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. The chapter discusses relationship between different forms of prolactin-inducible proteins (PIPs), and PIP in tumors and serum of breast cancer patients.

MicroRNAs and lung development

MicroRNAs (miRNAs) constitute a large group of small (∼22 nucleotides), non‐coding RNA sequences that are highly conserved among animals, plants and microorganisms, suggesting that microRNAs represent a highly conserved and important regulatory mechanism. They have been demonstrated to play an important role in gene regulation. Recently, miRNAs have become a major focus of interest for research in organ development. Research focusing on the potential role of microRNAs during lung development is slowly starting to emerge. A number of miRNAs have been demonstrated to play important roles during early and late lung development. Several studies have begun to profile miRNA expression at various stages of lung development and this article provides an overview of the various miRNAs that have been implicated in lung organogenesis. Pediatr Pulmonol. 2013; 48:317–323.

Unique Tracheal Fluid MicroRNA Signature Predicts Response to FETO in Patients With Congenital Diaphragmatic Hernia

Objective and Background: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. Methods: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-&bgr;2 in postnatal lung sections. We investigated miR-200b effects on TGF-&bgr; signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. Results: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-&bgr;2 expression was lower in CDH lungs. miR-200b inhibited TGF-&bgr;-induced SMAD signaling in cultures of human bronchial epithelial cells. Conclusions: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-&bgr;/SMAD signaling.

Pulmonary development considerations in the surgical management of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia remains a clinical challenge for both neonatologists and pediatric surgeons. Advancements in mechanical ventilation strategies and neonatal intensive care have improved survival and transformed treatment of congenital diaphragmatic hernia from emergent surgery to early stabilization of the newborn followed by delayed repair of the diaphragmatic defect. Surgical technique has evolved and minimally invasive surgical approaches to close the diaphragmatic defect in these babies will likely improve with increasing experience. Finally, as more patients are diagnosed prenatally, attempts have been made to close the diaphragmatic defect prenatally. Unfortunately, this approach did not change the outcome of affected babies. Recently, progress has been made with prenatal tracheal plugging to improve prenatal lung development. In the near future experimental studies will start to explore new ways of treating affected babies prior to birth. This article reviews the evolution of the current treatment strategies in congenital diaphragmatic hernia and its future directions.

Developmental changes in insulin-like growth factor I receptor gene expression in the mouse mammary gland.

The insulin‐like growth factor I receptor (IGF‐IR), which mediates the mitogenic action of IGF‐I, has been shown to play an essential role in normal growth and development. However, the precise role of IGF‐IR in the growth and differentiation of the mammary gland has not been elucidated. This study examines the profile of the IGF‐IR gene and protein expression during normal postnatal mammary gland development in order to gain further insight into the role of the IGF‐I/IGF‐IR during mammary gland morphogenesis. Gene and protein expression were examined in developing mouse mammary glands (virgin, pregnant, lactating, involuting) by real time PCR analysis and Western blotting. Both IGF‐IR gene and protein expression levels were high during early pregnancy. Interestingly, the level of gene expression was significantly down‐regulated during late pregnancy (5.4 fold) and lactation (9–13 fold) and significantly up‐regulated (3.9 fold) during late involution, to the level observed in the virgin mammary gland. By in situ hybridization, the IGF‐IR transcripts were localized to the proliferating ductal epithelium of the mammary glands of virgin mice and to the differentiating ductal and alveolar epithelium of the mammary glands during pregnancy and lactation. In the involuting gland, the transcripts were localized to the regressing ductal epithelium. These data are direct evidence that IGF‐IR expression is important for alveolar cell proliferation and suggest that the progression of involution may require the down‐regulation of IGF‐IR gene expression. Altogether, these results demonstrate that a developmental IGF‐IR gene expression pattern exists in the mouse mammary gland and that increases in gene expression at specific phases of development may reflect an important role for IGF‐I/IGF‐IR at those phases of development.

MicroRNAs in Lung Development and Disease

MicroRNAs (miRNAs) are small (∼22 nucleotides), non-coding RNA molecules that regulate gene expression post-transcriptionally by inhibiting target mRNAs. Research into the roles of miRNAs in lung development and disease is at the early stages. In this review, we discuss the role of miRNAs in pediatric respiratory disease, including cystic fibrosis, asthma, and bronchopulmonary dysplasia.

The transcriptome of nitrofen-induced pulmonary hypoplasia in the rat model of congenital diaphragmatic hernia

Background:We currently do not know how the herbicide nitrofen induces lung hypoplasia and congenital diaphragmatic hernia in rats. Our aim was to compare the differentially expressed transcriptome of nitrofen-induced hypoplastic lungs to control lungs in embryonic day 13 rat embryos before the development of embryonic diaphragmatic defects.Methods:Using next-generation sequencing technology, we identified the expression profile of microRNA (miRNA) and mRNA genes. Once the dataset was validated by both RT-qPCR and digital-PCR, we conducted gene ontology, miRNA target analysis, and orthologous miRNA sequence matching for the deregulated miRNAs in silico.Results:Our study identified 186 known mRNA and 100 miRNAs which were differentially expressed in nitrofen-induced hypoplastic lungs. Sixty-four rat miRNAs homologous to known human miRNAs were identified. A subset of these genes may promote lung hypoplasia in rat and/or human, and we discuss their associations. Potential miRNA pathways relevant to nitrofen-induced lung hypoplasia include PI3K, TGF-β, and cell cycle kinases.Conclusion:Nitrofen-induced hypoplastic lungs have an abnormal transcriptome that may lead to impaired development.

Actions of pituitary prolactin and insulin-like growth factor II in human breast cancer

The pituitary gland plays a central role in the regulation of mammary gland development. It secretes a variety of hormones that directly or indirectly influence the mammary gland. The best known mammogenic hormone of the pituitary gland is prolactin which directly promotes development and differentiated function (e.g., milk synthesis) of the normal mammary gland. Also, the pituitary gland can indirectly influence mammary gland development via the production of gonadotropins which stimulate the ovary to produce steroids such as estrogen and progesterone; these ovarian steroids are indispensible in mammary gland development. Furthermore, pituitary hormones regulate the production of thyroid hormones and adrenal glucocorticoids which are important in the performance of the mammary gland.