Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Both hyaluronan and one of its receptors, CD44, can be demonstrated in the early human conceptus and in placental stroma. The variants of CD44 resulting from variable exon splicing are found in metastasizing human malignancies and are also involved in hyaluronan uptake and degradation. The resulting hyaluronan fragments are known to be highly angiogenic. We postulated that the self-limited process of trophoblast invasion of the uterine decidua results in part from the strategy of alternative splicing of CD44, similar to that used by invasive cancer cells in the course of metastatic spread and possibly angiogenesis. Monoclonal antibodies specific for CD44s and for an exon expressed during metastatic tumour progression, CD44v7, were used to examine this hypothesis. In this study we found human trophoblasts, for the first time, to express CD44. Intermediate trophoblasts of first and second trimester exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the invading characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7-8. Moreover, in the case of placenta accreta there was a prominent membrane staining of the trophoblasts that were embedded in the fibrin layer over the myometrium. The highly metastatic choriocarcinoma cells also expressed CD44v7-8. We propose, therefore, that the invading trophoblasts utilize the alternatively splicing machinery. These cells retain their invasive capabilities through the permissive ECM by carrying the CD44v7-8 isoform, which binds weakly to hyaluronan and thus prevents it from being degraded by intracellular hyaluronidase.
Mol Hum Reprod 1996 Sep
PMID:Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis. 923 83

The aims of this study were to establish whether both the cystic fibrosis (CF) and multidrug resistance (MDR1) genes are expressed in the human placenta during development and differentiation. To study their pattern of expression during development, RNA was extracted from first, second and third trimester human placentas. To investigate differentiation, RNA was extracted from cytotrophoblast cells isolated from human term placentas and maintained in culture for 18, 66, 90 and 114 h and from the undifferentiated choriocarcinoma cell line JAr. Using the reverse transcriptase-polymerase chain reaction (RT-PCR) with gene specific, intron spanning primers, a cDNA product of 1 kb, as expected for CF expression, was detectable following 35 cycles of PCR from all RNA samples except those from JAr; in the latter a product was only detected in one sample out of four separate passages and this was only just detectable after 40 cycles of PCR. RT-PCR using MDR1 specific primers resulted in a product from all samples at 0.34 kb as expected if this gene is expressed. These results demonstrate that both the CF and MDR1 genes are expressed in the human placenta at all stages of development and differentiation, although the expression of the CF, but not the MDR1, gene appears to be much weaker in the undifferentiated JAr cells in comparison with cytotrophoblast cells.
Mol Hum Reprod 1996 Sep
PMID:Expression of the cystic fibrosis (CF) and multidrug resistance (MDR1) genes during development and differentiation in the human placenta. 923 84

The human chorionic somatomammotropin gene enhancer (CSEn) is composed of multiple enhansons (Enh) that share sequence similarities with those of the simian virus, SV40 enhancer (SVEn). The sequence homology includes two GT-IIC-like (Enh1 and Enh4) and three SphI/II-like enhansons (Enh2, Enh3, and Enh5). We previously showed that transcription enhancer factor 1 (TEF-1) and a 30-kDa placental-specific factor, chorionic somatomammotropin enhancer factor 1 (CSEF-1), bind to Enh4, which plays an essential role in enhancer function. In this study, we demonstrate that TEF-1 and CSEF-1 bind specifically to all the other GT-IIC- and SphI/II-like elements within CSEn with a broad range of binding affinities that vary between 0.005 and 0.15 that of Enh4. Each individual concatenated enhanson was able to stimulate hCS promoter activity in an orientation-independent manner in choriocarcinoma cells (BeWo) with an observed stimulation that was directly proportional to its relative binding affinity for TEF-1 and CSEF-1. These results indicate that CSEn function results from the cooperative interaction of TEF-1 and/or CSEF-1 binding to multiple, low-affinity GT-IIC- and SphI/II-like enhansons within the enhancer.
Mol Endocrinol 1997 Aug
PMID:Human chorionic somatomammotropin enhancer function is mediated by cooperative binding of TEF-1 and CSEF-1 to multiple, low-affinity binding sites. 925 14

The human GH (GH) gene family includes the pituitary-specific hGH-1, placental-specific chorionic somatomammotropin (hCS-5, hCS-2, and hCS-1), and hGH-2 genes. These duplicated, nearly identical genes are localized on approximately 50 kb of DNA on chromosome 17q23-q24. An enhancer (CSEn2), located downstream of the hCS-2 gene, participates in mediating placental-specific hCS gene expression. In the preceding paper we demonstrated that CSEn2 activity derives from the cooperative binding of transcription factor-1, TEF-1, and a placental-specific factor CSEF-1 to multiple enhansons, Enh1-Enh5, that are related to the SV40 GT-IIC and SphI/SphII enhansons. Here we demonstrate that two copies of CSEn2 or a single copy of CSEn2 linked to either of the other two enhancers in the hGH/hCS locus, CSEn1 and CSEn5, act cooperatively to enhance hCS promoter activity in choriocarcinoma (BeWo) cells, but silence the promoter in pituitary GC cells. Mutation of Enh4, an essential GT-IIC-like enhanson in the context of the intact enhancer, abolishes silencer activity, and multimerized GT-IIC enhansons mimic the intact CSEn enhancer/silencer activities in BeWo and GC cells, respectively. By antibody-mediated supershift, Western, and far Western analyses, we identified TEF-1 as the GT-IIC-binding factor in pituitary cells. The data suggest that TEF-1 may be involved in pituitary-specific repression of placental GH/CS gene transcription through long-range interactions between the multiple CS enhancers present on the GH/CS gene locus.
Mol Endocrinol 1997 Aug
PMID:The human chorionic somatomammotropin enhancers form a composite silencer in pituitary cells in vitro. 925 15

The JAr choriocarcinoma cell cultures have demonstrated high levels of aromatase activity and have been useful for assaying a wide variety of aromatase inhibitors for aromatase inhibition in intact cells. Recently, several 7alpha-arylaliphatic androgens have shown effective inhibition of human placental microsomal aromatase in vitro, with apparent Ki values ranging from 10 to 20 nM. A series of 7alpha-arylaliphatic androst-4-ene-3,17-dione compounds demonstrated potent competitive inhibition, and 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were enzyme-activated irreversible inhibitors. Both series of these potent inhibitors were investigated for the ability to inhibit aromatase activity in JAr cells by measuring the conversion of [1beta-3H]-androstenedione to 3H2O and unlabelled estrone. JAr cell cultures were incubated for 2 h at 37 degrees C with the aromatase inhibitors at concentrations of 10 pM to 10 microM, the percentage of enzyme inhibition was determined, and IC50 values for inhibitors were calculated. Both series of synthetic compounds demonstrated good to excellent aromatase inhibition, and the most effective inhibitors in both series were those compounds with a phenylpropyl substituent at the 7alpha-position of the steroid nucleus. The 7alpha-arylaliphatic androst-4-ene-3,17-diones exhibited inhibition of JAr aromatase activity with IC50 values from 300 to 434 nM. More potent aromatase inhibition was observed with the 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones, which exhibited IC50 values from 64 to 232 nM. Enhanced efficacy of steroidal enzyme-activated irreversible inhibitors compared to competitive inhibitors was observed in these studies and is consistent with previous reports. These results suggest that JAr choriocarcinoma cells with high levels of aromatase activity may be useful in differentiating steroidal aromatase inhibitors exhibiting different mechanisms of enzyme inhibition. In summary, the 7alpha-phenylpropyl androsta-1,4-diene-3,17-dione analogs, which are enzyme-activated irreversible inhibitors, demonstrated the most effective inhibition of aromatase activity present in the JAr cell cultures among the various 7alpha-arylaliphatic androgens.
J Steroid Biochem Mol Biol 1997 Apr
PMID:Aromatase inhibition in JAr choriocarcinoma cells by 7alpha-arylaliphatic androgens. 932 12

CG is required for maintenance of the corpus luteum during pregnancy in higher primates. As CG is a heterodimeric molecule, some form of coordinated control must be maintained over the transcription of its two subunit genes. We recently found that expression of human CG beta-subunit (hCGbeta) in JAr human choriocarcinoma cells was almost completely silenced by the embryonic transcription factor Oct-3/4, which bound to a unique ACAATAATCA octameric sequence in the hCGbeta gene promoter. Here we report that Oct-3/4 is also a potent inhibitor of hCG alpha-subunit (hCGalpha) expression in JAr cells. Oct-3/4 reduced human GH reporter expression from the -170 hCGalpha promoter in either the presence or absence of cAMP by about 70% in transient cotransfection assays, but had no effect on expression from either the -148 hCGalpha or the -99 hCGalpha promoter. Unexpectedly, no Oct-3/ 4-binding site was identified within the -170 to -148 region of the hCGalpha promoter, although one was found around position -115 by both methylation interference footprinting and electrophoretic mobility shift assays. Site-directed mutagenesis of this binding site destroyed the affinity of the promoter for Oct-3/4, but did not affect repression of the promoter. Therefore, inhibition of hCGalpha gene transcription by Oct-3/4 appears not to involve direct binding of this factor to the site responsible for silencing. When stably transfected into JAr cells, Oct-3/4 reduced the amounts of both endogenous hCGalpha mRNA and protein by 70-80%. Oct-3/4 is therefore capable of silencing both hCGalpha and hCGbeta gene expression. We suggest that as the trophoblast begins to form, reduction of Oct-3/4 expression permits the coordinated onset of transcription from the hCGalpha and hCGbeta genes.
Mol Endocrinol 1997 Oct
PMID:Silencing of the gene for the alpha-subunit of human chorionic gonadotropin by the embryonic transcription factor Oct-3/4. 932 47

Interferon-tau (oIFNtau), the major secretory product of ovine conceptuses between days 13 and 21 (day 0=day of estrus) of pregnancy, is implicated in the process of maternal recognition of pregnancy. Culturing of day-14 and day-16 conceptus tissues in the presence of human granulocyte macrophage-colony stimulating factor (hGM-CSF) or interleukin-3 (IL-3) produces a marked increase in oIFNtau mRNA and protein expression. Since GM-CSF and IL-3 are localized at the luminal and glandular epithelia of the ovine endometrium, maternally derived GM-CSF and IL-3 may affect conceptus production of oIFNtau in a paracrine manner. However, the molecular mechanisms by which endometrial GM-CSF and IL-3 up-regulate oIFNtau production have not been defined. As an initial investigation of the signaling pathway regulating the GM-CSF induction of the oIFNtau gene, day-16 conceptuses were treated with an inducer, phorbol 12-myristate 13-acetate (PMA) and an inhibitor, calphostin C of the protein kinase C (PKC) pathway. Treatment with either 150 units/ml hGM-CSF (P<0.01) or 10 nM PMA (P<0.05) resulted in a significant increase in oIFNtau mRNA expression. Pretreatment of conceptuses with 1 microM PMA for 12 h to produce PKC-deficient tissues or treatment with 50 mM calphostin C abolished the hGM-CSF-induced increase in oIFNtau mRNA. An in vitro expression system was established for the analysis of oIFNtau gene regulatory sequences. The oIFNtau010 gene has been isolated previously and found to be the principal oIFNtau gene up-regulated during the preimplantation period. 5'-Flanking regions of the oIFNtau010 gene, 2 kb and 0.8 kb, were cloned into a basic chloramphenicol acetyltransferase reporter plasmid. These oIFNtau010 promoter constructs, along with expression controls, were transfected into human choriocarcinoma cells (JAR and JEG3) and their responsiveness to hGM-CSF and second messenger system activators including PMA, calcium ionophore (A23187) and 8-bromo-cAMP were characterized. The oIFNtau010 promoter constructs were up-regulated by hGM-CSF and PMA treatments (P<0.01). Combined treatment with PMA and A23187 prevented the promoter activation seen with PMA alone. The conceptus culture data, along with the results from the transfection experiments, suggest that the stimulatory effect of GM-CSF on oIFNtau is mediated through the PKC second messenger system.
J Mol Endocrinol 1997 Oct
PMID:Enhancement of ovine trophoblast interferon by granulocyte macrophage-colony stimulating factor: possible involvement of protein kinase C. 934 4

Aromatase cytochrome P450 catalyses the reaction to convert androgens to estrogens by coupling with NADPH-cytochrome P450 reductase in the endoplasmic reticulum. The human aromatase cytochrome P450 gene (CYP19) is expressed in a variety of tissues under regulation of tissue-specific promoters. Previously, we localized a cell-type specific transcriptional enhancer element between -242 and -166 relative to the major cap site of the gene, by transient expression analysis in human BeWo choriocarcinoma cells. In the present study, we demonstrate that the enhancer element consists of two subelements, element I (located between -238 and -200), and element II (located between -196 and -176) as analysed by DNase I footprinting using the nuclear extracts of BeWo cells. The gel mobility shift assay shows that each of these subelements binds specific nuclear factor(s). The transient expression of the bacterial chloramphenicol acetyltransferase gene constructs involving the subelements in BeWo cells reveals that the elements activate reporter gene expression synergistically when present together, nevertheless each of the elements by itself also has an enhancer activity. The transient expression analysis further shows that element I is responsible for the transcriptional synergism with the binding site of a nuclear factor-interleukin-6 (NF-IL-6) (also known as CCAAT enhancer/binding protein beta), which is located between -2141 and -2115 relative to the major cap site of the gene. These results suggest that the enhancer element plays important roles in sustaining the high levels of CYP19 expression in placental cells in cooperation with other cis-acting transcritional regulatory elements.
J Steroid Biochem Mol Biol 1997 Apr
PMID:Cooperative regulation of the human aromatase cytochrome P450 gene transcription by placenta-specific cis-acting elements. 936 92

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7alpha-substituted androstenediones. Numerous 7alpha-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7alpha-(4'-aminophenylthio)-androsta-1,4-diene-3,17-di one (7alpha-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7alpha-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7alpha-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7alpha position with a carbon-carbon linkage. Several 7alpha-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent Kis ranging between 13 and 19 nM. Extension of the research on these 7alpha-arylaliphatic androgens includes the introduction of a C1-C2 double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7alpha-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7alpha-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k(inact)s ranging from 4.4 x 10(-4) to 1.90 x 10(-3) s(-1). The best inactivator of the series was 7alpha-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T(1/2) of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC50 values of 64-328 nM. The 7alpha-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7alpha-position of the steroid in the active site of aromatase. The size of the 7alpha-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.
J Steroid Biochem Mol Biol 1997 Apr
PMID:Biochemistry and pharmacology of 7alpha-substituted androstenediones as aromatase inhibitors. 936 97

The conversion of C19 steroids to estrogens is catalysed by aromatase P450 (P450arom; product of the CYP19 gene). Tissue sites of expression include the gonads and brain; however, in a small subset of mammals, P450arom is also expressed in the placenta. In humans, gonadal expression employs a promoter proximal to the start site of translation, whereas expression in the placenta relies on a promoter which is distal to this site. We characterized the bovine CYP19 gene in both the ovary (OV) and placenta (PL), to determine if this method of regulation of tissue-specific expression is employed in other species. Both bovine and human species express P450arom in these tissues, however, the pattern of expression is very different. In both humans and cattle, P450arom is expressed in the granulosa cells of the ovary, however, after ovulation only the luteinized granulosa cells of the human continue to express P450arom. There is a high degree of sequence identity (>70%) shared between humans and cattle in the OV-specific 5'-flanking DNA, whereas little identity (<40%) was found between humans and cattle in the PL-specific DNA. Promoters and 5'-flanking regions of human and bovine CYP19 genes were subcloned into a luciferase (LUC) vector. Bovine PL-specific constructs transfected into JEG-3 human choriocarcinoma cells failed to express LUC activity. When OV-specific constructs were transfected into luteinized bovine granulosa cells, there was no change in LUC activity in cells transfected with the bovine constructs after treatment with forskolin, whereas all of the corresponding human constructs expressed LUC activity. The bovine 5'-flanking DNA lacks a cAMP-responsive element-like sequence (CLS) critical for cAMP-stimulated transcription of P450arom in the human ovary. With the absence of this CLS sequence in the bovine gene, there appears to be little enhancement of transcription by cAMP in the portion of the 5'-flanking region studied so far. When the analogous region of the bovine promoter was mutated to the human CLS, only a partial restoration of LUC activity was observed. An additional element therefore appears to be important in preventing the full expression of the bovine CYP19 gene in luteinized granulosa cells.
J Steroid Biochem Mol Biol 1997 Apr
PMID:Regulation of aromatase expression in the ovary and placenta: a comparison between two species. 936 17


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