tretinoin and Choriocarcinoma

Choriocarcinoma is malignant cancer originating from placental trophoblast. The incidence of this cancer is estimated at 0.57-1.1 per 1000 births in the United States of America, Australia, Europe, and New Zealand. The rate is much higher in South East Asia and Japan with two occurrences per a thousand births. Telomerase activity is an important part of the apoptotic process. Increased telomerase activity will result in cellular immortality and poor prognosis in cancer. Vitamin A possess an essential role in cell proliferation and differentiation. One of the active metabolites of vitamin A is All-Trans Retinoic Acid (ATRA).. In this study, we examined the role of ATRA against telomerase activity in choriocarcinoma cell. This cell was derived from BeWo cell line (ATCC CCL-98) and were given different doses of ATRA.. From this study, Choriocarcinoma cell that was given ATRA in dosage of 50μg/ml inhibit telomerase activity by extending the cycle time of 39.51±0.09, compared to the control group with a cycle time of 37.62±0.43. Cycle length change consistently with higher dose of ATRA.. This study has proven that ATRA could inhibit telomerase activity by lengthening the cycle. Changes in the increase of ATRA doses in this experimental test need to be studied further on experimental animals, either administered as a single agent or as an addition to standard treatment of trophoblastic disease.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Differentiation; Choriocarcinoma; Dose-Response Relationship, Drug; Female; Humans; Pregnancy; Telomerase; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Arsenic Trioxide; Arsenicals; Choriocarcinoma; Female; Humans; Kidney Neoplasms; Leukemia, Promyelocytic, Acute; Male; Middle Aged; Neuroectodermal Tumors; Ovarian Neoplasms; Oxides; Prognosis; Tretinoin

The high expression of folate receptor (FR) on cancer cells might be a potential target for cancer therapy. In this study, the FR-β expression and the modulation effect of all-trans retinoic acid (ATRA) in a number of cancer cell lines were analyzed. The gateway of ATRA activity on FR-β expression was further studied by a panel of retinoid activators and inhibitors. The results revealed that ATRA was capable of upregulating the expression of FR-β protein in KG-1 cells in a dosage-dependent manner, not in KG-1a, NB4, HL60, 293, L1210, JAR, and Hela cells. FR-β mRNA expression in KG-1 cells was higher when ATRA was present in culture medium at 10⁻⁶ mol/L for 5 days, and it went down to baseline when ATRA was removed from the medium, vice versa. The upregulation of FR-β expression in KG-1 cells by ATRA was not associated with cell proliferation and differentiation. In addition, activators of retinoid acid receptor (RAR)α and RARγ, CD336, and CD2781 also induced FR-β expression. The induction of FR-β expression by CD336 could be inhibited by RARγ antagonist CD2665; RARβ agonist CD-417 and CD-2314 as well as retinoid X receptor (RXR) agonist LG100364 could not induce FR-β expression. These results indicate that ATRA within a certain range of concentration could reversibly induce the expression of FR-β in a dosage- and cell type-dependent manner, and its action in KG-1 cells might be associated with the signal transduction of retinoid receptor RARα and RARγ, rather than RARβ and RXRs.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cervix Uteri; Choriocarcinoma; Female; Folate Receptor 2; Folate Receptors, GPI-Anchored; Humans; Kidney; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Uterine Neoplasms

All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death.. To investigate the tissue dynamic and cellular invasion effects of ATRA in choriocarcinoma (CCA), an aggressive trophoblastic tumour, by using a three-dimensional organotypic culture model system and cell invasion assay, respectively.. An organotypic culture model of two CCA cell lines, JAR and JEG, was established. The effects of 1 microM ATRA on proliferation, differentiation and apoptosis on this CCA model were assessed by morphological assessment of the mitotic and apoptotic figures as well as by Ki-67 and caspase-related M30 cytoDeath antibody immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The effect of ATRA on p53 and its regulated protein product, WAF1/Cip1, was also evaluated with DO7 and p21(WAF1) antibodies, respectively. Moreover, the effect of ATRA on cellular (CCA) invasion was also investigated with Cell Invasion Kit on the JEG cell line.. ATRA was found to induce marked apoptosis in organotypic cultures of both cell lines, as evidenced by increased M30-positive cells (p<0.0001) and increased TUNEL-positive cells (p<0.0001) in treated cultures; to decrease proliferation, as evidenced by decreased Ki-67-positive cells (p<0.0001); and to decrease p53-DO7 immunoreactivity (p<0.0001) and increase p21(WAF1) (p<0.0001) immunoreactivity. 1.5 microM ATRA was found to effectively inhibit JEG cell invasion in the cell invasion assay.. ATRA treatment was found to inhibit invasion and proliferation and enhance apoptosis, probably by the activation of caspases and induction of differentiation. ATRA and synthetic retinoids may be alternative agents for the treatment of CCA.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Proliferation; Choriocarcinoma; Dose-Response Relationship, Drug; Humans; In Situ Nick-End Labeling; Tretinoin; Tumor Suppressor Protein p53

The retinoid X receptor (RXR) is essential as a common heterodimerization partner of several nuclear receptors (NRs). However, its function as a bona fide receptor for endogenous ligands has remained poorly understood. Such a role would depend on the existence of RXR activating ligands in vivo and on the ability of such ligands to influence relevant biological functions. Here we demonstrate the presence of endogenous RXR ligands in the embryonic central nervous system (CNS) and show that they can activate heterodimers formed between RXR and the orphan NR Nurr1 in vivo. Moreover, RXR ligands increase the number of surviving dopaminergic cells and other neurons in a process mediated by Nurr1-RXR heterodimers. These results provide evidence for a role of Nurr1 as a ligand-independent partner of RXR in its function as a bona fide ligand-activated NR. Finally, our findings identify RXR-Nurr1 heterodimers as a potential target in the treatment of neurodegenerative disease.

Topics: Animals; Anticholesteremic Agents; Antineoplastic Agents; Brain; Cell Survival; Cells, Cultured; Choriocarcinoma; DNA-Binding Proteins; Dopamine Agents; Female; Fungal Proteins; Ligands; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Neurons; Nuclear Receptor Subfamily 4, Group A, Member 2; Organic Chemicals; Rats; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; Transcription Factors; Tretinoin

The biosynthesis of 17beta-estradiol (E(2)) in human placenta involves the actions of aromatase and 17beta-hydroxysteroid dehydrogenase type 1 (17HSD1). Aromatase, an enzyme complex comprised of P450aromatase (P450arom) and NADH-cytochrome P450 reductase, converts androgens to estrogens, whereas 17HSD1 catalyzes the reduction of estrone to E(2). In the present study, the effects of retinoic acids (RAs) on P450arom and 17HSD1 expression in placental cells were investigated. Treatment with all-trans-RA (at-RA) or 9cis-RA increased E(2) production in JEG-3 choriocarcinoma cells and cytotrophoblast (CTB) cells isolated from normal early placentas. Meanwhile, the activity of aromatase and expression of P450arom mRNA were induced by at-RA in JEG-3 cells. Northern blot analysis showed that the effect on P450arom mRNA expression occurs in a dose- and time-dependent fashion. Similar to at-RA and 9cis-RA, Ro40-6055, the retinoic acid receptor alpha (RARalpha)-selective activator, increased the expression of P450arom and 17HSD1 mRNA in JEG-3 cells. On the other hand, Ro41-5253 (Ro41), the RARalpha-selective antagonist, blocked the stimulatory effect of RAs on P450arom expression. Surprisingly, Ro41 induced the activity and mRNA expression of 17HSD1 in JEG-3 cells, which is in contrast to the expected inhibitory effect and, moreover, remarkably potentiated the induction by at-RA and 9cis-RA. However, reporter gene analysis revealed that the influence of Ro41 on the transcription of the HSD17B1 gene, which encodes 17HSD1, is considerably milder in JEG-3 cells, and it only additively enhanced the effect of at-RA. Finally, it was found that at-RA and 9cis-RA increased the expression of P450arom and 17HSD1 mRNA in CTB cells, but to a lesser extent. The data suggest that RAs may play a role in promoting the biosynthesis of E(2 )in the placenta. In addition, Ro41 has divergent effects on gene expression in JEG-3 cells.

Topics: 17-Hydroxysteroid Dehydrogenases; Aromatase; Blotting, Northern; Cell Line; Choriocarcinoma; Dose-Response Relationship, Drug; Enzyme Activation; Estradiol; Female; Humans; Placenta; Pregnancy; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured

The syncytiotrophoblasts of the human placenta express high levels of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme responsible for the inactivation of glucocorticoids. It has been proposed that the placental 11beta-HSD2 serves as a barrier to protect the fetus from high levels of maternal cortisol. To examine the hypothesis that nutritional signals regulate the expression of 11beta-HSD2 in placental syncytiotrophoblasts, we investigated the effects of retinoic acids (RAs), the major metabolites of vitamin A, on the expression of 11beta-HSD2 using human choriocarcinoma JEG-3 cells as a model. This trophoblast-like cell line displays a number of functional similarities to the syncytiotrophoblast. Treatment for 24 h with all-trans RA (1-1000 nM) resulted in a dose-dependent increase in 11beta-HSD2 activity with a maximal effect (increase to 3-fold) at 100 nM. The effect of all-trans RA (100 nM) was also time-dependent in that the effect was detectable at 6 h and reached its maximum by 48 h. Similar increases in 11beta-HSD2 activity were observed when the cells were treated with 9-cis RA. Results from semi-quantitative reverse transcription-polymerase chain reaction demonstrated that there was a corresponding increase in 11beta-HSD2 mRNA after RA treatment. Moreover, treatment with actinomycin D (100 ng/ml) abrogated the increase in 11beta-HSD2 mRNA induced by RA, indicating an effect on transcription. In conclusion, the present study has demonstrated for the first time that RA, at physiological concentrations, induces 11beta-HSD2 gene expression and enzyme activity in JEG-3 cells. If this occurs in vivo, the present finding suggests that high expression of 11beta-HSD2 in the human placenta may be maintained, at least in part, by dietary intake of vitamin A.

Topics: 11-beta-Hydroxysteroid Dehydrogenases; Choriocarcinoma; Dactinomycin; Gene Expression; Humans; Hydroxysteroid Dehydrogenases; Isoenzymes; Nucleic Acid Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA), a retinoid metabolite, acts as a gene regulator via ligand-activated transcription factors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs), both existing in three different subtypes, alpha, beta and gamma. In the intracellular regulation of retinoids, four binding proteins have been implicated: cellular retinol binding protein (CRBP) types I and II and cellular retinoic acid binding protein (CRABP) types I and II. We have used in situ hybridization to localize mRNA species encoding CRBP- and CRABP I and II as well as all the different nuclear receptors in the developing and adult rat and mouse central nervous system (CNS), an assay to investigate the possible presence of RA, and immunohistochemistry to also analyse CRBP I- and CRABP immunoreactivity (IR). RXRbeta is found in most areas while RARalpha and -beta and RXRalpha and -gamma show much more restricted patterns of expression. RARalpha is found in cortex and hippocampus and RARbeta and RXRgamma are both highly expressed in the dopamine-innervated areas caudate/putamen, nucleus accumbens and olfactory tubercle. RARgamma could not be detected in any part of the CNS. Using an in vitro reporter assay, we found high levels of RA in the developing striatum. The caudate/putamen of the developing brain showed strong CRBP I-IR in a compartmentalized manner, while at the same time containing many evenly distributed CRABP I-IR neurons. The CRBP I- and CRABP I-IR patterns were closely paralleled by the presence of the corresponding transcripts. The specific expression pattern of retinoid-binding proteins and nuclear retinoid receptors as well as the presence of RA in striatum suggests that retinoids are important in many brain structures and emphasizes a role for retinoids in gene regulatory events in postnatal and adult striatum.

Topics: Animals; Animals, Newborn; Brain Chemistry; Choriocarcinoma; Corpus Striatum; Gene Expression Regulation, Developmental; Hippocampus; Humans; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Inbred Strains; Olfactory Pathways; Oligonucleotide Probes; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RAR alpha and RXR alpha, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RAR alpha and RXR alpha. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 +/- 2%) being observed after 4 days of treatment with Ro 25, a RXR alpha specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 +/- 10%) occurred at 48 h with the RXR alpha-specific ligand. The RAR alpha-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXR alpha in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA.

Topics: Antibodies; Blotting, Northern; Blotting, Western; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Gene Expression Regulation; Nuclear Proteins; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.

Topics: 17-Hydroxysteroid Dehydrogenases; Breast Neoplasms; Choriocarcinoma; Cyclic AMP; Drug Synergism; Epidermal Growth Factor; Female; Humans; Isoenzymes; Pregnancy; RNA, Messenger; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

To determine the activities of all-trans retinoic acid (RA) on choriocarcinoma cells in vitro.. The antiproliferative effect of all-trans RA on 4 choriocarcinoma cell lines was measured by the MTT assay. The effect of all-trans RA combined with methotrexate or actinomycin-D was then examined. The effect of all-trans RA on hCG secretion was also studied. The gene expression of retinoic acid receptors (RARs) was examined by RT-PCR.. All-trans RA inhibited cell proliferation dose- and time-dependently; a 6-day exposure to 1 microM all-trans RA suppressed the cell growth by 67.8%-82.0% compared to the controls. An enhanced effect was observed in the combined administration of all-trans RA and methotrexate or actinomycin-D. The secretion of hCG increased 4-fold to 9-fold by the addition of 1 microM all-trans RA. RARs genes were expressed in all cell lines.. The anticancer activity presented here appears to warrant further evaluation of all-trans RA as adjuvant therapy for choriocarcinoma.

Topics: Antineoplastic Agents; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Dactinomycin; Methotrexate; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Testicular germ cell tumors are the most common form of cancer in young adult males. They result from a derangement of primordial germ cells, and they grow out from a noninvasive carcinoma-in-situ precursor. Since carcinoma in situ can readily be cured by low-dose irradiation, there is a great incentive for non- or minimally invasive methods for detection of carcinoma in situ. We have recently shown that human Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor, show alternative splicing and alternative promoter use of the platelet-derived growth factor alpha-receptor gene, giving rise to a unique 1.5-kb transcript. In this study we have set up a reverse transcriptase-polymerase chain reaction strategy for characterization of the various transcripts for this receptor. Using this technique, we show that a panel of 18 seminomas and II nonseminomatous testicular germ cell tumors all express the 1.5-kb transcript. In addition, a panel of 27 samples of testis parenchyma with established carcinoma in situ were all found to be positive for the 1.5-kb transcript, while parenchyma lacking carcinoma in situ, placenta, and control semen were all negative. These data show that the 1.5-kb platelet-derived growth factor alpha-receptor transcript can be used as a highly selective marker for detection of early stages of human testicular germ cell tumors.

Topics: Adult; Alkaline Phosphatase; Base Sequence; Biomarkers, Tumor; Carcinoma, Embryonal; Choriocarcinoma; Clone Cells; DNA Primers; Gene Expression; Germinoma; Humans; Male; Molecular Sequence Data; Polymerase Chain Reaction; Receptor, Platelet-Derived Growth Factor alpha; Receptors, Platelet-Derived Growth Factor; Seminiferous Tubules; Seminoma; Teratoma; Testicular Neoplasms; Testis; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

The controlled invasiveness of the trophoblast is based on the balance between invasive properties at implantation and the differentiation program of the developing placenta. During placental development in rats a switch of connexin gene expression has been observed in parallel to the switch from the invasive to the differentiated phenotype of trophoblast cells. To investigate the role of connexin expression for trophoblast invasion, proliferation, and differentiation, we studied one rat trophoblast (HRP-1) and one rat choriocarcinoma cell line (Rcho-1). The choriocarcinoma cells were characterized by expression of cx31 and a lack of E-cadherin, corresponding to the invasive trophoblast in vivo, whereas HRP-1 cells expressed cx43, normally found in the spongiotrophoblast and in late giant cells, and E-cadherin. Upon retinoic acid treatment, Rcho-1 cells irreversibly lost cx31 expression, accompanied by a loss of functional coupling. No changes in regard to connexin expression and cell-cell communication could be observed in HRP-1 cells. In addition, treatment of Rcho-1 cells with retinoic acid for 7 days upregulated expression of cx43 transcript, but no protein could be found. Proliferation was clearly reduced and the mean volume of cells doubled from Day 4 to Day 7 of retinoic acid treatment in Rcho-1 cells, while both parameters were not affected in HRP-1 cells. Both cell lines showed a similar invasion rate using a Matrigel invasion assay, and invasion was equally suppressed upon retinoic acid treatment. Thus the different connexin expression appears more likely to play a role in regulating proliferation and differentiation along the multilineage pathway than invasiveness of rat trophoblast cells.

Topics: Animals; Cadherins; Calcium; Cell Communication; Cell Division; Cell Movement; Choriocarcinoma; Collagen; Connexin 43; Connexins; Drug Combinations; Fluorescent Dyes; Gap Junctions; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Isoquinolines; Laminin; Proteoglycans; Rats; RNA, Messenger; Tretinoin; Trophoblasts; Tumor Cells, Cultured

Three regions of the human placental lactogen (hPL) promoter that contain several half-site motifs that closely resemble the responsive elements for thyroid hormone (TR), all trans retinoic acid (RAR) and 1,25 dihydroxyvitamin D3 (VDR) have been identified and characterized. Transfection studies in BeWo choriocarcinoma cells indicate that site A (nt -979 to -954) is responsive to RAR alpha but not TR beta. Site B (nt -1140 to -1170) is responsive to both RAR alpha and TR beta, and site C (nt -550 to -580) is not responsive to either RAR alpha or TR. These findings, together with the observation that placental cells express retinoid receptors and TRs, strongly suggest a role for these receptors in the regulation of the hPL gene. Site B on the hPL promoter is able to integrate the responses to RA and T3 through a single element.

Topics: Base Sequence; Binding Sites; Calcitriol; Chloramphenicol O-Acetyltransferase; Choriocarcinoma; DNA; Humans; Molecular Sequence Data; Placental Lactogen; Promoter Regions, Genetic; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Repetitive Sequences, Nucleic Acid; Retinoid X Receptors; Thyroid Hormones; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Human 17 beta-hydroxysteroid dehydrogenase type 1 (17HSD type 1) catalyzes primarily the reductive reaction of estrone to the biologically more active form, estradiol. The enzyme is highly expressed in the human placenta and the ovary and, in addition, in certain estrogen target cells, such as breast epithelial cells. To elucidate the transcriptional control of the EDH17B2 gene, the gene encoding 17HSD type 1, we fused a series of 5'-deletion mutants of the EDH17B2 gene into chloramphenicol acetyl transferase reporter gene vectors. An enhancer region was identified within the bases -661 to -392 and it increased, in both orientations, thymidine kinase promoter activity more than 200-fold in JEG-3 choriocarcinoma cells. This enhancer region was also functional in another choriocarcinoma cell line, JAR, although to a lesser extent. In BT-20 and T-47D breast cancer cells the enhancer region increased thymidine kinase promoter activity to some degree but not as efficiently as expected on the basis of endogenous enzyme expression. No such enhancer activity was observed in 17HSD type 1 nonexpressing cell lines. The retinoic acid responsive element, which was located between bases -503 and -487 in the EDH17B2 enhancer, bound retinoid acid receptor alpha retinoid X receptor alpha complex and transmitted retinoic acid induction on transcription in JEG-3 and T-47D cells. Finally, a silencer, functional in all the cell lines tested, was localized in the region from -392 to -78. Deletion of the region lad to a 4-fold increase in reporter gene expression. Altogether, our findings suggest that transcriptional control of the EDH17B2 gene is coordinated by the cell-specific enhancer and the silencer.

Topics: 17-Hydroxysteroid Dehydrogenases; Base Sequence; Breast Neoplasms; Chloramphenicol O-Acetyltransferase; Choriocarcinoma; Enhancer Elements, Genetic; Female; Gene Deletion; Gene Expression Regulation, Enzymologic; Humans; Male; Molecular Sequence Data; Promoter Regions, Genetic; Prostatic Neoplasms; Recombinant Fusion Proteins; Transcription, Genetic; Transfection; Tretinoin; Tumor Cells, Cultured

Interferon-tau (IFN tau) is produced exclusively by the trophectoderm during the peri-implantation stage of pregnancy in ruminant ungulate species. Human choriocarcinoma cells (Jar) stably transfected with 1.8 kilobases of promoter from a bovine IFN tau gene ahead of a human GH (hGH) reporter gene constitutively synthesize hGH, but expression is not increased further by exposure to Newcastle disease virus. This and earlier experiments suggest that the transcriptional cues regulating IFN tau expression are distinct from those operating on other type I IFN genes. Transient transfection experiments reveal that two distinct promoter regions are required for full constitutive expression: one proximal (to position -126), which directs basal expression, and a more distal promoter region (positions -280 to -400), which acts as an enhancer. Nuclear extracts prepared from ovine conceptuses during the period of IFN tau expression interact with the proximal promoter region (positions -34 to -126) to form several complexes of high electrophoretic mobility. Although nucleotide sequence motifs potentially capable of binding the transcription factor IRF-1 are present in this region, IRF-1 does not transactivate the IFN tau gene. The distal part of the promoter contains only one region (-322 to -358) that forms a complex with these conceptus nuclear extracts. Both proximal and distal gel shift patterns become dramatically different when IFN tau gene expression ceases, perhaps reflecting the appearance of transcriptional repressors. Together these experiments support the conclusion that the control of IFN tau gene expression is very different from that of other type I IFN genes and that trophoblast-specific expression depends upon distal as well as proximal promoter regulatory elements.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Base Sequence; Binding Sites; Cattle; Chlorocebus aethiops; Choriocarcinoma; Consensus Sequence; DNA-Binding Proteins; Ectoderm; Enhancer Elements, Genetic; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Interferon Type I; L Cells; Mice; Molecular Sequence Data; Neoplasm Proteins; Newcastle disease virus; Phosphoproteins; Pregnancy Proteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Sequence Alignment; Sequence Homology, Nucleic Acid; Sheep; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Tretinoin; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

Fusion gene constructs containing the human choline acetyltransferase 5' flanking region are stimulated by thyroid hormone (T3) in neuronal NG108-15 and NE1-115 cells but not in non neuronal COS-1 and JEG-3 cells. To identify potential T3 receptor binding elements (T3RE), chimeric plasmids containing various lengths of the 5' end of the hChAT gene linked to the CAT reporter gene were assayed by transient transfections into NG108-15, NE1-115 and COS-1 cells. We show that regulation is T3 specific as estrogen, dexamethasone, dihydrotestosterone, all-trans-retinoic acid and 9-cis-retinoic acid have no effect. We localized several potential T3REs and characterized the most proximal T3RE (position 3280-3291) which contains two hexameric half-sites arranged as a direct repeat without a base pair spacer. An oligonucleotide containing this sequence confers T3 responsiveness to a heterologous promoter. The transcriptional response of this T3RE is markedly reduced after mutation of the first or second half-site indicating that both half-sites are required for a maximal T3 response. We have found that RAR alpha, RXR alpha and COUP-TF do not enhance T3 responsiveness and therefore they may not interact with T3R alpha in NG108-15 cells on this regulatory sequence. T3R monomer and dimer specific binding to the proximal T3RE is demonstrated by gel-retardation DNA binding assays and by methylation interference experiments. In COS-1 cells, T3R inhibits transcriptional activation by the transcription factor AP-1 whereas in NE1-115 cells T3R enhances AP-1 mediated activation in a T3 dependant fashion. It is likely that these effects involve protein-protein interactions. These results suggest that the T3 receptor can act as a positive transcriptional regulatory factor on the hChAT gene.

Topics: Amino Acid Sequence; Animals; Base Sequence; Breast Neoplasms; Cells, Cultured; Chlorocebus aethiops; Choline O-Acetyltransferase; Choriocarcinoma; Enzyme Induction; Female; Humans; Molecular Sequence Data; Neoplasms, Hormone-Dependent; Neuroblastoma; Neurons; Organ Specificity; Proto-Oncogene Proteins c-jun; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Steroids; Transcriptional Activation; Tretinoin; Triiodothyronine; Tumor Cells, Cultured; Uterine Neoplasms

Retinoic acid (RA), an active metabolite of vitamin A, is an important mediator of cellular differentiation and has been shown to stimulate human CG (hCG) secretion by JEG-3 choriocarcinoma cells in vitro. In order to determine whether RA stimulates the hCG secretion by other trophoblastic cell lines, we evaluated the effect of RA on hCG, hCG-alpha subunit (hCG-alpha), and progesterone secretion in three choriocarcinoma cell lines: JEG-3, JAR, and BeWo. RA stimulated hCG and hCG-alpha secretion in a dose-dependent fashion by each of the three cell lines. The time required to give a statistically significant increment of hCG and hCG-alpha over control cells was 48 h. The addition RA to cholera toxin (10 micrograms/ml) resulted in an additive or synergistic stimulation of hCG and hCG-alpha secretion by the three cell lines. Cycloheximide (1 microM) abolished the effect of RA on hCG and hCG-alpha secretion in the BeWo cell line. Progesterone secretion in response to RA was inconsistent. Progesterone secretion by both JEG-3 and BeWo cell lines were stimulated at high concentrations of RA (1 x 10(-6], whereas progesterone secretion by JAR cells was not stimulated. Intracellular levels of cAMP were not affected by RA treatment in the JEG and JAR cells. In the dosages used, RA did not significantly alter cell number in any of the cell lines. RA in physiologic concentrations stimulates hCG and hCG-alpha secretion by three choriocarcinoma cell lines in vitro. Whether RA is a physiologic mediator of placental hormone production is unknown.

Topics: Cell Line; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Cyclic AMP; Cycloheximide; Female; Glycoprotein Hormones, alpha Subunit; Humans; Kinetics; Pregnancy; Progesterone; Tretinoin; Uterine Neoplasms

The effects of biologic response modifiers such as interferon-gamma, tumor necrosis factor alpha (TNF), and retinoic acid on the human chorionic gonadotropin (hCG) secretion of cultured choriocarcinoma cells (JAR) and term placenta have been studied. Although the proliferation of JAR cells was not inhibited by these agents, retinoic acid and TNF markedly increased both the intracellular levels as well as the secreted amounts of hCG. In the case of the term placenta, only retinoic acid increased the hCG secretion into the culture medium, whereas interferon-gamma and TNF both markedly reduced secretion. The cytostatic agent etoposide (VP-16) was able to augment the hCG secretion on the choriocarcinoma cells but did not alter its production on term placenta. The The data presented indicate different mechanisms of regulation of hCG secretion in the normal and malignant trophoblast.

Topics: Choriocarcinoma; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Female; Humans; Immunologic Factors; Interferon-gamma; Placenta; Pregnancy; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The synthesis of human chorionic gonadotropin (HCG), the subunit of HCG (HCG alpha), and pregnancy-specific beta 1-glycoprotein (PS beta G) was studied in temperature sensitive (ts), simian virus 40 (SV40) tsA mutant-transformed human first trimester placental (SPA255-26) cells. Retinoic acid increased the production of HCG and PS beta G but inhibited the production of HCG alpha in these cells. Passage of SPA255-26 placental cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of HCG and PS beta G and a reduced level of HCG alpha. The retinoic acid induced phenotypic changes in these placental cells were reversible; removal of retinoic acid immediately decreased the production of HCG and PS beta G while increasing the production of HCG alpha. The ratio of HCG to HCG alpha in control SPA255-26 cells was approximately 0.1; this ratio increased to 4.8 in cells maintained in medium containing retinoic acid. Similarly, the HCG-to-HCG alpha ratio increased in choriocarcinoma cells maintained in retinoic acid containing medium. Our data suggest that retinoic acid may be needed to maintain a balanced production of HCG, HCG alpha, and PS beta G in placental cells in vitro. Retinoic acid may also play a role in modulating placental protein production during pregnancy.

Topics: Cell Differentiation; Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Female; Humans; Macromolecular Substances; Placenta; Pregnancy; Pregnancy Proteins; Pregnancy-Specific beta 1-Glycoproteins; Tretinoin

Choriocarcinoma cells maintain multiple hormonal functions in culture. We have found that these cells secreted no immunoreactive pregnancy-specific beta 1-glycoprotein (PS beta G), a placental protein. Choriocarcinoma cells can be induced to synthesize low levels of PS beta G by retinoic acid, 8-bromo-cAMP (8BrcAMP), cholera toxin, methyl-isobutylxanthine (MIX), and 5-bromo-2'-deoxyuridine (BrdUrd). The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave synergistic induction of PS beta G. The simultaneous addition of retinoic acid and BrdUrd failed to give even additive induction. In addition to stimulating PS beta G production, retinoic acid increased the production of hCG and its alpha-subunit (hCG alpha) by choriocarcinoma cells. The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave additive induction for hCG and hCG alpha. Passage of choriocarcinoma cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of PS beta G, hCG, and hCG alpha. These retinoid-treated choriocarcinoma cells remained responsive to 8BrcAMP or compounds that increase intracellular cAMP concentrations and to BrdUrd; the production to PS beta G and hCG was greatly stimulated by 8BrcAMP, cholera toxin, or MIX, and the production of hCG and hCG alpha was greatly inhibited by BrdUrd. However, the production of hCG alpha was only slightly induced by these cAMP modulators, and the production of PS beta G was not increased by BrdUrd.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Cell Line; Cell Transformation, Neoplastic; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Cyclic AMP; Deoxyuracil Nucleotides; Female; Fluorodeoxyuridylate; Humans; Pregnancy; Pregnancy-Specific beta 1-Glycoproteins; Tretinoin; Uterine Neoplasms