calcimycin and Uterine-Neoplasms

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 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

Calcyclin is a member of the S-100 family of calcium-binding proteins, whose expression is enhanced when quiescent cells are exposed to mitogenic signals. The function of calcyclin is unknown, but it is thought to be involved in modulating the intracellular calcium concentration following mitogenic stimuli. Since activation of protein kinase C (PKC) also occurs following stimulation of quiescent cells by a variety of mitogens, we have investigated the relationship between calcyclin expression and PKC activation in three human endometrial adenocarcinoma cell lines. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cell cultures resulted in a change in cell morphology, an inhibition of proliferation, an increase in calcyclin transcription rate, and an increase in calcyclin mRNA and calcyclin protein levels. In contrast, PMA had no effect on cell morphology or cell proliferation in the Ishikawa adenocarcinoma cell line but enhanced calcyclin expression. Another bioactive phorbol ester had the same effect, whereas the calcium ionophore A23187 and the non-phorbol-ester-type tumor promoter thapsigargin had no effect on calcyclin expression. The effect of PMA on calcyclin expression was blocked by the simultaneous addition of the PKC inhibitor staurosporine and by protein synthesis inhibition with cycloheximide. RNase protection assays and primer extension analysis demonstrated that PMA enhanced transcription from all three of the previously identified transcription start sites in the calcyclin gene. These data clearly demonstrate a dissociation between calcyclin expression and cellular proliferation and suggest that the enhanced calcyclin expression which is seen in quiescent cells following mitogenic stimuli may result from activation of the PKC system.

Topics: Adenocarcinoma; Alkaloids; Calcimycin; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Division; Cycloheximide; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein A6; S100 Proteins; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Uterine Neoplasms

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