calcimycin and Choriocarcinoma

In this study, the NUCC-3 choriocarcinoma cell line was used as an in vitro placental cell model to investigate the effects of follistatin on basal and GnRH-stimulated hCG secretion and/or its subunit mRNA levels. Follistatin (1.5-100 ng/ml; 48 h) alone did not affect basal hCG secretion or its subunit mRNA levels. GnRH increased hCG secretion and hCG beta-subunit mRNA levels in a dose-dependent manner, with maximal effects (2.37- and 2.4-fold increases, respectively) at a dose of 10(-8) M after 24 h of culture (P < 0.001). The time-course study showed that the increase in hCG secretion induced by GnRH occurred between 6-48 h after treatment. Follistatin (6-100 ng/ml; 48 h) significantly suppressed GnRH-stimulated hCG secretion and hCG alpha- and beta-subunit mRNA levels, with maximal suppression of 73.1%, 106.9%, and 129.1%, respectively (P < 0.001). In addition, follistatin (25 ng/ml) inhibited hCG secretion in response to phorbol 12-myristate 13-acetate (100 nM) by 90.3%. However, follistatin had no effect on hCG secretion evoked by forskolin (10 microM), and no change in hCG secretion was observed after treatment with a calcium ionophore (A23187; 10 microM) alone or in combination with follistatin. Furthermore, there was no significant difference in the half-lives of hCG alpha and -beta mRNA induced by GnRH alone compared with those induced by GnRH plus follistatin (P > 0.1), indicating that follistatin did not affect the stability of hCG alpha and -beta mRNA. Our data suggest that follistatin inhibits GnRH-stimulated hCG secretion as well as hCG alpha- and beta-subunit mRNA levels in the NUCC-3 choriocarcinoma cell line by decreasing the rate of transcription through the second messenger transduction system-protein kinase-C, rather than by affecting the stability of mRNA. These findings indicate that follistatin may play an important role in the regulation of hCG production in the placenta during pregnancy.

Topics: Calcimycin; Choriocarcinoma; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Colforsin; Follistatin; Gene Expression Regulation; Glycoprotein Hormones, alpha Subunit; Glycoproteins; Gonadotropin-Releasing Hormone; Half-Life; Humans; Kinetics; Peptide Fragments; Placenta; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

To examine the mechanism regulating trophoblastic aromatase, we studied the effects of various agents on aromatase activity and the aromatase cytochrome P-450 (P-450arom) concentration in human choriocarcinoma JEG-3 cells. Aromatase activity was assessed by radioassay with [1 beta-3H] androstenedione. The P-450arom concentration was determined by an enzyme-linked immunosorbent assay with specific antibodies to P-450arom. Human chorionic gonadotropin (hCG) stimulated aromatase activity and increased the P-450arom concentration in a concentration-dependent (0.1-100 IU/ml) manner. Cholera toxin (CT), an adenylate cyclase activator, stimulated aromatase activity and the P-450arom concentration in a concentration-dependent (0.1-10ng/ml) and a time-dependent (12-72h) manner. 12-O-tetradecanoyl phorbol 13-acetate (TPA) (0.1-100ng/ml), a protein-kinase C activator, also stimulated aromatase activity and increased the P-450arom concentration. On the other hand, Ca2+ ionophore A23187, an agent increasing intracellular Ca2+ accumulation, inhibited aromatase activity and reduced the P-450arom concentration. The effects of CT, TPA and Ca2+ ionophore were additive. Aromatase activity was correlated with the P-450arom concentration. These results suggest that in JEG-3 cells the signal transduction system modulates aromatase activity by changing the P-450arom concentration.

Topics: Aromatase; Calcimycin; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Cytochrome P-450 Enzyme System; Enzyme-Linked Immunosorbent Assay; Female; Humans; Pregnancy; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein [adrenodoxin reductase (AdRed)] and an iron sulfur protein [adrenodoxin (Adx)]. The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins. Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins. These, plus purified human Adx similarly prepared by Dr. L. Vickery, were injected into rabbits to raise antiserum to each of the proteins. Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting. Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo-cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 microM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively. In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed. The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187. In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx. The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187. These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steroidogenesis in these cell systems is regulated principally at the level of mRNA abundance.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Cortex Neoplasms; Adrenodoxin; Animals; Calcimycin; Cholesterol Side-Chain Cleavage Enzyme; Choriocarcinoma; Cloning, Molecular; Escherichia coli; Ferredoxin-NADP Reductase; Genetic Vectors; Humans; Kinetics; Mice; Mitochondria; Plasmids; Pregnenolone; Protein Kinase C; Protein Kinases; Recombinant Proteins; Restriction Mapping; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Base Sequence; Calcimycin; Cholesterol Side-Chain Cleavage Enzyme; Choriocarcinoma; Colforsin; Cyclic AMP; Cycloheximide; DNA; Ethanol; Female; Gene Expression Regulation, Enzymologic; Genes; Humans; Molecular Sequence Data; Organ Specificity; Placenta; Pregnancy Proteins; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription Factors; Tumor Cells, Cultured; Uterine Neoplasms

The BeWo cell line, derived from choriocarcinoma, produces and releases human chorionic gonadotropin (hCG) and its alpha- and beta-subunits. The authors have already reported that cAMP and EGF stimulated the production and secretion of hCG and its subunits by cultured BeWo cells. Therefore, in order to elucidate the role of signal transduction systems (cAMP-A-kinase system, DG-C-kinase system and Ca(2+)-calmodulin system) in the regulation of hCG (alpha, beta) synthesis by human choriocarcinoma cells, effects of cholera toxin (CT), an activator of adenylate cyclase, phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, and Ca2+ ionophore A23187, an activator of Ca2+ modulation on hCG (alpha, beta) production and secretion by BeWo cells cultured in a serum-free condition were evaluated. Immunoreactive hCG alpha, hCG beta and hCG in the media and cultured cells were measured by each homologous RIA for hCG alpha, hCG beta and hCG, respectively. Addition of CT at a concentration of 100 ng/ml into the medium caused extreme increases in the cellular levels of hCG alpha, hCG beta and hCG together with remarkable increases in hCG alpha, hCG beta and hCG levels in the medium. This stimulatory effect of CT was first observed on the increase of hCG alpha levels in cultured BeWo cells and medium at 3h, then observed on the increase of hCG beta levels at 6h and was last detectable on the increase of hCG levels in the cultured cells and medium at 12h. Addition of PMA at a concentration of 100 ng/ml into the medium caused an increase in the cellular and medium levels of hCG alpha, hCG beta and hCG shortly (3h) after the exposure to PMA. Addition of A23187 at a concentration of 100 ng/ml into the medium caused a slight increase in hCG alpha levels in the medium at 6h without accompanying the increase in those cellular levels. When added together, PMA potentiated the stimulatory effect of CT on hCG alpha, hCG beta and hCG levels in the cultured BeWo cells and medium, while PMA did not potentiate the effect of A23187 in this experimental condition. These findings suggest that cAMP-A-kinase system plays a major role in the signal transduction of hCG (alpha, beta) synthesis and secretion by BeWo choriocarcinoma cells, and that DG-C-kinase system interacts synergistically with cAMP-A-kinase system in the regulation of hCG (alpha, beta) synthesis and secretion by BeWo cells. Ca(2+)-calmodulin system appears to participate in the regulation of hCG alpha secretion

Topics: Calcimycin; Cholera Toxin; Choriocarcinoma; Chorionic Gonadotropin; Humans; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We have investigated the functional ability of a choriocarcinoma-cell-derived factor to block human T cell responses and the factor's immunoregulatory site of action on the T cell signal transduction pathway. The factor completely suppressed human T cell responses activated by phorbol ester and calcium ionophore, reagents which strongly stimulate IL-2-mediated T cell responses. It failed to inhibit CD 25 expression and IL-2 production by T cell blasts in the T cell activation phase, but completely blocked recombinant IL-2-induced proliferation of T cell blasts in the T cell proliferation phase. Absorption experiments with the factor and Con A-induced T cell blasts as well as [125I]IL-2 binding experiments with T cell blasts revealed that the factor acted on the physiological events occurring after IL-2-mediated stimulation of IL-2 receptor complexes, demonstrating no interaction of the factor with either IL-2 molecules or IL-2 receptor complexes. Moreover, it suppressed murine IL-2 dependent T cell line proliferation, suggesting the presence of common pathways in human and murine T cell proliferation. The biological and immunological significance of the factor during pregnancy and in the immunosuppressed tumor-bearing hosts are discussed.

Topics: Calcimycin; Cell Differentiation; Cell Division; Choriocarcinoma; Concanavalin A; Humans; Immune Tolerance; Interleukin-2; Receptors, Interleukin-2; Recombinant Proteins; Suppressor Factors, Immunologic; T-Lymphocytes; Tetradecanoylphorbol Acetate; Trophoblasts; Tumor Cells, Cultured

An immunosuppressive factor released by choriocarcinoma cell lines was analyzed in the present study. It inhibited the proliferative responses of human T cells stimulated by lectins or alloantigens. It also blocked the generation of alloreactive cytotoxic T cells. The suppressive activity of the factor was detected in the responses of the T cells costimulated with 1 nM 12-O-tetradecanoyl phorbol 13-acetate and 1 microM A23187, suggesting the possibility that the factor acted on the intracellular signal transduction in T cells rather than interfering with early events such as T cell receptor signal transduction through cell membranes. Moreover, the factor acted directly on T cell proliferation pathways without activation of suppressor cells but did not act on T cell activation pathways. Taken together, all these findings expanded our previous reports on a factor released by normal trophoblasts, indicating the possible identity of the two factors. The physicochemical properties of the choriocarcinoma-derived factor were examined, and the biological significance of the factor during pregnancy was discussed in this paper.

Topics: Calcimycin; Choriocarcinoma; Female; Humans; Lymphocyte Activation; Suppressor Factors, Immunologic; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms