Identification of tissue of origin in body fluid specimens using a gene expression microarray assay.

Background: Body fluid specimens may be the first and only pathologic specimen for clinical evaluation in metastatic cancer cases. The challenge of identifying the tissue of origin in metastatic cancer has led to the emergence of molecular-based assays, such as the microarray-based Pathwork Tissue of Origin gene expression test. The ability to use body fluid specimens in this test would be valuable in providing diagnoses to cancer patients without clearly identifiable primary sites.

Identification of a novel estrogen-regulated gene, EIG121, induced by hormone replacement therapy and differentially expressed in type I and type II endometrial cancer.

Purpose: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer.

Experimental Design: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy.

p53-dependent inhibition of progestin-induced VEGF expression in human breast cancer cells.

VEGF, a potent angiogenic growth factor, is up-regulated in many tumors including human breast tumors and stimulates growth of vascular networks that support tumor growth and metastasis. We previously reported that natural and synthetic progestins (P) increased VEGF mRNA and protein levels in progesterone receptor (PR) containing T47-D human breast cancer cells in a PR dependent manner, but not in PR positive ZR-75 and MCF-7, or in PR negative MDA-MB-231 cells. This indicated that factors beside PR are involved in progesterone-dependent VEGF regulation.

Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium.

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.

Inhibition of progesterone-induced VEGF production in human breast cancer cells by the pure antiestrogen ICI 182,780.

The 'pure' antiestrogen ICI 182,780 (Faslodex) is in clinical trials for treatment of human breast cancer. Recently, we showed that ICI 182,780 exhibits a novel antiprogestin activity in transient transfection assays in the total absence of estrogen receptors. In this work, we determined if ICI 182,780 displays antiprogestin activity for an endogenous progesterone responsive gene.

Summary of the National Toxicology Program's report of the endocrine disruptors low-dose peer review.

At the request of the U.S. Environmental Protection Agency (U.

Pharmacological and endogenous progestins induce vascular endothelial growth factor expression in human breast cancer cells.

Tumor expansion is dependent on angiogenesis, which is regulated by peptide growth factors of which vascular endothelial growth factor (VEGF) is one of the most selective and potent. VEGF expression is regulated by steroid hormones in a number of systems, including T47-D human breast cancer cells in which VEGF protein levels are elevated by progestins. In the present study, we investigated the effect of progestins on VEGF mRNA levels in human breast cancer cells.

Regulation of vascular endothelial growth factor expression by estrogens and progestins.

Estrogens increase the expression of vascular endothelial growth factor (VEGF) mRNA in the rodent uterus. This regulatory effect is rapid, beginning within 1 hr after hormone treatment, dose dependent, and blocked by the pure antiestrogen ICI 182,780. The induction of the transcript is blocked by inhibitors of RNA but not of protein synthesis, and we have recently identified estrogen response elements in the VEGF gene.

Estrogen receptor-mediated processes in normal and cancer cells.

The role of estrogens in breast and other cancers has been extensively investigated for many years, and historically most of these studies have focused on the hormonal regulation of cell proliferation. The most recent work in this area has focused on the expression of genes likely to mediate proliferation (e.g.

Identification of functional estrogen response elements in the gene coding for the potent angiogenic factor vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and a prognostic factor for many tumors including those of endocrine-responsive tissues such as the breast and uterus. We and others have previously shown that VEGF is regulated by estradiol and tamoxifen in the uterus and by estradiol in human breast cancer cells, and pharmacological evidence has suggested that this regulation was mediated by transcriptional activation of the estrogen receptor (ER). This prompted us to investigate whether the VEGF gene contains sequences that bind the ER and confer hormonal inducibility to reporter constructs in the presence of the two ER subtypes.

Induction of the angiogenic factor VEGF in the uterus by the antiprogestin onapristone.

Onapristone (also referred to as ZK 98,299) is an antiprogestin that shares a number of structural similarities to mifepristone (RU-486) and other drugs in this class. While investigating the actions of antiprogestins on steroid hormone induced gene expression of angiogenic factors such as vascular endothelial growth factor (VEGF), we noted that onapristone alone induces VEGF transcript levels in the immature, ovariectomized rat uterus. In addition, onapristone induces expression of c-fos mRNA, which is induced by estrogens but not progestins in this target tissue.

Regulation of VEGF in the reproductive tract by sex-steroid hormones.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. In adults, angiogenesis is an infrequent event in the normal tissue except in the female reproductive tract where angiogenesis occurs frequently during the cyclical repair and regeneration of the endometrium as well as in the ovary. Little is known about angiogenesis in the male reproductive tract.

An approach to the development of quantitative models to assess the effects of exposure to environmentally relevant levels of endocrine disruptors on homeostasis in adults.

The workshop "Characterizing the Effects of Endocrine Disruptors on Human Health at Environmental Exposure Levels" was held to provide a forum for discussions and recommendations of methods and data needed to improve risk assessments of endocrine disruptors. This article was produced by a working group charged with determining the basic mechanistic information that should be considered when designing models to quantitatively assess potential risks of environmental endocrine disruptors in adults. To reach this goal, we initially identified a set of potential organ system toxicities in males and females on the basis of known and/or suspected effects of endocrine disruptors on estrogen, androgen, and thyroid hormone systems.

Synthetic estrogen 17alpha-ethinyl estradiol induces pattern of uterine gene expression similar to endogenous estrogen 17beta-estradiol.

17alpha-Ethinyl estradiol is one of most widely prescribed estrogens. We compared the effects of this synthetic estrogen to those of the endogenous ovarian hormone 17beta-estradiol on the expression of four estrogen-inducible genes in the rat uterus. The genes examined include c-fos, c-jun, vascular endothelial growth factor, and creatine kinase B, which are all known to be primary responses to estrogen administration.

Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins.

Proper regulation of angiogenesis and vascular permeability is essential for the physiological functioning of the female reproductive tract, and major health problems in women, such as dysfunctional uterine bleeding, endometriosis, and uterine cancer, involve a vascular component. There is a large body of literature that describes the effects of sex steroids on the vasculature of the reproductive tract, but far less is known about the molecular mechanisms that regulate these important actions. We hope that this minireview will help emphasize the need for mechanistic studies in this area to improve treatment and prevention of these major health problems in women.

Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements.

Estrogen receptors are derived from two different gene products referred to as estrogen receptor-alpha (ER-alpha) and ER-beta. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-beta binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-alpha and confer estrogen inducibility to reporter constructs.

The pure antiestrogen ICI 182,780 inhibits progestin-induced transcription.

The pure antiestrogen ICI 182,780 binds to the estrogen receptor with high affinity and is currently in clinical trials for the treatment of human breast cancer. We now show for the first time that ICI 182,780 also exhibits potent antiprogestin activity at doses frequently used in laboratory investigations. The antiprogestin activity of ICI 182,780 was detected in HeLa, HepG2, and CV1 cells transiently transfected with either the A or B forms of the human progesterone receptor and a luciferase construct driven by a progesterone-response element.

The 3-flanking region of the mouse c-fos gene contains a cluster of GGTCA hormone response-like elements.

We previously identified an estrogen response element in the 3'-flanking region of the c-fos protooncogene [1, 2]. This element, GGTCAnnnCAGCC, has one half-site identical to that of the consensus ERE (GGTCAnnnTGACC) but only limited homology to the second half-site. Because of this non-canonical sequence and atypical location in the 3'-untranslated region of an estrogen target gene, we decided to analyze sequences adjacent to this element for the possible presence of other regulatory elements.

A case of a laboratory animal feed with high estrogenic activity and its impact on in vivo responses to exogenously administered estrogens.

We recently noted that immature rats failed to exhibit a normal uterine response to exogenously administered estradiol as assessed by both biochemical (induction of gene expression) and morphological (altered uterine and vaginal histology and size) end points. An initial analysis suggested that this was due to a high degree of estrogenization from a dietary source which was producing a near maximal uterotrophic response prior to hormone treatment. Subsequent chemical analysis indicated that the feed in question contained high amounts of two well-known phytoestrogens, genistein (210 mg/kg) and daidzen (14 mg/kg), and the lot of feed in question produced a large uterotrophic effect when fed to immature ovariectomized rats.

Phorbol ester inhibition of estrogen-induced uterine deoxyribonucleic acid synthesis.

Protein kinase C (PKC) is a key regulatory enzyme in the control of growth and differentiated function in many cell types. Recently it has become clear that cross talk occurs between PKC and steroid hormone-activated signaling pathways. In this work we have thus used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to investigate the relationship between PKC activation and estrogen-induced proliferation in an in vivo model of hormone action, the immature rat uterus.

Triphenylethylene antiestrogens induce uterine vascular endothelial growth factor expression via their partial estrogen agonist activity.

Estradiol induces vascular endothelial growth factor (VEGF) expression in the rat uterus and this may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue. Triphenylethylene antiestrogens such as tamoxifen have mixed agonist/antagonist activity and their specific effects are tissue and gene specific. These drugs exhibit primarily antiestrogenic actions in mammary tissue and are thus used for the treatment of breast cancer.

Progestin regulation of vascular endothelial growth factor in human breast cancer cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the degree of vascularity, progression, and metastasis of breast cancer, and cases of this disease with increased vascular density have a poor prognosis. We show that in T47-D human breast cancer cells, progesterone induces a dose-dependent increase of 3-4-fold in media VEGF levels, with a maximum response occurring at a concentration of 10 nM. This effect is blocked by the antiprogestin RU 486.

Selective inhibition of estrogen-regulated gene expression in vivo by the pure antiestrogen ICI 182,780.

Estrogen-stimulated cell proliferation is thought to be mediated by the induction of growth-regulatory factors. Abnormal regulation of such factors may be associated with tumorigenesis. Two such factors, vascular endothelial growth factor and c-fos, are rapidly induced in the uterus by estrogens.

Cultured human uterine smooth muscle cells are retinoid responsive.

Primary cultures of human uterine smooth muscle cells have been widely used as a model system to evaluate agents that may play a role in the regulation of both normal and abnormal proliferative responses. We have used this in vitro system to determine if human uterine smooth muscle cells are responsive to treatment with a potent natural derivative of vitamin A, all-trans retinoic acid (ATRA). These studies were also designed to determine if there is a difference in retinoid responsiveness between normal smooth muscle and adjacent leiomyoma (a benign tumor of uterine smooth muscle).

Neonatal exposure to diethylstilbestrol permanently alters the basal and 17 beta-estradiol induced expression of c-fos proto-oncogene in mouse urethroprostatic complex.

Perinatal estrogen exposure induces permanent structural and functional changes in the male reproductive tract. We have studied the effect of neonatal estrogenization on the estrogen-responsive c-fos proto-oncogene expression in mouse prostate. Fos is involved in growth and differentiation, and may play a central role in regulating diverse estrogen-related cellular differentiation.