A system for detecting high impact-low frequency mutations in primary tumors and metastases.

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

A conditionally replicative, Wnt/beta-catenin pathway-based adenovirus therapy for anaplastic thyroid cancer.

Thyroid cancer affects between 10,000 and 15,000 people per year in the US. Typically, this disease can be controlled with surgical resection and radioiodide treatment. However, resistance to these conventional therapies is observed in some patients, who develop intractable anaplastic thyroid cancer (ATC), for which no effective therapies exist. Recently, a sizable fraction of undifferentiated or poorly differentiated thyroid cancers were shown to contain mutations in beta-catenin, an oncogenic protein involved in the etiology of cancers of many tissues. We developed a conditionally replicative adenovirus (named 'HILMI') which, by virtue of TCF response elements drives E1A and E1B expression, replicates specifically in cells with an active Wnt/beta-catenin pathway. We show that several thyroid cancer cell lines, derived from undifferentiated or anaplastic tissues and possessing an active Wnt/beta-catenin pathway, are susceptible to cell killing by HILMI. Furthermore, viral replication in ATC cells as xenograft tumors in nude mice was observed, and prolonged survival of mice with ATC tumors was observed following administration of the HILMI therapeutic vector. The results warrant further development of this therapeutic approach for ATC patients.

NF-κB-HOTAIR axis links DNA damage response, chemoresistance and cellular senescence in ovarian cancer.

The transcription factor nuclear factor kappa B (NF-κB) and the long non-coding RNA (lncRNA) HOTAIR (HOX transcript antisense RNA) have diverse functional roles in cancer. In this study, we show that upregulation of HOTAIR induced platinum resistance in ovarian cancer, and increased HOTAIR levels were observed in recurrent platinum-resistant ovarian tumors vs primary ovarian tumors. To investigate the role of HOTAIR during DNA damage induced by platinum, we monitored double-strand breaks and show that HOTAIR expression results in sustained activation of DNA damage response (DDR) after platinum treatment. We demonstrate that ectopic expression of HOTAIR induces NF-κB activation during DDR and interleukin-6 and interleukin-6 expression, both key NF-κB target genes. We show that HOTAIR regulates activation of NF-κB by decreasing Iκ-Bα (NF-κB inhibitor) and establish that by inducing prolonged NF-κB activation and expression of NF-κB target genes during DNA damage, HOTAIR has a critical role in cellular senescence and platinum sensitivity. Our findings suggest that an NF-κB-HOTAIR axis drives a positive-feedback loop cascade during DDR and contributes to cellular senescence and chemotherapy resistance in ovarian and other cancers.

Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

Estrogen differentially affects c-jun expression in uterine tissue compartments.

Estrogen rapidly induces expression of the jun immediate early gene family in mature and immature rodent uteri, suggesting that these protooncogenes are directly involved in the proliferative response of the uterus to estrogen. The jun family mRNAs, however, have not been localized to specific uterine cell types. Furthermore, it is necessary to differentiate between the response of the immature vs. the mature rat uterus to 17 beta-estradiol (E2-17 beta), because in the former, all uterine cell types respond to estrogen with increased DNA synthesis, but in the latter, the proliferative response is restricted to the uterine epithelial cells. In the present study, in situ hybridization was used to determine the cell type-specific location of mRNA encoding the immediate early genes c-jun, jun-B, and jun-D after the administration of E2-17 beta to mature and immature rats. Estradiol stimulated jun-B and jun-D expression primarily in the uterine luminal and glandular epithelium. The pattern of c-jun expression, however, was strikingly different; E2-17 beta repressed c-jun mRNA levels in the uterine luminal epithelium and simultaneously increased c-jun expression in the uterine myometrium. In mature vs. immature uteri, the general cell type-specific patterns of jun-B and jun-D expression were similar after estrogen administration. The expression of c-jun was increased by estrogen in the uterine glands as well the uterine myometrium of immature rats; however, in mature rats, uterine glandular epithelial cells did not respond to E2-17 beta administration with increased c-jun expression. These experiments demonstrate for the first time positive and negative regulatory actions of estrogen on c-jun expression and suggest a role for tissue-specific factors in the control of c-jun expression. The lack of maturational effects on jun gene expression implies that the differential response of the immature vs. the mature uterus to estrogen, in terms of cell proliferation, involves a point of control other than that at the level of the jun protooncogene family.

Cellular localization of estradiol-induced c-fos messenger ribonucleic acid in the rat uterus: c-fos expression and uterine cell proliferation do not correlate strictly.

Estrogens stimulate DNA synthesis and cell proliferation in the uterus. All major uterine cell types (luminal and glandular epithelium, stroma, and myometrium) respond to 17 beta-estradiol in the immature animal, whereas primarily epithelial cells of the uterine endometrium respond in the mature animal. Rapid activation of the c-fos protooncogene by estrogen precedes the uterine growth, suggesting that c-fos plays a role in amplifying the hormonal signal. The specific uterine cell types in which estrogen induces c-fos messenger RNA (mRNA) expression, however, have not been identified in either mature or immature animals. In this study, in situ hybridization was used to determine the cell type-specific location of mRNA encoding c-fos in the uterus. In both immature and mature castrated rats at 3 h after 17 beta-estradiol administration, c-fos expression was detected primarily in uterine luminal and glandular epithelia. Expression of c-fos returned to baseline levels by 24 h post 17 beta-estradiol treatment. There was no apparent difference in the uterine cell type-specific pattern of c-fos expression stimulated by estradiol in mature vs. immature animals. Nuclear run-on transcription assay in isolated luminal epithelial cell nuclei showed that c-fos gene transcription increased rapidly in the uterus after estradiol stimulation. Treatment of adult rats with a single injection of 16 alpha-estradiol, a short-acting, nonmitogenic estrogen, induced c-fos primarily in the uterine glandular epithelia. Progesterone is known to modify the action of estrogen on the uterus by redirecting the proliferative response from epithelia to stroma. To determine if progesterone modulation of estrogen action involves shifting of c-fos expression to stromal cells, rats were treated with progesterone for 48 h and then killed 0, 3, 6, or 12 h after an estradiol injection. In situ hybridization analysis revealed that c-fos mRNA remained localized in the uterine luminal and glandular epithelia, and expression was not shifted to the stroma. Although these results support the idea that c-fos plays a role in proliferation of uterine epithelial cells, they also invite reassessment of the role played by c-fos in both epithelial and nonepithelial uterine cell types.

Tamoxifen-induced proto-oncogene expression persists in uterine endometrial epithelium.

The use of the antiestrogen tamoxifen for breast cancer management, although generally well tolerated, is linked to an increase in uterine pathologies in a high number of postmenopausal women receiving the drug. This effect is thought to be due to estrogenic stimulation of the uterine endometrium by the antiestrogen; however, the molecular mechanism underlying the uterotrophic activity of tamoxifen and the uterine cellular compartments that respond to the drug have not been clearly established. In this study, we determined which of the several uterine tissues (myometrium, stroma, and luminal and glandular epithelium) demonstrated chronic overexpression of c-fos and the jun proto-oncogenes in response to tamoxifen. Uteri from tamoxifen-treated, castrated rats were examined histologically, and cell type-specific expression of c-fos, c-jun, jun-B, and jun-D was assessed using in situ hybridization. Treatment with tamoxifen resulted in uterine luminal and glandular epithelial hypertrophy and basally located nuclei by 36 h. Extreme uterine glandular and luminal epithelial cell hypertrophy persisted 7 days after administration of the drug. Expression of c-fos and jun-B messenger RNA was first detected in the luminal and glandular epithelial at 12-36 h post tamoxifen injection. Seven days after tamoxifen treatment, c-fos and jun-B messenger RNA levels were lower but still evident in the uterine endometrial epithelium. Tamoxifen completely repressed constitutive expression of c-jun in the uterine luminal epithelial cells by 12 h but, unlike estrogen, did not induce c-jun expression in the uterine myometrium. Expression of jun-D in the uterine glandular and luminal epithelia was observed at 12 h but not at 24 h post tamoxifen. These results support our working hypothesis that persistent overexpression of cellular oncogenes c-fos and jun-B in the uterine endometrial epithelium may contribute to the molecular mechanism underlying the uterine toxicity associated with chronic tamoxifen treatment.

The antiestrogen tamoxifen induces c-fos and jun-B, but not c-jun or jun-D, protooncogenes in the rat uterus.

Tamoxifen is the leading therapeutic agent in the management of breast cancer. Although classified as an antiestrogen, tamoxifen displays partial estrogen agonist activity in the rat uterus. The molecular mechanism of the uterotrophic action of tamoxifen is unclear. The purpose of the present study was to investigate the effect of tamoxifen on activation of the c-fos, c-jun, jun-B and jun-D protooncogenes in rat uteri in vivo. These immediate early response genes are transcription regulatory factors that can regulate the expression of genes containing an AP-1 recognition site. Northern blotting analysis of total uterine RNA revealed that treatment of adult, ovariectomized rats with tamoxifen elevated levels of c-fos mRNA 2.4-, 5.3- and 6.2-fold over expression in untreated rats by 6, 12 and 24 h post-tamoxifen injection, respectively. Tamoxifen induction of c-fos was still apparent by 48 h post injection. The effect of tamoxifen on expression of the jun gene family was also examined. Tamoxifen markedly induced uterine expression of jun-B mRNA by approximately 10-fold over expression in untreated rats by 24 h after administration. By 48 h, levels of jun-B transcripts had declined to 2.3 fold over control values. In contrast, only slight induction of c-jun and jun-D expression (1.3- and 1.6-fold, respectively) by 12 h after tamoxifen treatment was observed. The kinetic pattern of tamoxifen-induced expression of these genes was strikingly different from what has been reported for other stimuli, e.g., estrogen. This is the first report to examine the effects of tamoxifen on protooncogene expression in the rat uterus. Our results indicate that the activation of immediate early response genes may play a role in the uterotrophic actions of tamoxifen. However, based on the kinetics of induction, we suggest that tamoxifen is much less effective at transcriptional activation of protooncogenes than other uterine agonists, e.g., estrogen. The lack of synchronous expression of the jun genes may indicate that distinct control mechanisms exist among members of this protooncogene family.

A novel role for GM-CSF: enhancement of pregnancy specific interferon production, ovine trophoblast protein-1.

Transient and massive production of ovine trophoblast protein-1 (oTP-1) by preimplantation conceptuses seems to be a critical event required for the establishment of successful pregnancy. We have previously demonstrated that one of several oTP-1 genes is predominantly expressed between days 13 and 20 of pregnancy and that this oTP-1 gene contains an AP-1 site, a transcription enhancer element, in the 5'-flanking region. We have obtained evidence, indicating a linkage between cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and conceptus production of the trophoblast interferon (IFN), oTP-1. These are: 1) oTP-1 production (both polypeptide and mRNA) is enhanced by the addition of GM-CSF in vitro and 2) GM-CSF mRNA is localized in the luminal and glandular epithelium of the uterine endometrium. Based on these observations, we propose that the massive amounts of oTP-1 produced during the period of pregnancy establishment is stimulated at least in part by maternal GM-CSF.

Cell-specific induction of c-fos expression in the pituitary gland by estrogen.

Estrogens regulate many functions of pituitary lactotrophs, including PRL gene expression, release, storage, and cellular proliferation. The mechanism by which estrogens exert such a variety of functions is poorly understood. In the uterus, estrogens rapidly and transiently induce the expression of the immediate early genes c-fos and c-jun in specific cell types. The Fos/Jun proteins form the activating protein-1 (AP1) transcription factor that mediates ligand-activated cell proliferation, differentiation, and secretion. Here we used Fischer 344 (F344) rats that develop hyperprolactinemia and prolactinomas in response to estrogens. The objectives were to: 1) determine whether estrogen induces c-fos expression in the pituitary gland and identify the responsive cells; 2) compare the dynamics of c-fos induction in the pituitary and uterus; and 3) examine the temporal relationship between c-fos expression and PRL release. Ovariectomized F344 rats were injected with 1 microg estradiol and killed at different times thereafter. Pituitaries were subjected to in situ hybridization for c-fos and immunostaining for selected pituitary cells. Estradiol stimulated c-fos expression in lactotrophs and folliculo-stellate cells within the anterior lobe without affecting either the intermediate or neural lobes. In a second experiment, c-fos messenger RNA levels were measured by solution hybridization in anterior pituitaries and uteri from estradiol-treated rats. Trunk blood was analyzed for PRL by RIA. The estrogen-induced c-fos rise in the uterus was rapid, robust, and transient, whereas that in the anterior pituitary was delayed, lower, and sustained. The profile of serum PRL levels resembles that of c-fos induction in the anterior pituitary. We conclude that: 1) both lactotrophs and folliculo-stellate cells increase c-fos expression in response to estrogens; 2) induction of c-fos expression may mediate some estrogenic effects on PRL synthesis and release and lactotroph proliferation in F344 rats; and 3) the atypical dynamics of c-fos induction in the pituitary could be due to indirect effects of estrogens on PRL-regulating factors within the hypothalamo-pituitary complex as well as to pituitary-specific estrogen receptor isoforms, coactivators, or repressors.

Activation of transcription factors activator protein-1 and nuclear factor-kappaB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), the prototype agonist of the aromatic hydrocarbon (Ah) receptor, is a potent tumor promoter as well as a complete liver carcinogen that produces an oxidative stress response in rodents and in cultured cell lines. It has been proposed that TCDD promotes neoplastic transformation through oxidative signal transduction pathways, which results in activation of immediate-early response transcription factors. To set the stage for a test of this hypothesis, we evaluated the effect of TCDD treatment on the activation of several transcription factors, including those in the nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) families, which are activated by changes in the redox state of cells. In an extension of prior results, we found that TCDD treatment produced a sustained overexpression of AP-1 for at least 72 hr in wild-type mouse hepatoma Hepa-1 cells, but not in the Ah receptor-deficient derivative c35 or in cytochrome P450-1A1 (CYP1A1)-negative c37 cells. In addition, TCDD treatment caused a significant increase in the DNA binding activity of NF-kappaB, but not in the activities of the other transcription factors tested. AP-1 and NF-kappaB activation were blocked by the thiol antioxidant N-acetylcysteine and by nordihydroguaiaretic acid, an antioxidant and lipooxygenase inhibitor and an inhibitor of the epoxygenase activity of CYP1A1, and did not take place in c35, c37, or in Ah nuclear translator-deficient c4 cells. Hence, sustained activation of these two transcription factors by TCDD is likely to result from a CYP1A1-dependent and Ah receptor complex-dependent oxidative signal. Electrophoretic mobility supershift analyses with specific antibodies showed that most of the increase in NF-kappaB binding activity could be accounted for by increases in p50/p50 complexes. Since these complexes are known to repress NF-kappaB-dependent gene transcription, our results delineate a second molecular mechanism, in addition to the recently found block of tumor necrosis factor-alpha-mediated p50/p65 activation, that may be responsible for the immunosuppresive effects of TCDD.

Cell density influences hormonal responsiveness but not lipoprotein utilization in cultured bovine luteal cells.

The objective of the present study was to investigate the effect of cell density on hormonal responsiveness and lipoprotein utilization by cultured bovine luteal cells. Luteal cells obtained from regularly cycling dairy cows were plated at three culture densities: 0.5 X 10(6), 1 X 10(6) and 2 X 10(6) cells/flask in serum-free Ham's F-12 culture medium, and maintained for 9 days. Basal steroidogenesis was unaffected by cell density, while LH responsiveness was greatest in low density cultures. Progesterone produced in response to LH (10 ng/ml) was greater than control levels throughout the culture period in low density cultures. Luteal cells cultured at medium and high densities became responsive to LH only later in the culture period (days 5 and 9, respectively). In contrast, prostaglandin F2 alpha (PGF2 alpha, 100 ng/ml) was more effective in high density cultures, causing a complete inhibition of LH stimulation and returning progesterone levels to basal values. In low density cultures, treatment with PGF2 alpha + LH resulted in progesterone levels that were not significantly different from LH-treated cultures. There was no effect of cell density on utilization of either low density lipoprotein (LDL) or high density lipoprotein (HDL) for steroidogenesis. However, a synergistic effect of LH with either lipoprotein was observed in low and medium density, but not high density cultures. From these results, it is concluded that culture density can influence the responsiveness of bovine luteal cells to either LH or PGF2 alpha, but has no effect on lipoprotein utilization by these cells.

Strain differences in vaginal responses to the xenoestrogen bisphenol A.

Bisphenol A (BPA) is the monomer component of polycarbonate plastics and epoxy resins; human exposure derives from leachate in foodstuffs packaged in certain plastics or from epoxy-based dental appliances. BPA stimulates prolactin secretion in Fischer 344 (F344) rats but not in Sprague-Dawley (S-D) rats. The present studies were performed to determine if another classic estrogen target tissue, the rat vagina, responds to BPA in a strain-specific manner. In F344 rats BPA increased DNA synthesis in vaginal epithelium with a median effective dose (ED(50)) of 37.5 mg/kg body weight; DNA synthesis was not stimulated in S-D rats by any dose tested. Clearance of (3)H-BPA from blood followed the same time course in both strains of rats, with a half-life of 90 min. Scatchard analysis of [(3)H]estradiol binding showed no strain differences in concentration or affinity of the vaginal estrogen receptor. BPA increased the level of mRNA for the immediate early gene, c-fos, with similar dose-response curves in both rat strains. Thus, F344 and S-D rats exhibit differences in sensitivity to BPA at the level of cell proliferation in the vaginal epithelium. However, metabolic clearance of BPA and the early events that lead to the proliferative response, receptor-ligand interaction and induction of immediate early genes, show no strain differences. These observations suggest that differences in intermediate effects must account for the difference in sensitivity of the proliferative response to the xenoestrogen. Furthermore, these results point to the need for caution in choosing a suitable end point and animal model when seeking to test the estrogenic effects of xenobiotics.

Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast.

Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol. Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear. The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified. Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro. We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast. DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity. DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2. Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2. Ligand-receptor interaction for other C19-steroids was also examined. While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not. We have investigated whether DHEA can interact with the human ER in vivo. Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist.

Hormonal regulation and expression of the jun-D protooncogene in specific cell types of the rat uterus.

Steroid hormone regulation and cell-type specific expression of the jun-D protooncogene in rat uterus was examined. Adult, ovariectomized rats were injected with progesterone, testosterone, 17beta-estradiol (E2-17beta), 16alpha-estradiol (E2-16alpha), dexamethasone or cycloheximide. Uteri were collected between 0 and 6 h post-treatment. Northern blot analysis of uterine RNA revealed that induction of jun-D was specific for estrogenic steroids, as progesterone and testosterone had no effect on expression of this member of the jun gene family. Treatment with E2-17beta increased jun-D mRNA levels by approx. 5-fold, with expression reaching peak levels at 3 h after treatment and declining thereafter. Administration of E2-16alpha, a short-acting estrogen that does not cause uterine cell proliferation, increased expression of jun-D but with different kinetics compared to the long-acting E2-17beta. The mRNA levels of jun-D increased by 3-fold 1 h after administration of E2-16alpha but declined soon after. Slight induction of jun-D mRNA by dexamethasone was apparent, but to a much lesser extent compared to estrogen. The protein synthesis inhibitor, cycloheximide, did not block jun-D induction, indicating that this is an "immediate early" response. Expression of Jun-D protein was examined by immunohistochemical methods. E2-17beta treatment activated jun-D primarily in the nuclei of luminal and glandular epithelial cells of the endometrium. These results demonstrate that hormonal induction of jun-D is specific for estrogens and that uterine expression of this protooncogene occurs in a cell-type specific manner.

Effects of the xenoestrogen bisphenol A on expression of vascular endothelial growth factor (VEGF) in the rat.

Bisphenol-A (BPA) is used to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances. BPA binds to estrogen receptors and induces estrogenic activity in a number of biological systems. We recently reported that although Fisher 344 (F344) and Sprague-Dawley (S-D) rat strains exhibit different sensitivities to BPA at the level of vaginal epithelial cell proliferation, there was no difference in immediate early proto-oncogene expression between the two animal strains. In the present study we investigated the effects of BPA on expression of another estrogen-target gene, vascular endothelial growth factor (VEGF), in the uterus, vagina, and pituitary of F344 and S-D rats. Adult rats were ovariectomized and treated with BPA by intraperitoneal injection at concentrations of 0.02 to 150 mg/kg body wt. Expression of VEGF was monitored by RNase protection assay at 2 hr after treatment. There was a significant effect of dose of BPA on the type of VEGF isoform expressed in the uterus, vagina, and pituitary. BPA induced greater (P < 0.01) levels of VEGF164 and VEGF120+188 than VEGF110 levels. The lowest BPA dose that had a significant (P< 0.05) effect on VEGF expression compared with vehicle treatment was 37.5 mg/kg body wt.; dose-response curves did not differ between strains. This is the first report that the primary response of the uterus, vagina, and pituitary to BPA includes rapid induction of VEGF expression. Due to the capacity of VEGF to engage pleiotropic signaling pathways in other cellular systems, we suggest that modulation of VEFG may play a role in establishing the response of estrogen-target organs to estrogenic xenobiotics.

Evidence for expression of estrogen receptor cofactor messenger ribonucleic acid in the ovary and uterus of domesticated animals (sheep, cow and pig).

Expression levels of estrogen receptor cofactors (coactivators or corepressors) in specific tissue compartments and cells are thought to influence the expression of estrogen responsive genes and thereby influence overall hormonal responsiveness of target tissues. To date, the presence of cofactors has been reported in tissues from humans, rats and mice. We analyzed the presence and distribution of messenger ribonucleic acids (mRNAs) encoding several transcriptional cofactors in the ovary and uterus of three domestic animal species, the sheep, cow and pig. Northern analysis for cofactors SRC-1, GRIP1, RAC3, p300, RIP140, and SPA showed expression in ovaries from all three species. In addition, lower expression of SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs was observed during the luteal phase (day 10-12 of the estrous cycle) than at estrus (day 0); however, SPA transcript levels remained unchanged. We then examined expression of mRNAs for changing (SRC-1, RIP140) and constitutively expressed (SPA) cofactors in ovine ovaries. SRC-1 and RIP140 transcripts in corpus luteum were lower compared to the surrounding ovarian tissue. SPA mRNA expression, however, was similar in corpus luteum and surrounding tissues. To determine which ovarian cell types express SRC-1, RIP140, and SPA, in situ hybridization was performed on sheep ovaries. Silver grains corresponding to these cofactors were seen in ovarian granulosa, theca and stromal cells, but appeared to be most abundant in the granulosa cells. Expression of SRC-1 and RIP140 in corpus luteum, however, was reduced compared to expression in follicular cells. Finally, we examined cofactor expression in ovine, bovine, and porcine uterus. Northern blot analysis for SRC-1, GRIP1, RAC3, p300, and RIP140 mRNAs showed higher expression in extracts of the endometrium compared to whole uterus. We provide the first evidence for the presence of estrogen receptor cofactor mRNAs in the ovary and uterus of three domestic animal species. We suggest that coactivators are conserved among species and associated with hormonal responsiveness of reproductive tract tissues in sheep, cow and pig.

Extension of short cycles in postpartum beef cows by intrauterine treatment with catecholestradiol.

Eight multiparous beef cows were used to examine the effects of intrauterine infusion of catecholestradiol (4-hydroxylated estradiol) on development and function of the first corpus luteum after parturition. Calves were weaned on day 1 (day 0 = parturition) to initiate formation of a corpus luteum (CL) by approximately day 10 or 11. Before CL formation, on days 5 to 9, cows received twice daily infusions of catecholestradiol (4 micrograms; n = 4) or vehicle (n = 4) into the uterine horn opposite the previous pregnancy. Plasma progesterone during the first estrous cycle was elevated longer (P less than .001) and reached a higher (P less than .001) concentration in cows treated with catecholestradiol. The decline in progesterone was associated with an increase in plasma 13,14-dihydro, 15-keto-prostaglandin F2 alpha (PGFM) in all cows infused with catecholestradiol. In contrast, a rise in PGFM at the end of the first short cycle was detected in only one of four cows treated with vehicle. Furthermore, PGFM concentrations were linearly related (R2 = .870; P less than .001) to concentrations of progesterone. Estradiol-17 beta concentrations were not different during the infusion period, but after formation of the first CL, estradiol remained elevated (P less than .01) in cows that received vehicle. Results of this experiment suggest that exposure of postpartum beef cows to catecholestradiol extended luteal function in association with enhanced PGFM release.

Influence of catecholestradiol on short-lived corpora lutea in beef cows.

The influence of intrauterine administration of catecholestradiol (4-hydroxylated estradiol) on lifespan of the initial postpartum corpus luteum was evaluated in suckled beef cows. In experiment 1, postpartum cows (n = 23) were untreated (CONTROL) or received intrauterine infusions (0700 and 1700 hr) of either vehicle (SAL) or catecholestradiol (CATE; 4 micrograms) from day 15 to 22 (day 0 = parturition). Blood samples were collected three times weekly (day 15 to 100) and analyzed for progesterone. In experiment 2, cows received twice daily intrauterine infusions of either vehicle (n = 18), or catecholestradiol (n = 19), from day 25 +/- .5 to day 30 +/- .5. Following the final infusion, calves were temporarily weaned from all cows for 48 hr. At the end of the 48 hr weaning period, cows in each infusion group received either an i.m. injection of 1,000 IU hCG (SAL+hCG, n = 9; CATE+hCG, n = 9) or no further treatment (SAL, n = 9; CATE, n = 10). Blood samples were collected daily for 21 d following calf removal and 3 times weekly through 100 d postpartum. In both experiments, the initial postpartum elevation in peripheral progesterone concentrations was characterized as either a short (< 5 d) or extended (> 8 d) luteal phase. In experiment 1, postpartum anestrous interval (60 +/- 3.4 d) and incidence of short luteal phases (77%) were similar among CONTROL, SAL and CATE treatments. In experiment 2, luteal phases were induced within 10 d of onset of weaning in 90, 100, 56 and 60% of cows in SAL+hCG, CATE+hCG, SAL and CATE treatments, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in follicular endocrinology during final maturation of porcine oocytes.

Thirty-one gilts were ovariectomized between 21 and 34 hr after the onset of estrus to compare changes in follicular endocrinology with stages of oocyte maturation. Oocytes were recovered from 6 to 8 mm follicles and classified by stage of meiosis. Remaining follicular fluid was assayed for steroids and dermatan sulfate. Amounts of prostaglandin F2 alpha (PGF2 alpha) and E2 (PGE2) were measured in intramural tissues. Coincident with germinal vesicle breakdown, the follicular content of all steroids except testosterone decreased (P less than .05). As oocytes approached metaphase II, the amount of progesterone within follicles increased (P less than .05), and estradiol continued to decrease (P less than .05). The pattern of dermatan sulfate content was biphasic and peaked at germinal vesicle breakdown and anaphase stages. Amounts of PGF2 alpha and PGE2 within intramural tissues increased (P less than .05) throughout oocyte maturation. Follicular atresia was evident during estrus; however, more (P less than .05) atretic follicles were recovered at germinal vesicle than metaphase II stages (20 vs 3%, respectively). Follicular development, within a gilt, was skewed (P less than .05) and classification of follicles by hormone content demonstrated that a majority were more mature than a minority of less mature follicles. These data suggest that follicular maturation and oocyte development are highly correlated in swine. Furthermore, partitioning the follicular variability by hour and stage of oocyte maturation allowed for more precise assessment of follicular endocrinology than previously reported.