transforming-growth-factor-alpha and Choriocarcinoma

Pregnancy in the rat and mouse is characterized by a developmental switch in expression at midgestation between the related placental lactogens I and II (PLI, PLII) suggesting that these proteins have important but different roles. The factors that control this differential gene expression are poorly understood. In this paper we describe experiments which investigate the effects of EGF and TGFalpha on rPLI and rPLII mRNA levels in the rat choriocarcinoma cell line, Rcho. This cell line has been widely used as a model system for studying genes expressed in the rodent placental giant cell. We find that in these cultures both growth factors produce a two fold increase in endogenous rPLI mRNA levels, and a two fold decrease in rPLII mRNA levels. Using DNA transfection assays we have tested the effects of TGFalpha on the expression of hybrid rPLI and rPLII 5'flanking/luciferase reporter constructs in Rcho cells. The transfected rPLI/luciferase reporter constructs produce a similar two fold increase in reporter expression to that seen for the endogenous mRNA, suggesting that EGF/TGFalpha positively regulate rPLI mRNA levels by effects on gene transcription. Unlike the endogenous gene, however, the rPLII/ luciferase constructs show a five fold increase in rPLII-directed luciferase expression in the presence of TGFalpha. The effect of EGF/TGFalpha on placental rPLII mRNA levels appears to involve negative response element(s) located elsewhere in the gene to those regions tested.

Topics: Animals; Choriocarcinoma; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Luciferases; Placental Lactogen; Rats; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

The role of transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor (EGFR) interactions in regulating benign and malignant trophoblast proliferation were examined. Benign cytotrophoblast (CT) demonstrated mitogenic stimulation in response to TGF-alpha; BeWo and JAr choriocarcinoma cell lines failed to respond. EGFR levels in BeWo and JAr were determined by enzyme linked immunoassay (ELISA) to be at least 10-fold higher than those in benign CT. EGFR isolated from BeWo and JAr also demonstrated functional tyrosine kinase activity. Using a combination of immunoperoxidase (IP) and ELISA techniques, choriocarcinoma cells were found to produce significant quantities of TGF-alpha that were comparable with those reported previously by this laboratory for benign CT, and were felt to be stimulating their own proliferation in an autocrine fashion. EGFR blocking and TGF-alpha neutralizing antibodies inhibited JAr proliferation whereas an EGF neutralizing antibody did not. The data presented here and in our previous report indicate that a TGF-alpha-EGFR autocrine loop may regulate normal and malignant CT proliferation. Choriocarcinoma cells may be proliferating at a maximal rate due, in part, to EGFR overexpression and are therefore unable to respond further to exogenous growth factor. Thus, EGFR overexpression may contribute to the uncontrolled proliferation of choriocarcinoma cells in general.

Topics: Antibodies, Blocking; Cell Division; Choriocarcinoma; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Pregnancy; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor alpha; Trophoblasts; Tumor Cells, Cultured

17 beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) is a steroidogenic enzyme that catalyzes the reversible interconversion of estrone and estradiol. In this study, we investigated the roles of epidermal growth factor (EGF) and tumor growth factor-alpha (TGF alpha) in the regulation of 17HSD type 1 gene expression and catalytic activity in cultured JAR, JEG-3, and BeWo choriocarcinoma cells. EGF and TGF alpha increased 17HSD type 1 protein concentrations in JAR and JEG-3 cells, as measured by time-resolved immunofluorometric assay, and 17HSD catalytic activity, as determined by production of estradiol from estrone. These increases were accompanied by parallel increases in concentrations of the 1.3-kilobase messenger RNA coding for 17HSD type 1 in these cells. EGF receptor tyrosine kinase activity inhibitors, tyrphostins, inhibited EGF action in JEG-3 cells, indicating that tyrosine kinase activity is needed for stimulation of the 17HSD type 1 gene. Treatment with 8-bromo-cAMP or phorbol 12-myristate 13-acetate increased the amount of 17HSD type 1 protein. Furthermore, phorbol 12-myristate 13-acetate potentiated the stimulatory effect of EGF. These results suggest that EGF and/or TGF alpha may play an important role in 17HSD type 1 regulation and, consequently, in estrogen production in the human placenta.

Topics: 17-Hydroxysteroid Dehydrogenases; 8-Bromo Cyclic Adenosine Monophosphate; Catechols; Choriocarcinoma; Epidermal Growth Factor; Humans; Nitriles; Protein-Tyrosine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

Trophoblastic cells abundantly express epidermal growth factor (EGF) receptors, which, when activated by EGF or transforming growth factor-alpha, can influence cellular growth and metabolism. Various lymphocyte and macrophage cytokines have been found to influence the proliferation of human choriocarcinoma (CCA) cells in vitro. In the current study we investigated the possibility that certain cytokine effects are mediated by changes in EGF receptor expression. JEG-3 human CCA cells were incubated with varying concentrations of interleukin 1-alpha (IL-1 alpha), interleukin 1-beta (IL-1 beta), interleukin 2, gamma-interferon, granulocyte-macrophage colony stimulating factor and tumor necrosis factor-alpha (TNF), and the expression of EGF receptor was measured by radioimmunoassay using a murine monoclonal antibody with specificity for the EGF receptor. Proliferative or growth suppressive effects of the cytokines were assessed by quantitative analysis of the DNA in the cell culture wells. Macrophage-derived cytokines IL-1 alpha, IL-1 beta and TNF significantly suppressed cell growth; this was associated with a significant increase in EGF receptor expression. The other cytokines had no significant effect on either EGF receptor expression or cell growth. We also studied the expression of EGF mRNA in JEG-3, Jar and BeWo CCA cell lines. By reverse transcription followed by polymerase chain reaction, low levels of EGF mRNA were detected in all three cell lines. Therefore, EGF may be synthesized by JEG-3, Jar and BeWo CCA cell lines to participate in an autocrine growth pathway. Our findings support the concept that cytokines may act as paracrine mediators of autocrine processes involved in CCA cell growth regulation by modulating growth factor receptor expression.

Topics: Blotting, Northern; Cell Count; Cell Division; Choriocarcinoma; Cytokines; DNA, Neoplasm; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Polymerase Chain Reaction; Radioimmunoassay; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Neoplasms