Robert O. Hussa

CONTROL MECHANISMS FOR GONADOTROPHIC HORMONE PRODUCTION IN VITRO

SummaryAn internal control mechanism capable of controlling gonadotrophic hormone secretion in the human trophoblast has been identified by incubation of an established cell line with a precursor of progesterone.Biological, biochemical, and electron microscopic findings verify inhibition of gonadotrophic hormone synthesis, depletion of glycogen, activation of phosphorylase, the glycogen-metabolizing enzyme, and ultrastructural appearance of liquid-containing cytoplasmic granules in single cells, indicative of steroid synthesis.

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

SummaryThe secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N6- and O2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1α and F2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.

Effects of methotrexate on established cell lines of human choriocarcinoma

Abstract Cell lines of human choriocarcinoma (BeWo and Jar lines) were incubated in vitro for up to 7 days with 0·10, 10 , or 1000 μM methotrexate (MTX). The cell population did not change significantly for up to 6 days after daily addition of 0·10 to 1000 μM MTX to the culture medium, that was also changed each day. During the same time period the cell population quadrupled in untreated control flasks. The drug caused an increase in amount of protein per cell and in the protein: DNA ratio. Incubation for 5 hr with MTX at concentrations of 0·10 and 10 μM resulted in 83 and 93% inhibition, respectively, of deoxyuridine- 6 - 3 H incorporation into DNA. After 3 days of incubation with either 0·10 or 10 μM MTX, no incorporation of labeled deoxyuridine into DNA was observed. Incubation in the presence of the antifolate resulted in a rapid dose-dependent compensatory increase in incorporation of thymidine- 2 - 14 C into DNA, rising to 135 and 197% of untreated control levels after 24 hr of incubation with 0·10 and 10 μM MTX, respectively. At later times a decrease occurred in absolute rate of thymidine incorporation into DNA, but not in the relative rate compared to untreated cells of the same age. MTX did not significantly alter the incorporation of leucine- 3 H or uridine- 3 H into the trichloroacetic acid precipitate, nor of glucose- 3 H into DNA, RNA, protein, or lipid in the trophoblastic cells. Omission of serum from the daily medium did not change the above results. Unsuccessful attempts were made to subculture cells previously incubated with concentrations of 0·10 μM MTX or greater, although successful subculturing was possible in the presence of 0·01 μM MTX. The deleterious effects of MTX observed in both the BeWo and Jar cell lines were consistent with the suggestion that the major effect of the drug is to inhibit dihydrofolate reductase.

Early Stimulation of Human Chorionic Gonadotropin Secretion by Dibutyryl Cyclic AMP and Theophylline in Human Malignant Trophoblast Cells in vitro: Inhibition by Actinomycin D, α-Amanitin, and Cordycepin

Human malignant trophoblast cells (BeWo line) in culture were employed to investigate the early stimulation of human chorionic gonadotropin (hCG) secretion by 1 mM dibutyryl cyclic AMP and 1 mM theophylline (dbT). The earliest increase in secreted immunoreactive hCG occurred at 3 1/2 h following addition of dbT, and was preceded by an increase in intracellular hCG. These results suggested that dbT stimulated hCG synthesis, rather than release. Addition of either 0.062 mug/ml actinomycin D or 100 mug/ml cordycepin along with dbT, or 3 mug/ml alpha-amanitin 9 h prior to addition of dbT, prevented the increase in hCG secretion at 3 1/2-8 h. It was concluded that synthesis of RNA (possibly messenger RNA) is required for the early stimulation of hCG secretion by dbT.

Effects of antimicrotubule agents, potassium and inhibitors of energy production on hCG secretion.

SummaryThe BeWo trophoblastic cell line was employed to assess the requirement for microtubules and cellular energy in human chorionic gonadotropin (hCG) secretion. In contrast to the general inhibitory effect of colchicine and vincristine on hormone secretion in systems involving exocytosis, wide concentration ranges of these antimicrotubule agents caused enhancement of hCG secretion. Similarly, cytochalasin B, an agent that interferes with microfilament function, doubled both basal hCG secretion, and secretion of hCG stimulated by 1mm dibutyryl cyclic AMP plus 1mm theophylline (dbT). Inhibitors of cellular energy production (2,4-dinitrophenol, malonate, azide) decreased both secreted and cellular levels of hormone. High concentrations of K+ gave no enhancement of basal or dbT-stimulated hCG secretion, nor any reduction of cellular hCG levels. These findings contrasted with observations of others in secretory systems involving exocytosis, in which high K+ potentiated basal or stimulated hormone release and depleted cellular stores of hormone. It was concluded that the process of hCG secretion in the malignant trophoblast is fundamentally different from the mechanism of protein hormone secretion in other tissues.

Trophoblast estrogen synthetase stimulation by dibutyryl cyclic AMP and theophylline: Increase in cytochrome P-450 content☆

Abstract The inclusion of both dibutyryl cyclic AMP and theophylline in the culture medium of human malignant trophoblast cells (JAr line) for 72 hours results in an enhanced estrogen secretion through the increased specific activity of estrogen synthetase (aromatase), a cytochrome P-450 mono-oxygenase enzyme system. The data described here suggest that this increased aromatase activity is due to an increase in the concentration of only one component of the mono-oxygenase system, cytochrome P-450.

Glycogen metabolism in human hormone-producing trophoblastic cells in continuous culture

SummaryGlycogen metabolism was studied in human hormone-producing trophoblastic cells (BeWo line). Cells supplemented daily with high glucose (3 g per liter in medium) contained 5.5% glycogen and utilized glucose at an initial rate of 12.2 mμmoles per min per mg of protein. In cells supplemented daily with low glucose (1 g per liter), the initial rate of glucose consumption was 23 mμmoles per min per mg of protein and the glycogen content reached only 0.4% of wet weight 24 hr after medium replenishment. When glycogen-depleted cultures were refed glucose, an accumulation of glycogen was observed, with initial deposition occurring in areas near the cell surface. After exhaustion of extracellular glucose, cytoplasmic glycogen was utilized at a rate of 2.8 mμmoles per min per mg of protein. Addition of either low or high glucose to glycogen-depleted cells resulted in the same rate of glycogen synthesis (approximately 8 mμmoles per min per mg of protein). It was suggested that unique regulatory mechanisms function in the control of glycogen metabolism in glycoprotein hormone-producing cytotrophoblastic cells.

Electron microscopic and biochemical patterns of the normal and malignant trophoblast.

Abstract The malignant trophoblast in cell culture (BeWo line) known to produce human chorionic gonadtropin (HCG) consists of single cells with cytoplasmic ultrastructures similar to that of the placental cytotrophoblast. One of the striking features of the trophoblastic cells is the excessive accumulation of cytoplasmic glycogen, which may be depleted by epinephrine, and, to a lesser extent, by glucose “starvation.” Glycogenolysis is accompanied by a concomitant increase of endoplasmic reticulum. Hence, the development of this cytoplasmic organelle may reflect at least two different phases of functional activity in trophoblastic cells: (1) hormone synthesis and (2) carbohydrate metabolism. It is cocluded that the cytotrophoblast, in addition to its role as progenitor of syncytium, also actively synthesizes HCG. Hormone synthesis may be reflected by the development of endoplasmic reticulum but is not determined by the phase of differentiation of trophoblastic cells at which syncytium is formed. The enzyme pattern of the BeWo cells resembles that of other malignant tumors. A mucoprotein layer surrounding the BeWo cells is revealed by colloidal iron stain, and its significance is discussed. Present studies indicate that the placenta at term is still an actively functioning organ.

Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture

The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [(Bu)2 cAMP]. Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively. Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.

Production of Tumor-Associated Antigen, TA-4, by the CaSki Cervical Carcinoma Cell Line

Previous Japanese studies described the purification of TA-4 from homogenates of tumor tissues excised from squamous cell carcinomas of the uterine cervix, and the development of a radioimmunoassay to detect TA-4 in sera of patients with this disease. The aim of the present investigation was to determine if TA-4 was produced by the CaSki cell line, established in culture ten years ago from epidermoid carcinoma of the uterine cervix. The radioimmunoassay detected the TA-4 antigen in the CaSki cells, but not in cell lines derived from either choriocarcinoma or breast carcinoma. The TA-4 concentration in the CaSki cell lysate exceeded that in the CaSki culture fluid by more than twentyfold, and exceeded the concentration of human chorionic gonadotropin beta-like immunoreactive material by nearly two orders of magnitude. Antiserum to TA-4 was used to immunoprecipitate biosynthetically labeled TA-4 from CaSki cultures that had been incubated with [3H]leucine. After electrophoresis and autoradiography, the immunoprecipitated material showed a major band corresponding in apparent molecular weight (48,000 daltons) to TA-4 originally isolated from squamous cell carcinoma tumors. It is concluded that the CaSki cell line constitutes an ideal model with which to investigate the biosynthesis and regulation of TA-4, and a source for large-scale production of TA-4 for characterization studies as well as development of clinical diagnostic reagents.