tretinoin and Uterine-Neoplasms

Recent clinical trials with a combination of interferon (IFN alpha) and 13 cis-retinoic acid resulted in high response rates among women with locally advanced and metastatic carcinoma of the uterine cervix. The authors sought to amplify these observations by employing the isomer of 13 cis-retinoic acid, all-trans retinoic acid (tRA), in combination with IFN alpha.. Sequential clinical trials were initiated by the New York Gynecologic Oncology Group to test the combination of tRA and IFN alpha in women with metastatic or recurrent carcinoma of the cervix who had failed primary therapy. IFN alpha was administered at 6 MU subcutaneously 3 times per week. In the first trial, tRA was administered at 50 mg/m2 orally 3 times per day on a daily schedule (daily regimen), whereas in the second trial, tRA was administered at the same dose 3 times per day, but only on Days 1-3 each week (intermittent schedule). Clinical outcomes included response to therapy and survival. Plasma pharmacokinetic studies of tRA were performed in both trials to assess the effects of different schedules on plasma levels of the drug.. Fourteen women with metastatic or recurrent squamous cell carcinoma of the cervix were enrolled in the daily trial and 12 women in the intermittent trial. There was no clinical activity for either regimen, and both studies were terminated according to an early stopping rule. Because tRA has been reported to induce its own metabolism, plasma levels of tRA were measured on Days 1, 8, and 28. The change in the area under the time versus tRA concentration curve (AUC) was significantly different between the two groups. The average AUC on Day 8 was 14% of that observed on Day 1 for the daily treatment group; in contrast, it was 107% on Day 1 in the intermittent treatment group. In 6 of 8 patients studied in the daily trial, the AUC decreased at least 60% by either Week 2 or Week 4. In contrast, in the intermittent trial, only 3 of 9 patients experienced >60% decrease in plasma levels of the drug at either Day 8 or Day 28.. The combination of tRA + IFN alpha was inactive in patients with advanced carcinoma of the cervix when employed at these doses on either the daily or intermittent schedule. The failure of activity of this regimen did not result from induction of metabolism of tRA, suggesting that intrinsic mechanisms of resistance to tRA at the cellular level may be of greater importance.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Interferon alpha-2; Interferon-alpha; Middle Aged; Multicenter Studies as Topic; Neoplasm Recurrence, Local; Recombinant Proteins; Tretinoin; Uterine Neoplasms

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

Leiomyomas, monoclonal tumors developed by the transformation of myometrium somatic stem cells, are a major health concern that can severely impair quality of life. Pathological alterations of signaling pathways have been recognized as a key feature in a variety of human diseases. Our objective was to analyze treatment with all-trans-retinoic acid (ATRA) by suppression of the phosphoinositide 3-kinase (PI3K) pathway on growth, signaling pattern and interactions among PI3K/B-cell lymphoma 2 (Bcl2)/retinol leiomyoma proteins.. Cultures of paired myometrium and leiomyoma cells from premenopausal women undergoing hysterectomy were collected. Western blot and analysis of variance were used for analysis.. Significant differences were detected between treatment with ATRA alone or with LY294002 (a PI3K growth suppressor) in response to treatment and among cell samples and cell numbers. Leiomyoma cells were less affected. Immunochemical analysis of signaling patterns demonstrated that treatments affected most of the examined protein levels differently. Significant differences between the cell type responses to treatment in pyruvate phosphate dikinase 1 (pPDK1), Bad and pβ-catenin levels were identified. The pβ-catenin level showed highly significant interaction between response to treatment and cell type.. ATRA treatment on PI3K pathway suppression significantly affected growth, signaling pattern and interactions among PI3K/Bcl2/retinol proteins involved in the growth, survival and apoptosis of leiomyomas. Interpretation of our results suggests that increasing knowledge of the role of signaling interplay in the pathogenesis of leiomyomas may present an opportunity to use specific signal transduction inhibitors for treating and preventing this disorder.

Topics: Adult; bcl-Associated Death Protein; beta Catenin; Cell Line, Tumor; Chromones; Female; Humans; Hysterectomy; Leiomyoma; Middle Aged; Morpholines; Myometrium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Premenopause; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Tretinoin; Uterine Neoplasms

Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1, in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.

Topics: Adult; beta Catenin; Blood Vessels; Cells, Cultured; COUP Transcription Factor II; Female; Follicular Phase; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Leiomyomatosis; Luteal Phase; Middle Aged; Myometrium; Neoplasm Proteins; Peptide Fragments; Progesterone; Protein Structure, Tertiary; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

To study the influence of liarozole on leiomyoma cell proliferation and extracellular matrix (ECM) gene expression in immortalized leiomyoma cells.. Laboratory study.. University hospital.. None.. Tissue culture, real-time reverse transcription-polymerase chain reaction, Western blot.. Proliferation, messenger RNA (mRNA), and ECM protein expression.. Proliferation of leiomyoma cells was inhibited by treatment with liarozole at suprapharmacologic concentrations. The mRNA and protein expression of COL1A1, COL4A2, versican, fibromodulin, and fibronectin was increased in untreated leiomyoma cells compared with untreated patient-matched myometrial cells. Extracellular matrix mRNA expression was decreased in a dose-dependent manner in leiomyoma cells treated with pharmacologic concentrations of liarozole. In addition, myometrial cells treated with liarozole demonstrated no statistically significant alteration in ECM regulation.. Liarozole inhibited ECM protein production at pharmacologic concentrations in immortalized human leiomyoma cells. Retinoic acid metabolic blocking agents represent a potential therapeutic drug family for human leiomyomas.

Topics: Antineoplastic Agents, Hormonal; Cell Proliferation; Dose-Response Relationship, Drug; Extracellular Matrix; Extracellular Matrix Proteins; Female; Humans; Imidazoles; Leiomyoma; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

To detect changes induced by all-trans-retinoic acid (ATRA) on the expression and activation of target proteins of the retinoic acid (RA) and PI3K/Akt pathways involved in leiomyoma growth.. A study on human tissue cultures.. Hadassah University Hospital.. Premenopausal women with uterine leiomyomas.. Paired cultures of normal myometrium and leiomyomas, from women undergoing hysterectomy, were obtained.. The effect of ATRA was examined on the expression and phosphorylation of relevant RA, PI3K/Akt, and Bcl2 proteins (immunochemical analysis), cell proliferation, cell cycle distribution, and apoptosis.. Applying our cell culture model, we demonstrated that ATRA induced changes in the expression and activation of the RA and PI3K/Akt pathway proteins in leiomyoma cells, with significant increases of alcohol dehydrogenase 1 and cyclin D2 protein levels. In part of the leiomyoma cells, ATRA induced a relative increase of Bax (proapoptotic) as well as a relative decrease of phosphorylated glycogen synthase kinase 3β (proapoptotic).. Our results highlight the involvement of ATRA in the RA and PI3K/Akt pathways, whose specific signaling products may influence the outcome of leiomyoma growth by regulating cell proliferation, apoptosis, and survival. These results might be useful for the on-going research into alternative methods for treating and preventing this disorder.

Topics: Adult; Apoptosis; Cell Cycle; Cell Proliferation; Cells, Cultured; Drug Evaluation, Preclinical; Female; Humans; Leiomyoma; Matched-Pair Analysis; Middle Aged; Models, Biological; Phosphatidylinositol 3-Kinases; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Uterine Neoplasms

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

Uterine leiomyomas are clinically significant tumours that may develop due to an altered differentiation pathway. We have previously identified a dysregulated retinoic acid (RA) pathway that reduced retinoic exposure in human leiomyoma surgical specimens, and have shown that the leiomyoma phenotype was characterized by excessive and disorganized extracellular matrix (ECM).. The goal of this study was to determine the impact of RA exposure on the disrupted ECM phenotype of leiomyomas.. Study of immortalized and molecularly confirmed cells generated from surgical specimens of spontaneous uterine leiomyoma and matched myometrium.. Immortalized leiomyoma and myometrial cells retained the molecular characteristics of their progenitor tissue. Proliferation of leiomyoma cells was inhibited by all-trans retinoic acid (ATRA). Furthermore, there was a dose-dependent decrease in soluble extracellular collagen protein in ATRA-treated leiomyoma cells. Exposure of leiomyoma cells to ATRA resulted in a dose-dependent inhibition of templates for specific ECM protein production including collagen 1, collagen 4, fibronectin and versican. Notably, expression levels in treated leiomyoma cells approached those found in myometrial cells. These mRNA alterations translated into altered protein. Down-regulation was also observed among the RA pathway genes such as CYP26A1 with exposure to ATRA. Finally, ATRA down-regulated TGF-beta3 mRNA expression and the TGF-beta regulated genes in leiomyoma cells.. Exposure of leiomyomas to ATRA down-regulated cell proliferation, ECM formation, RA metabolism and TGF-beta regulation, suggesting that RA exposure can alter the leiomyoma phenotype to one that more closely approximates normal myometrium.

Topics: Cell Differentiation; Cell Proliferation; Cells, Cultured; Collagen; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; Myometrium; Phenotype; Signal Transduction; Tretinoin; Uterine Neoplasms

An alteration of the retinoid pathway can influence the development of uterine leiomyomas in animal models, and retinoids have shown efficacy in inhibiting the growth of this benign tumor both in vitro and in vivo. However, the underlying mechanisms and biological implications are unclear. The present study was based on the demonstration of an accumulation of full-length retinoid X receptor alpha (RXRalpha) in leiomyomas that was not associated with a modification of its gene expression. This accumulation was shown to increase the transcription of the RXR-responsive gene cellular retinoic acid binding protein II (CRABP-II) and to be linked to the cellular redistribution of the receptor and to its retarded degradation via the ubiquitin/proteasome pathway. Accordingly, treatment with a specific proteasome inhibitor but not with protease inhibitors strongly inhibited the degradation of full-length RXRalpha in cells deriving from both myometrium and leiomyoma, but the formation of RXRalpha/ubiquitin conjugates was differentially regulated between the two cell types. Moreover, full-length RXRalpha accumulated in leiomyomas was abnormally phosphorylated at serine/threonine residues relative to myometrial tissue. The ligand to RXRalpha, 9-cis-retinoic acid, induced the receptor breakdown in smooth muscle cells deriving from both normal and tumor tissue, whereas a MAPK-specific inhibitor was able to reduce RXRalpha levels only in leiomyoma cells. These results suggest that switching of the ubiquitin/proteasome-dependent degradation of RXRalpha by phosphorylation in leiomyomas may be responsible for the accumulation of the receptor and the consequent dysregulation of retinoic acid target genes. The ability of retinoids to modify this molecular alteration may be the rationale for their use in the treatment of leiomyomas.

Topics: Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; Myocytes, Smooth Muscle; Phosphorylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Retinoid X Receptor alpha; Transcriptional Activation; Tretinoin; Uterine Neoplasms

To analyze expression of the retinoic acid signaling pathway genes that are involved in retinol metabolism, transport, transcriptional activation, and transcriptional products in spontaneous human leiomyomas.. Laboratory study of human leiomyoma and patient-matched myometrial tissue.. Eight women undergoing hysterectomy for symptomatic leiomyomas.. Confirmation of an altered retinoic acid pathway analyzed by microarray, real time reverse transcription-polymerase chain reaction, Western blot, immunohistochemistry, and high-performance liquid chromatography (HPLC).. Gene and protein expression.. Regardless of patient demographics and leiomyoma location and size, we found decreased expression of the major genes involved in retinoic acid pathway including alcohol dehydrogenase-1 (-3.97- +/- 0.03-fold), aldehyde dehydrogenase-1 (-3.1- +/- 0.07-fold), cellular retinol binding protein-1 (-2.62- +/- 0.04-fold), and cellular retinoic acid binding protein-1 (-2.42- +/- 0.20-fold). Cytochrome P450 (CYP 26A1), which is responsible for retinoic acid metabolism, was highly up-regulated in leiomyomas (+5.4- +/- 0.53-fold). Nuclear receptors demonstrated a complex pattern of under-expression (RARalpha, RARbeta, RXRalpha, RXRgamma) and over-expression (RARgamma, RXRbeta) at both the mRNA and protein level. Differences in protein amounts were confirmed by Western blot. Finally, a reduced amount of cellular ATRA and 9-cis retinoic acid was confirmed by HPLC in leiomyomas compared with myometrial tissues.. Molecular alterations in the retinoic acid pathway of leiomyomata result in a decrease in retinoic acid exposure.

Topics: Adult; Female; Gene Expression Regulation, Neoplastic; Humans; Hysterectomy; Immunohistochemistry; Leiomyoma; Menstrual Cycle; Middle Aged; Myometrium; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Uterine Neoplasms

Fibroids are benign neoplasms of myometrial smooth muscle cells (SMC). Despite being the most common tumor in humans, their etiology is poorly understood. Recent microarray studies have demonstrated that multiple members of the retinoid pathway are differentially expressed between myometrium and fibroids. The aim of this present study was to investigate gene expression of members of the retinoid pathway in matched myometrium and fibroids. We have demonstrated differential gene expression of two binding proteins [cellular retinol-binding proteins (CRBP) 1 and 2], three enzymes [alcohol dehydrogenase 1 (ADH1), aldehyde dehydrogenase (ALDH1) and retinol dehydrogenase (RODH)] and two receptors [retinoid X receptors (RXR) alpha and gamma] involved in the retinoid pathway by real-time PCR. There were no differences in gene expression for retinoid receptors RARalpha, beta, gamma and RXRbeta, and for the metabolizing enzyme cytochrome P450, family 26 subfamily A. We confirmed results for ADH1, ALDH1, CRBP1 and CRABP2 at the protein level by western blot. Using immunohistochemistry these proteins were mostly localized to myometrial and fibroid SMC. An exception to this was ALDH1 protein, which displayed strong staining localized to cells of the connective tissue, presumably fibroblasts, with a striking differential expression pattern between myometrium and fibroids. These results demonstrate that the retinoid pathway is altered in fibroids when compared with normal myometrium and specifically identify ALDH1 in fibroid fibroblasts. These alterations can lead to aberrant retinoic acid (RA) production and signaling, and alter the expression of RA target genes, which may be an important step in fibroid development.

Topics: Adult; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Female; Gene Expression; Histone-Lysine N-Methyltransferase; Humans; Isoenzymes; Leiomyoma; Middle Aged; Myometrium; Retinal Dehydrogenase; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Uterine Neoplasms

Uterine leiomyomata are the main indication for a hysterectomy in the United States and occur in 25% of women >35 years. Because uterine leiomyomata can form when ovariectomized guinea pigs are exposed to estradiol and retinoic acids, we tested whether human leiomyomata had high levels of retinoic acids and related nuclear receptors. Compared with normal human myometrium, leiomyomata had 3- to 5-fold higher levels of peroxisome proliferator-activated receptor gamma (PPARgamma), retinoid X receptor alpha proteins, and all-trans retinoic acid, but only during the follicular phase of the menstrual cycle. 9-cis Retinoic acid was undetectable in either leiomyomata or myometrium. PPARgamma mRNA levels were lower in leiomyomata than myometrium, but only during the luteal phase of the cycle. A PPARgamma agonist, troglitazone, was given to guinea pigs along with estradiol and all-trans retinoic acid and produced the largest leiomyomata seen to date in this model. By contrast, no tumors formed when troglitazone was given alone or with estradiol or when troglitazone was given with estradiol and 9-cis retinoic acid. New therapies for human leiomyomata may emerge by combining antagonists for PPARgamma and retinoid X receptor alpha with selective estrogen receptor modulators.

Topics: Alitretinoin; Animals; Carcinogens; Chromans; Drug Implants; Estradiol; Female; Guinea Pigs; Humans; Leiomyomatosis; Menstrual Cycle; Myometrium; Neoplasm Proteins; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin; Troglitazone; Uterine Neoplasms

We have shown previously that treatment of human endometrial adenocarcinoma (RL95-2) cells with either 13-cis or all-trans retinoic acid results in reorganization of actin filaments, indicating reversion to a stationary phenotype. In the present study, we investigated the role of protein kinase C (PKC) in this process. Treatment of cells with PKC inhibitors (staurosporine, bisindolylmaleimide, or G¿6976) resulted in morphological alterations and reorganization of actin filaments similar to retinoic-acid-treated cells. For example, RL95-2 cells treated with staurosporine flattened, exhibited cell surface extensions and some actin filaments. Bisindolylmaleimide-treated cells flattened, and actin filaments reorganized similar to retinoic-acid-treated cells. RL95-2 cells treated with G¿6976, which inhibits only PKC, alpha, beta and gamma, exhibited many cell surface extensions and some actin filament reorganization. We then investigated whether retinoic acid affected the subcellular localization of PKC-alpha. In control cells, PKC-alpha was mainly evident as diffuse cytoplasmic immunostaining, with a small percentage of total PKC-alpha also evident in the plasma membrane. Retinoic acid treatment dramatically altered PKC-alpha localization, since a more distinct cytoplasmic and perinuclear staining pattern was apparent. Western blot analysis confirmed these results, since the amount of cytosolic PKC-alpha increased following retinoic acid treatment. Thus, retinoic-acid-induced endometrial differentiation may be associated with alterations in PKC-alpha localization and signaling.

Topics: Adenocarcinoma; Antineoplastic Agents; Carbazoles; Cell Differentiation; Cytoskeleton; Enzyme Inhibitors; Female; Humans; Immunohistochemistry; Indoles; Isoenzymes; Maleimides; Phenotype; Protein Kinase C; Protein Kinase C-alpha; Staurosporine; Tretinoin; Tumor Cells, Cultured; Uterine Neoplasms

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

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

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

Blinded analyses of the concentrations of binding proteins for retinol and retinoic acid (CRABP) in homogenates of cancer and normal tissue aliquots obtained from human cervix, endometrium, ovary, breast, and lung were carried out by the sucrose gradient ultracentrifugation technique. In carcinomas of the cervix and endometrium, CRABP mean values of 50.4 and 123.2 pmol/g tissue, respectively were detected. Such concentrations represent a 3- and 4-fold increase over the mean values of CRABP in the normal cervix (16.9 pmol/g) and normal endometrium (30.8 pmol/g), respectively. In carcinomas of the ovary, the mean CRABP level was 128.6 pmol/g compared to the maximal mean value of less than or equal to 0.46 pmol/g in the normal ovary. Elevated levels of CRABP were also found in breast and lung carcinomas compared to the amounts detected in the same patient in normal tissue aliquots of the same organ. The differences between CRABP concentrations in cervical, endometrial, ovarian, and breast carcinomas and those in normal tissue are statistically significant. In contrast, cellular retinol-binding protein concentrations were reduced in the endometrial, ovarian, breast, and lung carcinomas compared to normal tissues. There were no significant differences between the log-mean concentrations of cellular retinol-binding proteins in the cytosols from tissue aliquots of carcinoma of the cervix and those in the cytosols from tissue aliquots of normal cervix.

Topics: Carcinoma; Carcinoma, Squamous Cell; Carrier Proteins; Endometrium; Female; Genital Neoplasms, Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin; Uterine Cervical Neoplasms; Uterine Neoplasms