epidermal-growth-factor and Uterine-Neoplasms

Topics: Diphosphonates; Drug Therapy, Combination; Epidermal Growth Factor; ErbB Receptors; Estrogens; Female; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Leuprolide; Uterine Neoplasms

It is now evident that the use of levonorgestrel-releasing intrauterine system (LNg-IUS) is effective for long-term management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. This prompted us to characterize the effects of progesterone (P4) on the growth and apoptosis of uterine leiomyoma cells. On the other hand, we have recently noted that epidermal growth factor (EGF) and IGF-I play a crucial role in prompting uterine leiomyoma growth through stimulating the proliferative potential and inhibiting apoptosis of cultured human leiomyoma cells. In the present review, attention was paid to evaluate the effects of P4 on the expression of growth factors (EGF, IGF-I) and apoptosis-related factors (TNFalpha, Bcl-2 protein) in cultured uterine leiomyoma cells. Treatment with P4 augmented EGF and Bcl-2 protein expression, but inhibited IGF-I and TNFalpha expression in cultured leiomyoma cells. It is known that TNFalpha induces apoptosis in a variety of cell types and Bcl-2 protein is an apoptosis-inhibiting gene product. Thus, the results obtained suggest that P4 has dual actions on uterine leiomyoma growth: one is to stimulate leiomyoma cell growth and survival through up-regulating EGF and Bcl-2 protein expression as well as down-regulating TNFalpha expression in those cells, and the other is to inhibit leiomyoma cell growth through down-regulating IGF-I expression in those cells. This may explain why the size of uterine myomas during use of LNg-IUS increases in some but decreases in other instances. This may also explain why the size of uterine myomas during pregnancy does not increase despite the overwhelming increase in circulating concentrations of sex steroid hormones.

Topics: Apoptosis; Blotting, Western; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; Female; Growth Substances; Humans; Insulin-Like Growth Factor I; Leiomyoma; Progesterone; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factor-alpha; Up-Regulation; Uterine Neoplasms

Recent investigations, using DNA technology, of the molecular biology of smooth muscle tumours of the uterus have confirmed their monoclonality and have strengthened the view that oestrogen and oestrogen receptors play a major role in the pathogenesis of fibromyomata. In addition, increasing evidence suggests that progesterone, insulin-like growth factors, epidermal growth factors and other proteins are also involved. The mechanisms whereby gonadotrophin-releasing hormone agonists cause shrinkage of fibromyomata remain controversial but both vascular changes and cellular atrophy appear to play a role. A shift of emphasis in the study of fibromyomata has resulted from the demonstration that the myometrium adjacent to fibromyomata is not normal and shows some similarities to the tumours themselves.

Topics: Cytogenetics; Epidermal Growth Factor; Estrogens; Female; Humans; Leiomyoma; Molecular Biology; Progesterone; Receptors, Estrogen; Somatomedins; Uterine Neoplasms

Topics: Animals; Diethylstilbestrol; Epidermal Growth Factor; Estrogens; Female; Receptors, Estrogen; Uterine Neoplasms

Topics: Epidermal Growth Factor; Female; Humans; Immunoblotting; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Transforming Growth Factors; Uterine Neoplasms; Uterus

To study the effects of mifepristone on epidermal growth factor gene expression in human uterine leiomyoma.. 20 patients with leiomyoma were divided into two groups. One was control group who underwent hysterectomy because of leiomyoma, the other was experimental group who underwent hysterectomy after pretreatment with mifepristone 10 mg/daily for 3 months. Epidermal growth factor (EGF) mRNA was semiquantified in samples of leiomyoma and adjacent normal myometrium from patients untreated in different phases of menostrual cycle and from those treated with mifepristone. Semiquantitative reverse transcriptase polymerase chain reaction, using beta-actin as internal standard, was applied to determine levels of EGF mRNA.. Leiomyoma untreated with mifepristone had significantly greater amounts of EGF mRNA than adjacent normal myometrium of uterus only in the luteal phase, but not in the follicular phase of cycle. Similarly, leiomyoma untreated with mifepristone also had significantly larger amount of EGF mRNA than treated leiomyoma in the luteal phase of the cycle, whereas, no difference in the follicular phase of the cycle.. These findings suggested that: (1) EGF mRNA levels in leiomyoma were increased only in the luteal phase, therefore, maybe mainly controlled by progesterone; (2) mifepristone inhibited EGF gene expression in leiomyoma. This may be one of regression mechanism of uterine leiomyoma in response to the antiprogesterone mifepristone.

Topics: Epidermal Growth Factor; Female; Gene Expression; Hormone Antagonists; Humans; Leiomyoma; Mifepristone; RNA, Messenger; Uterine Neoplasms

MUC1 is a large cell surface mucin glycoprotein that plays diverse roles in both normal and tumor cell biology. These roles include mucosal hydration and protection, inhibition of embryo implantation, protection of tumor cells from the immune system and reduction of cytotoxic drug uptake. Similarly, the EGFR family of cell surface receptors drives many normal developmental processes as well as various aspects of tumor growth and gene expression. EGFR family members have been demonstrated to form complexes with MUC1 in various cellular contexts. Nonetheless, the role that EGFR activation plays in modulating MUC1 levels has not been considered. In this study, we demonstrate that activated EGFR drives high level MUC1 expression in multiple cell lines of uterine adenocarcinoma and pancreatic cancer origins. In some cells, addition of exogenous EGFR ligands (EGF or HB-EGF) elevates MUC1 levels while addition of the EGFR tyrosine kinase inhibitor, AG1478, reduces MUC1 levels. The thiazolidinedione, rosiglitazone, previously shown to reduce progesterone-stimulated MUC1 expression, also blocks EGFR ligand-driven MUC1 expression. This activity was observed at relatively high rosiglitazone concentrations (above 10 µM) and appeared to be largely PPARγ independent indicating a novel utility of this drug to reduce mucin-expression in various tumor settings. Collectively, these data demonstrate that: (1) activation of EGFR stimulates MUC1 expression in multiple cellular contexts and (2) it may be possible to develop useful interventions to reduce MUC1 expression as a complementary strategy for tumor therapy.

Topics: Blotting, Western; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Humans; Mucin-1; Pancreatic Neoplasms; Quinazolines; Rosiglitazone; Thiazolidinediones; Tyrphostins; Uterine Neoplasms

Uterine leiomyomas are the most common benign tumours in women, which arise from smooth muscle cells of the uterine myometrium and usually are multicentric. In spite of their frequency pathogenesis is widely unknown, mainly due to the absence of a suitable model system. We describe the systematic optimization of culturing leiomyoma tissue explants in an economical and effective ex vivo system.. Different concentrations of oxygen, different media, sera, hormones, and growth factor supplements were tested. Immunohistochemical stainings with antibodies against hormone receptors as well as specifying proliferation and apoptotic indices and real-time PCR were performed.. Main parameters for culturing myoma tissue explants were tested for finding an optimal protocol. Standard medium D-MEM-F12 in combination with the use of horse serum in a reduced concentration of 1% turned out to be optimal for these tissue cultures as well as the addition of estradiol and epidermal growth factor EGF to media. Reduced oxygen content in the incubator air showed no positive effect.. For culturing tissue explants of uterine leiomyoma several conditions were optimized. The established tissue culture model allows examining the effects of known and potential therapeutic substances and the influence of immune competent cells in the process of tumour formation to find new targets for medical treatment.

Topics: Culture Media; Epidermal Growth Factor; Estradiol; Female; Humans; Immunohistochemistry; Leiomyoma; Progesterone; Real-Time Polymerase Chain Reaction; RNA, Messenger; Uterine Neoplasms

Uterine leiomyomas are benign uterine tumors characterized by extracellular matrix remodeling, increased collagen deposition, and increased smooth muscle cell (SMC) proliferation. The reactive oxygen species (ROS) producing NADPH oxidase complex has been shown to be involved in the signaling pathways of several growth factors, cytokines, and vasoactive agents that stimulate proliferation of a variety of cell types. Our objective was to test the hypothesis that ROS derived from NADPH oxidase is a necessary component of the MAP kinase mitogenic pathway activated by platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in leiomyoma SMCs (LSMCs). Primary cell cultures of LSMCs were used as our experimental model. Our results showed that stimulation of these cells with PDGF or EGF caused a marked increase in intracellular ROS production and that the NADPH oxidase inhibitor, DPI, blocks ROS production. In addition, inhibition of ROS production by NADPH oxidase inhibitors blocked, in a dose-dependent manner, the EGF- and PDGF-induced increase in [(3)H]thymidine incorporation by LSMCs. Furthermore, an exogenous source of ROS, hydrogen peroxide, was sufficient to stimulate [(3)H]thymidine incorporation in LSMCs but did not affect COL1A2 and COL3A1 mRNA levels. Inhibition of the NADPH oxidase complex decreased PDGF-induced MAPK1/MAPK3 activation, whereas exogenous hydrogen peroxide induced MAPK1/MAPK3 activation. This article is the first report suggesting the presence of the NADPH oxidase system and its importance in mitogenic signaling pathways in LSMCs. The necessity of NADPH oxidase-derived ROS for EGF and PDGF signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.

Topics: Cell Division; Cell Line, Tumor; DNA; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Hydrogen Peroxide; Leiomyoma; Mitogen-Activated Protein Kinases; Muscle, Smooth; NADPH Oxidases; Platelet-Derived Growth Factor; Reactive Oxygen Species; Signal Transduction; Uterine Neoplasms

Human type II deiodinase is a master regulator of thyroid hormone activation in several tissues. In placenta, type II deiodinase mRNA levels and enzymatic activity are elevated only during the first trimester of pregnancy and then progressively decline. During this early stage, mitogens such as epidermal growth factor (EGF) have been shown to promote the proliferation of the trophoblast by acting through multiple mechanisms. Here we show that EGF modulates transcription of human type II deiodinase gene (Dio2) through distinct signaling pathways, leading to the assembly of a heterogeneous transcription factor complex. Gene expression and deiodination assays have shown that EGF promptly induces a short-lived Dio2 mRNA and enzymatic activity. The induction is mediated by ERK and p38 kinases, as demonstrated by selective inhibition or overexpression of different mitogen-activated kinases. Reporter assays of mutant constructs indicate that EGF-induced transcriptional activity on Dio2 promoter is mediated by the cAMP response element (CRE) and does not involve the activating protein 1 site. With functional and biochemical approaches, we have demonstrated that the EGF stimulation culminates with the assembly and recruitment over the Dio2 CRE of a composite complex, which consists of c-Jun, c-Fos, and CRE-binding protein. These results further support the hypothesis that placental iodothyronine metabolism is critical during early pregnancy.

Topics: Cell Line, Tumor; Choriocarcinoma; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Humans; Iodide Peroxidase; Iodothyronine Deiodinase Type II; JNK Mitogen-Activated Protein Kinases; Placenta; Pregnancy; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Thyroid Hormones; Transcription, Genetic; Uterine Neoplasms

Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3beta, and dephosphorylated (DP)-beta-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of beta-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3beta and increased nuclear localization of beta-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3beta and dephosphorylated (DP)-beta-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or beta-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3beta, DP-beta-catenin, and increased nuclear localization of beta-catenin in human and MMTV-CR-1 mice leiomyosarcomas.

Topics: Animals; Blotting, Western; Cell Movement; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Leiomyosarcoma; Mammary Tumor Virus, Mouse; Membrane Glycoproteins; Mice; Mice, Transgenic; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins; Signal Transduction; Uterine Neoplasms; Wnt1 Protein

Human uterine leiomyomas are very common smooth muscle cell tumors that occur in reproductive-age women and are the leading reason for performing hysterectomies. The present study was conducted to explore the potential mechanism behind the effects exerted by dominant-negative estrogen receptors (DNERs) delivered by adenovirus to leiomyoma cells to ascertain the utility of DNERs as a novel strategy for treatment of uterine fibroids.. We investigated the ability of DNER to affect estrogen response element (ERE) activity induced by wild-type estrogen receptor (ER) by using the adenovirus ERE luciferase (Ad-ERE-luc) system in ELT3 cells and the effect of graded doses of DNER (10, 50, and 100 plaque-forming units/cell) on the expression of some selected genes controlling cultured human leiomyoma cell proliferation (cyclin D1, Cox2, PCNA, VEGF, and EGF), apoptosis (Bcl2 and Bax), estrogen metabolism (COMT), and extracellular matrix formation (MMP(1)) as well as progesterone receptors (A and B) were assessed using Western blot analysis. These genes are all regulated by estrogen and/or progesterone.. DNER has the ability to suppress the ERE luc activity induced by wild-type ER (P < 0.01) and significantly (P < 0.05) reverse the expression of all estrogen- and progesterone-regulated genes in this study.. These results suggest that interruption of the estrogen signaling pathway using DNER results in modulation of both estrogen- and progesterone-regulated genes that control leiomyoma cell apoptosis, proliferation, extracellular matrix formation, progesterone receptors, and estrogen metabolism, which might account for the DNER mechanism of action.

Topics: Adenoviridae; Animals; Apoptosis; Catechol O-Methyltransferase; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genetic Therapy; Leiomyoma; Matrix Metalloproteinase 1; Progesterone; Rats; Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; Transcriptional Activation; Transfection; Up-Regulation; Uterine Neoplasms; Vascular Endothelial Growth Factor A

Progesterone plays a pivotal role in controlling uterine leiomyoma growth. The authors review studies they conducted to evaluate the comparative effects of asoprisnil on proliferation, apoptosis, and growth factor expression in cultured leiomyoma and normal myometrial cells. Treatment with asoprisnil decreased the proliferating cell nuclear antigen-positive rate and the number of viable cells and increased the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling- positive rate in cultured leiomyoma cells in a dose-dependent manner ( P < .05). Similarly, asoprisnil decreased Bcl-2 expression and increased cleaved caspase-3 and cleaved poly(adenosine 5'-diphosphate-ribose) polymerase in leiomyoma cells but not in normal myometrial cells. Similarly, asoprisnil decreased epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor (TGF) beta mRNA and protein expression, as well as EGF receptor, IGF-IR alpha, and TGF RII protein expression in leiomyoma cells but not in cultured normal myometrial cells. These results suggest that asoprisnil selectively inhibits proliferation by downregulating the growth factors and their receptor expression and induces apoptosis in leiomyoma cells without affecting proliferation and apoptosis in normal myometrial cells.

Topics: Apoptosis; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Estrenes; Female; Humans; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Leiomyoma; Myometrium; Oximes; Phosphorylation; Progesterone; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Receptor, IGF Type 1; Receptor, Transforming Growth Factor-beta Type II; Receptors, Progesterone; Receptors, Transforming Growth Factor beta; RNA, Messenger; Time Factors; Transforming Growth Factor beta3; Tumor Cells, Cultured; Uterine Neoplasms

This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells.. The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay.. Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation.. Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.

Topics: Adult; Blotting, Western; Cell Proliferation; Cells, Cultured; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Estrenes; Female; Gene Expression; Humans; Insulin-Like Growth Factor I; Leiomyoma; Middle Aged; Myometrium; Oximes; Proteoglycans; Receptor, IGF Type 1; Receptors, Growth Factor; Receptors, Progesterone; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

The objective of this study was to elucidate the effects of GnRH antagonist Cetrorelix on proliferation and apoptosis in human leiomyoma cells cultured in vitro. Isolated leiomyoma cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions in the presence or absence of graded concentrations of Cetrorelix (10(-5) to 10(-8) mol/liter) for 6 d. Cultured leiomyoma cells were used for semiquantitative RT-PCR, immunocytochemistry, Western blot analysis, and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay. RT-PCR analysis revealed the presence of mRNAs encoding for GnRH receptor and epidermal growth factor (EGF) in cultured leiomyoma cells. The number of viable cultured leiomyoma cells was significantly (P < 0.01) decreased by treatment with Cetrorelix compared with untreated control cultures. Immunocytochemical examination demonstrated that treatment with Cetrorelix attenuated the expression of proliferating cell nuclear antigen (PCNA) and EGF in cultured leiomyoma cells. Western blot analysis revealed that treatment with 10(-5) mol/liter Cetrorelix significantly (P < 0.01) decreased PCNA expression. In addition, treatment with 10(-5) mol/liter Cetrorelix remarkably increased the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling-positive rate and poly(ADP-ribose) polymerase expression at 24 h of treatment compared with untreated control cultures (P < 0.01). Furthermore, treatment with 10(-5) mol/liter Cetrorelix decreased immunoreactive EGF protein and EGF mRNA expression in cultured leiomyoma cells at 4 d of treatment. GnRH antagonist Cetrorelix may directly inhibit leiomyoma cell growth by down-regulating proliferation in association with a decrease in EGF mRNA expression and by up-regulating apoptosis in those cells.

Topics: Adult; Apoptosis; Base Sequence; Cell Survival; DNA Primers; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Poly(ADP-ribose) Polymerases; Premenopause; Proliferating Cell Nuclear Antigen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Uterine Neoplasms

The development of the human placenta involves a complex process of tightly regulated proliferation and invasion by extravillous trophoblast into the uterine decidua. Inadequate placentation is a feature of intrauterine growth restriction and other gestational pathology. There is some evidence that T(3) plays a role in the regulation of these processes and that T(3) may act synergistically with epidermal growth factor (EGF). The aim of this study was to define the expression of thyroid hormone receptors in extravillous trophoblast, elucidate the effects of T(3) on both proliferation and differentiation of human trophoblast cells of varying origins, and define the potential interaction between EGF and T(3) on these processes. Using immunohistochemistry, specific thyroid hormone receptor isoforms were localized in extravillous trophoblast in first- and second-trimester placental bed biopsies, indicating potential sensitivity to T(3). In studies of human trophoblast-derived cell lines and primary cultures of cytotrophoblast cells in vitro, T(3) and EGF exerted an antiproliferative effect on an extravillous-like cell line (SGHPL-4) but stimulated proliferation in JEG-3 choriocarcinoma cells. EGF enhanced survival of nonproliferative term primary cytotrophoblast cells and significantly enhanced invasion of fibrin gels by SGHPL-4 cells, an effect attenuated by T(3). Both T(3) and EGF also significantly enhanced SGHPL-4 motility. These results suggest that EGF and T(3) may act synergistically to regulate both proliferation and differentiated function of human trophoblast.

Topics: Cell Division; Cell Line, Transformed; Cell Movement; Choriocarcinoma; Epidermal Growth Factor; Female; Fibrin; Gels; Humans; In Vitro Techniques; Neoplasm Invasiveness; Pregnancy; Thyroid Hormone Receptors alpha; Thyroid Hormone Receptors beta; Triiodothyronine; Trophoblasts; Uterine Neoplasms

The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells.. Isolated cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay.. Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types.. The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.

Topics: Adult; Apoptosis; Cell Proliferation; Cells, Cultured; Deoxyuracil Nucleotides; Epidermal Growth Factor; ErbB Receptors; Female; Heparin-binding EGF-like Growth Factor; Humans; In Situ Nick-End Labeling; Intercellular Signaling Peptides and Proteins; Ki-67 Antigen; Leiomyoma; Myometrium; Proliferating Cell Nuclear Antigen; Uterine Neoplasms

Extracellular matrix is a place where various growth factors are bound and immobilised. It is expected that leiomyoma-associated remodelling of extracellular matrix in the uterus may be evoked by changes in the content of some growth factors.. The amount and distribution of EGF in the normal myometrium and in the leiomyoma during various growth stages were investigated.. The assay of EGF was carried out with the use of ELISA commercial kit. SDS/polyacrylamide gel electrophoresis of tissue extracts followed by Western immunoblot was performed.. It was found that all investigated tissues contained endogenous EGE Extractability of EGF depended on type of extracting solvent. Only slight amount of EGF could be extracted by 1 M acetic acid. Much more EGF could be solubilized in 0.05M Tris/HCl, pH 7.6. Our results showed that EGF bound to the uterus (normal and leiomyoma) components of different molecular mass. It is of interest that some components of both, acidic and neutral extracts (myometrium and leiomyomas) were not able to bind exogenous 125I-labelled EGF.

Topics: Adult; Blotting, Western; Epidermal Growth Factor; Extracellular Matrix; Female; Humans; Immunohistochemistry; Leiomyoma; Middle Aged; Myometrium; Uterine Neoplasms

Endometriosis, although it is a benign disorder, shares many similarities with cancer. There is increasing levels of evidence suggesting that some circulating factors involved in gynecologic cancers, such as alpha-fetoprotein (AFP), insulin-like growth factor binding protein-3 (IGFBP-3), c-erbB-2 (HER-2/neu), and epidermal growth factor (EGF), could also play a role in endometriosis. Hence, the present study was aimed at evaluating whether the levels of these molecules are modulated in the serum of patients with endometriosis.. Levels of AFP, IGFBP-3, c-erbB-2, and EGF were determined by enzyme-linked immunosorbent assay in serum from 36 subjects with surgically confirmed endometriosis and 36 controls with no surgical evidence of the disease. In addition, information such as demographic characteristics, personal habits, menstrual characteristics, and clinical profile was collected from each participating subject.. No significant difference was found between serum levels of AFP, IGFBP-3, c-erbB-2, and EGF in patients with endometriosis and controls, even when we adjusted for potential confounders and took into account the menstrual cycle. Moreover, no correlation was observed between the serum concentrations of these molecules and the stage of the disease. However, a correlation was detected between soluble levels of IGFBP-3 and presence of uterine leiomyoma.. Although AFP, IGFBP-3, c-erbB-2, and EGF are not altered in the circulation of patients with endometriosis, their involvement in the development of endometriotic lesions cannot be excluded.

Topics: Adolescent; Adult; alpha-Fetoproteins; Endometriosis; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor Binding Protein 3; Leiomyoma; Receptor, ErbB-2; Uterine Neoplasms

Parathyroid hormone-related peptide (PTHrP) have been found to be expressed in a variety of human tumors. Parathyroid hormone-related peptide is known as the major mediator of humoral hypercalcemia of malignancy, may also regulate placental calcium flux, uterine contraction and fetal tissue development. The purpose of this study is to evaluate the expression of PTH/PTHrP receptor in choriocarcinoma JAR cell line.. This study was carried out at the Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia, between November 2002 and August 2003. Choriocarcinoma JAR cell line treated for 12 and 72 hours with epidermal growth factor, (EGF) (20ng/ml), estradiol, E2 (10(-8) M), dexamethasone, (DEX) (10(-8) M) or 1,25 dihydroxycholecalciferol, 1,25 (DHCC) (10(-8) M). We investigated the expression of parathyroid hormone (PTH)/PTHrP receptor in JAR cell line with these treatments compared with untreated JAR cells. The PTH/PTHrP receptor expression were detected with 3.3nM 125I-PTHrP-34Tyrosine.. The expression of the receptors at 12 hours were increased following exposure to EGF, E2 or DEX, whereas 1,25 DHCC inhibited the receptor expression. In further experiments at 72 hours with the same treatments, the receptors expression were remarkably increased with EGF, E2 or DEX, whereas, 1,25 DHCC inhibited the receptor expression in these cells.. These data suggested that in JAR cells, The EGF, E2 and DEX upregulated the PTH/PTHrP receptor expression, whereas the 1,25 DHCC down-regulated the PTH/PTHrP receptor, and the 1,25 DHCC may play an important role as antiproliferative drug for choriocarcinoma.

Topics: Calcitriol; Cell Division; Cell Line, Tumor; Choriocarcinoma; Dexamethasone; Down-Regulation; Epidermal Growth Factor; Estradiol; Female; Humans; Parathyroid Hormone; Pregnancy; Pregnancy Complications, Neoplastic; Receptor, Parathyroid Hormone, Type 1; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

A case of epithelioid trophoblastic tumour (ETT), occurring in a fallopian tube of a 39-year-old woman, is reported. The patient presented with a positive pregnancy test, but continued to have 'periods'. A palpable right adnexal mass was noted that was confirmed on ultrasound. The mass was removed together with the uterus, omentum and associated ovary. Careful examination of the uterus revealed no evidence of either an antecedent tumour or intra-uterine pregnancy. Histologically, the tubal mass displayed sheets and islands of large, relatively uniform, mitotically active polyhedral cells, with surrounding necrosis. The immunoprofile of the tumour was atypical in that alpha-inhibin and epidermal growth factor were weakly positive, but other results were consistent with the diagnosis of ETT. The patient received a foreshortened course of standard EMACO (etoposide, actinomycin-D, methotrexate, vincristine, and cyclophosphamide) combination chemotherapy for high-risk gestational trophoblastic disease. Serum beta-hCG fell from a pre-operative level of 52 000 U/mL to non-pregnant levels within two courses and she remains well and disease-free 12 months post-diagnosis.

Topics: Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Chorionic Gonadotropin, beta Subunit, Human; Dactinomycin; Disease-Free Survival; Doxorubicin; Epidermal Growth Factor; Epithelioid Cells; Etoposide; Fallopian Tube Neoplasms; Female; Humans; Hysterectomy; Immunohistochemistry; Inhibins; Leucovorin; Methotrexate; Pregnancy; Treatment Outcome; Trophoblastic Neoplasms; Uterine Neoplasms; Vincristine

The trophoblast, an important component of the mammalian placenta, has several essential biological roles in the maintenance of pregnancy. First, trophoblast cells must attach to the uterine endometrium, and then they must invade to a depth at which the vascular network exists. Here, we investigated the effects of epidermal growth factor (EGF) on alpha2 integrin expression, adhesiveness to collagen, and invasive activity using human BeWo choriocarcinoma cells. EGF induced the expression of alpha2 integrin mRNA and protein, as shown by Northern blotting, Western blotting, and flow cytometry. Human chorionic gonadotropin (hCG) secretion was enhanced by the addition of EGF, which suggests that the BeWo cells functionally differentiated similarly to normal trophoblasts. EGF also dose-dependently stimulated the invasiveness of BeWo cells. Antibody against alpha2 integrin inhibited this effect, suggesting that it may be mediated by an increase of cell surface integrin. EGF had no effect on the adhesiveness of BeWo cells to collagen, whereas it stimulated the chemokinetic activity in a dose-dependent manner. The increase of chemokinetic activity was suppressed by antibody against alpha2 integrin. These results suggest that EGF may induce alpha2 integrin expression in trophoblast cells, thereby enhancing their invasiveness into the endometrium via an increase of their chemokinetic activity.

Topics: Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Chemotaxis; Choriocarcinoma; Collagen; Epidermal Growth Factor; Female; Humans; Integrin alpha2; Neoplasm Invasiveness; RNA, Messenger; Uterine Neoplasms

Immunolocalization of transforming growth factor alpha (TGF-Alpha), epidermal growth factor (EGF), insulinlike growth factor (IGF)-I, vascular endothelial growth factor (VEGF(165,189,121)), basic fibroblast growth factor (FGF)-2, EGF receptor (R), IGF-IRbeta, and FGFR-1 was studied in uterine leiomyomas and matched myometrial samples taken from seven women (42-47 years of age) in the proliferative phase of the menstrual cycle. Immunolocalization of growth factor peptides was accomplished with either monoclonal or polyclonal antibodies to the amino or carboxy terminus of growth factor peptides or their respective receptors, or against full-length recombinant growth factor. All reactions were conducted using the avidin-biotin complex method. Immunolocalization of TGF-alpha, EGF, EGF-R, IGF-I, IGF-IRbeta, FGF-2, FGFR-1, and VEGF was observed in the cytoplasm of smooth-muscle cells of leiomyomas and matched myometrium. The cytoplasm of vascular smooth-muscle cells expressed TGF-alpha, EGF, EGF-R, IGF-I, IGF-IRbeta, FGF-2, FGFR-1, and VEGF, whereas the vascular endothelium was positive for TGF-alpha, EGF, EGF-R, FGF-2, and FGFR-1 in both leiomyomas and matched myometria. Fibroblasts within the fibrous component of some leiomyomas were positive for IGF-I and FGF-2 and minimally positive for FGFR-1. In addition, the extracellular matrix of leiomyomas showed focal localization of FGF-2 and IGF-I in some tumors. When scores of intensity and percent positive staining were compared, IGF-IRbeta was significantly increased in the leiomyomas compared to matched myometria, whereas EGF was significantly decreased in the uterine leiomyomas compared to matched myometria. In summary, these data revealed growth factors to be expressed differentially in smooth muscle, vascular and fibroblastic cell types of leiomyomas and matched myometria. Specifically, IGF-IRbeta was significantly increased in leiomyomas; although a similar increase was seen with IGF-I peptide, statistical significance was not achieved. The EGF peptide was significantly decreased in the leiomyomas compared to matched myometrium. These data suggest that IGF-IRbeta and IGF-I peptide may be one of several growth factor/receptor pathways important in uterine leiomyoma growth during the proliferative phase of the menstrual cycle. In addition, decreased EGF may be secondary to the predominant estrogenic milieu present at time of sampling, as it has been proposed that progesterone, and not estrogen, may regulate E

Topics: Adult; Case-Control Studies; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 2; Growth Substances; Humans; Immunohistochemistry; Leiomyoma; Lymphokines; Menstrual Cycle; Middle Aged; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, IGF Type 2; Receptors, Fibroblast Growth Factor; Receptors, Growth Factor; Somatomedins; Transforming Growth Factor alpha; Uterine Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To study the effects of epidermal growth factor (EGF) on uterine leiomyoma and the relationship of EGF and estrogen, progesterone.. Fourty patients with uterine leiomyoma and twenty normal women were studied. Serum EGF level was determined by radioimumunoassay, estradiol (E2) and progesterone (P) levels were measured by enzyme marked monoclonal antibody assay.. Serum EGF, E2 and P levels in observed group were significantly higher than those in control group during secretive phase (P < 0.005). The serum EGF level in observed group during prolifrative phase was significantly higher than that of secretive phase (P < 0.05). The serum EGF level increased while uterus enlarged; EGF wasn't related to E2(r = 0.25, P > 0.05) and was related to P (r = 0.71, P < 0.005). The EGF level was the highest when E2 and P both increased.. EGF has the effect on growth of uterine leiomyoma. It is possible that EGF secretion can be stimulated by E2 and the effect of P is stronger than E2.

Topics: Adult; Epidermal Growth Factor; Estradiol; Female; Humans; Leiomyoma; Middle Aged; Progesterone; Uterine Neoplasms

In this work, we report the isolation of a factor from the culture supernatant of confluent fibroblasts from human cervix with the diagnosis of uterine myomatosis. This factor possesses the capacity to inhibit the proliferation of normal fibroblasts. The proliferation inhibitor factor (PIF) was purified from the culture supernatant by precipitation with 80% ammonium sulfate, and by molecular sieve chromatography. Our results indicate that PIF is a protein of 23 kDa, which is highly sensitive to trypsin treatment, and is thermolabile, since temperatures equal to, or above, 60 degrees C eliminate the protein activity in 15 to 20 min. Western blot analyses identified no cross reactions of the purified PIF with TGF-alpha, TNFalpha, IFNgamma, or IL-1beta, suggesting that PIF is a new protein belonging to the group of factors secreted by fibroblasts able to inhibit cellular proliferation.

Topics: Blotting, Western; Cell Count; Chromatography, Gel; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblasts; Gentian Violet; Growth Inhibitors; Humans; Interferon-gamma; Interleukin-1; Leiomyoma; Neoplasm Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Neoplasms

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. In order to characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined whether sex steroids could influence the proliferation of leiomyoma cells. As epidermal growth factor (EGF) has been shown to mediate estrogen action and play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on EGF and EGF receptor (EGF-R) expression in leiomyoma cells. In cultures of leiomyoma cells, the addition of either estradiol (E(2); 10 ng/ml) or progesterone (P(4); 100 ng/ml) resulted in an increase in proliferating cell nuclear antigen (PCNA) expression in the cells, whereas in cultures of normal myometrial cells, the addition of E(2) augmented PCNA expression in the cells, but P(4) did not. Immunoblot analysis revealed that leiomyoma cells contained immunoreactive EGF and that P(4) treatment resulted in an increase in EGF expression in the cells, whereas E(2) treatment resulted in a lower EGF expression in the cells. By contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) upregulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) upregulates the expression of PCNA and EGF-R in those cells. It is, therefore, conceivable that P(4) and E(2) act in combination to stimulate the proliferative potential of leiomyoma cells through the induction of EGF and EGF-R expression. We also found that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to that in normal myometrium and that Bcl-2 protein expression in leiomyoma cells was upregulated by P(4), but downregulated by E(2). It seems, therefore, likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells. The abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of a leiomyoma relative to that of normal myometrium in the uterus.

Topics: Adult; Apoptosis; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Leiomyoma; Myometrium; Progesterone; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Uterine Neoplasms

The role of the epidermal-growth-factor receptor (EGFR) in cervical cancer is controversial, due to technical difficulties in localizing or in quantifying EGFR by homogenate assays or immunohistochemistry. Our autoradiographic approach, in combination with morphometry, allowed cell-type-specific quantification of EGFR, leading to the following observations: (i) In normal cervical epithelium, EGFR levels per cell were high in non-dividing squamous cells of the upper layers of normal epithelium, where a mitogenic function of these EGFRs can be excluded. (ii) In contrast to earlier findings in tissue homogenates, but consistent with our observation in normal cervical epithelium that cells of the proliferating strata (basal and parabasal cells) express intermediate and comparatively reduced levels of EGFR per cell, cervical cancers displayed a significant reduction both of specific EGF binding and of EGFR levels per cell as compared with normal epithelium. (iii) A significant negative correlation of cell density and EGFR number per cell was obtained. In normal cervical epithelium, cervical intra-epithelial neoplasia and invasive cervical cancer (p = 0.002). This negative correlation was most evident in normal epithelium, where large changes of cell density occur within one slide (p < 0.001). (iv) Specific EGF-binding was also significantly reduced in endometrial cancers when compared with normal endometrium. It is proposed that in uterine tissues low or intermediate levels of EGFR do not exclude their function as mediators of cell proliferation.

Topics: Autoradiography; Carcinoma; Cervix Uteri; Down-Regulation; Endometrium; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Humans; Radioligand Assay; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Uterine Neoplasms

Uterine leiomyoma is the most common smooth muscle cell tumor of the myometrium. Estrogen and progesterone (P4) are believed to be physiological regulators of leiomyoma growth. We recently showed that Bcl-2 protein, an apoptosis-inhibiting gene product, was abundantly expressed in leiomyoma relative to its expression in the normal myometrium and that Bcl-2 protein expression in cultured leiomyoma cells was up-regulated by P4, but down-regulated by 17 beta-estradiol (E2). To further characterize the molecular mechanism of sex steroidal regulation of leiomyoma growth, we examined the effect of menstrual phase on proliferating cell nuclear antigen (PCNA) expression in leiomyoma and investigated whether sex steroids could influence PCNA expression in leiomyoma cells cultured under serum-free conditions by immunoblot and immunohistochemical analyses. As epidermal growth factor (EGF) has been shown to mediate estrogen action and to play a crucial role in regulating leiomyoma growth, we also investigated the effects of sex steroids on the expression of EGF and EGF receptor (EGF-R) in cultured leiomyoma cells. The PCNA labeling index in leiomyomas was much greater in the secretory, P4-dominated, phase than in the proliferative phase of the menstrual cycle and was significantly higher than that in the adjacent normal myometrium throughout the menstrual cycle. In monolayer cultures of leiomyoma cells, the addition of either E2 (10 ng/mL) or P4 (100 ng/mL) resulted in an increase in PCNA expression in the cells compared to that in control cultures, whereas in monolayer cultures of myometrial cells, the addition of E2 augmented PCNA expression in the cells, but P4 did not. Immunoblot analysis of proteins extracted from cultured leiomyoma cells revealed that leiomyoma cells contained immunoreactive EGF with a molecular mass of 133 kDa and that the addition of P4 resulted in a remarkable increase in the expression of 133- and 71-kDa immunoreactive EGF in the cells compared to that in control cultures, whereas the addition of E2 resulted in a somewhat lower expression of immunoreactive EGF in the cells. Furthermore, immunocytochemical analysis with a monoclonal antibody to human EGF-R demonstrated that the treatment with E2 augmented EGF-R expression in the cells compared to that in untreated cells, but P4 did not. The concentrations of sex steroids used were within the physiological tissue concentrations found in leiomyomas and myometria. These results indicate that P4

Topics: Adult; Blotting, Western; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Humans; Immunohistochemistry; Leiomyoma; Myometrium; Progesterone; Proliferating Cell Nuclear Antigen; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

In order to elucidate the regulation of placental growth, we have characterized the expression of proliferating cell nuclear antigen (PCNA), apoptotic DNA fragmentation and bcl-2 protein in human placenta during pregnancy. PCNA and bcl-2 protein expression were examined by immunohistochemical techniques, while the occurrence of apoptotic DNA fragmentation was assessed by in situ analysis of DNA 3'-end labeling method. Both PCNA expression and apoptotic DNA fragmentation were found in cytotrophoblasts (C-cells), being most abundant in early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, bcl-2 protein expression was found in syncytiotrophoblasts (S-cells), being least abundant in early placenta, less abundant in midterm placenta and most abundant in term placenta. These data indicate that early placenta is characterized by the highly proliferative activity of C-cells associated with the increased occurrence of apoptosis, whereas term placenta is characterized by the abundant expression of bcl-2 protein in S-cells. Furthermore, effects of EGF on the proliferative activity and differentiated function of trophoblasts were investigated using an organ culture system. Explants of trophoblastic tissues were cultured with or without EGF, in the presence or absence of 10(-8)M triiodo-L-thyronine (T3) in a serum-free condition. In 4-5 week placentas, EGF and EGF receptor were almost exclusively localized in C-cells, and EGF augmented the proliferative activity of C-cells without affecting the ability to secrete hCG and hPL. By contrast, in 6-12 week placentas, EGF and EGF receptor were predominantly localized in S-cells, and EGF stimulated hCG and hPL secretion without affecting the proliferative activity of C-cess. The addition of T3 (10(-8)M) resulted in an increased secretion of immunoreactive EGF by cultured placental explants. These findings suggest that EGF acts as a local factor in regulating early placental growth and function in synergy with thyroid hormone. On the other hand, progesterone selectively inhibited pleise hCG (alpha, beta) mRNAs expression and decreased hCG secretion in normal placental tissues, whereas choriocarcinoma did not respond to progesterone. This suggests that inhibitory regulation of hCG synthesis in choriocarcinoma is different from normal placenta. It was also found that in molar trophoblasts and choriocarcinoma cells PCNA expression was high, but both bcl-2 protein and apoptotic signal

Topics: Cell Differentiation; Cell Division; Choriocarcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hydatidiform Mole; Immunohistochemistry; In Vitro Techniques; Placenta; Pregnancy; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Trophoblasts; 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

We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B(0) (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657-18661, 1996). The presence of the amino acid transport system B(0) (ATB(0)) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na(+)-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0). Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB(0) activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB(0) activity and on ATB(0) mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB(0) activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB(0) activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B(0) and that the expression of the system in these cells is stimulated by EGF.

Topics: Alanine; Amino Acid Transport Systems; Amino Acid Transport Systems, Basic; Amino Acid Transport Systems, Neutral; Aurintricarboxylic Acid; Biological Transport; Carrier Proteins; Choriocarcinoma; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; HeLa Cells; Humans; Isoflavones; Leucine; Phosphorylation; Placenta; Tumor Cells, Cultured; Tyrosine; Uterine Neoplasms

Gestational Trophoblastic Disease (GTD) is an abnormal condition of the placenta, the incidence of which is very high in the State of Kerala, India. The biology of the disease is vague and methods to determine the malignant potentialis limited. Placentae of normal (50) and molar pregnancy (95) including 32 patients showing persistent disease were used in this study. EGF expression was analyzed by immunohistochemistry using monoclonal antibody to EGF and quantitated using isotope labelled antibody to EGF. EGF was expressed more strongly in molar placentae in all gestational ages in comparison with normal placentae of similar gestational age, the expression being restricted mainly to first and second trimesters in normal placentae. Moles with early presenting symptoms (< 12 weeks) were at a higher risk for developing persistent disease. In conclusion, the immunohistochemical study shows high expression of EGF in early normal placentae and moles linking its role to proliferative and differentiating activity of trophoblasts. Tumors with histological diagnosis of invasive moles and choriocarcinoma showed very strong binding of EGF and thus EGF has the potential of identifying high risk lesions.

Topics: Adolescent; Adult; Choriocarcinoma; Epidermal Growth Factor; Female; Humans; Hydatidiform Mole; Hydatidiform Mole, Invasive; Immunohistochemistry; Male; Middle Aged; Placenta; Pregnancy; Reference Values; Uterine Neoplasms

Hormone receptors and oncoproteins are receiving increased attention as possible prognostic factors in different carcinomas. Few data are available regarding quantification of their levels of expression in gynecologic malignancies.. Epidermal growth factor (EGF) receptor specific binding capacities and affinities were measured by ligand binding assay using [125I]EGF in a competition mode with Accufit software (Lundon Software, Inc., Middlefield, OH). HER-2/neu oncoprotein was extracted from membranes and measured using an enzyme-linked immunosorbent assay. Cathepsin D was measured by an immunoradiometric assay using cytosols for steroid receptor analyses.. EGF receptors in 23 nonmalignant uteri ranged from undetectable to 50 fmol/mg membrane protein (median, 0), with dissociation constant values of 1.2 x 10(-9) M to 8.5 x 10(-10) M, compared with EGF receptors in 76 endometrial cancers that ranged from undetectable to 7674 fmol/mg (median, 52). HER-2/neu oncoprotein ranged from undetectable to 2.9 HER-2/neu units (HNU)/microg protein (median, 0.6) in 41 nonmalignant uteri and from undetectable to 5.8 HNU/microg protein (median, 2.5) in endometrial cancers (n = 53). Cathepsin D ranged from 5 to 32 pmol/mg cytosol protein (median, 11) in 42 nonmalignant uteri and 18 to 144 pmol/mg protein (median, 42) in 29 endometrial cancers.. Determination of the frequency and levels of EGF receptors, HER-2/neu protein, and cathepsin D in uteri with and without cancer and the availability of reference materials developed in our laboratory, will allow evaluation of their prognostic value in cancers of the uterus.

Topics: Cathepsin D; Cell Membrane; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Leiomyoma; Prognosis; Radioimmunoassay; Receptor, ErbB-2; Recombinant Proteins; Uterine Neoplasms; Uterus

We examined four choriocarcinoma cell lines, NaUCC-1, NaUCC-3, NaUCC-4 and BeWo, for the presence of epidermal growth factor (EGF) by enzyme immunoassay and reverse transcription and polymerase chain reaction, and for EGF receptor (EGFR) by 125I-EGF binding assay. Specific EGF binding and EGF proteins were detected in these four choriocarcinoma cell lines. On the cell lines examined, NaUCC-4 had the greatest EGF binding capacity (18 x 10(5) sites/cell) and the highest amount of immunoreactive EGF (142 pg/ml). These results prompted us to assess the significance of EGF/EGFR autocrine mechanism in NaUCC-4 cells. Low doses of exogenous EGF stimulated 3H-thymidine incorporation, and monoclonal antibodies against EGF or EGFR dose-dependently inhibited 3H-thymidine incorporation. On the other hand, these antibodies did not significantly affect hCG production. These results suggested that EGF might function in an autocrine manner to stimulate proliferation rather than differentiation of NaUCC-4 choriocarcinoma cells.

Topics: Adult; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hydatidiform Mole; Pregnancy; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Neoplasms

The biosynthesis of human chorionic gonadotrophin (hCG) is a hallmark endocrine function of human choriocarcinoma cells. The present study investigated the consequences of greatly diminishing this synthesis in JAR cells by stably transfecting them with pRSV-antisense hCG-alpha cDNA expression vector. The vector directs the synthesis of antisense hCG-alpha subunit mRNA which would then bind to sense hCG-alpha subunit mRNA, thus blocking its translation and consequently dimer hCG protein synthesis. The transfection with pRSV-antisense hCG-alpha cDNA resulted in a dramatic decrease in hCG secretion as compared with untransfected parental cells or those transfected with an empty vector used for the selection of clones. The decreased secretion was due to a decreased synthesis which in turn was due to a fall in steady-state hCG-alpha and -beta subunit mRNA levels. The decrease of hCG-beta subunit transcripts was unexpected and it was not due to contamination of antisense hCG-alpha cDNA construct with hCG-beta sequence. The transcription of hCG-alpha and -beta subunit genes was not altered in transfected cells suggesting that increased degradation was responsible for decreased steady-state hCG subunit mRNA levels. Despite the decreased hCG levels, the transfected cells maintained normal hCG receptor levels, responded to epidermal growth factor stimulation of hCG synthesis and secretion and grew at the same rate as the control parental cells and those transfected with an empty vector.

Topics: Biomarkers, Tumor; Cell Division; Choriocarcinoma; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; DNA, Antisense; DNA, Complementary; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Glycoprotein Hormones, alpha Subunit; Humans; Neoplasm Proteins; Peptide Fragments; Receptors, LH; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Uterine Neoplasms

Uterine leiomyomas (fibroids) are benign, smooth muscle cell (SMC) tumors of the myometrium containing abundant extracellular matrix (ECM). Heparin-binding growth factors present in leiomyoma and normal myometrial fresh tissue were isolated using heparin-affinity fast protein liquid chromatography. Purification of these growth factors was monitored by the stimulation of [3H]thymidine incorporation into BALBc-3T3 cells and myometrial SMC. Western blot analysis confirmed that two consistent peaks of growth factor activity (eluting at 0.5 M NaCl and 1.7 M NaCl) were platelet-derived growth factor (PDGF), 31 kDa, and basic fibroblast growth factor (bFGF), 18 kDa, respectively. Northern blot analysis of leiomyoma and myometrial tissue revealed three RNA transcripts (2.8, 2.3, and 1.9 kb) for PDGF-A chain, one RNA transcript (4.0 kb) for PDGF-B chain, and two RNA transcripts (3.7 and 3.5 kb) for bFGF. RNase protection assay showed elevated expression of the bFGF mRNA transcript in leiomyomas in 3 out of 5 patients. Immunoperoxidase staining of paraffin-embedded tissue showed that PDGF was predominantly intracellular in both vascular and myometrial SMC. Basic FGF, by contrast, was found primarily bound to the ECM of myometrium and fibroids. Leiomyomas showed much stronger staining for bFGF due to the large areas of ECM in these tumors. A third mitogenic peak eluting at 1.1 M NaCl was also seen in both myometrial and leiomyoma tissue. This peak was not definitively identified by Western blotting. However, Northern analysis for heparin binding-epidermal growth factor (HBEGF), which also elutes at 1.1 M NaCl, detected one RNA transcript for HBEGF (2.5 kb) in normal myometrium but little or no expression in the corresponding leiomyoma tissue. Immunoperoxidase staining showed that HBEGF was a cell-membrane-associated protein in both normal myometrial and leiomyoma SMC with more intense staining in normal myometrium. These results show that both leiomyomas and myometrium synthesize a number of heparin-binding growth factors. The enhanced growth of leiomyomas may be due, in part, to the presence of large quantities of bFGF that are stored in the ECM of these tumors. In addition, the level of HBEGF mRNA declines during the transformation of myometrial SMC into leiomyomas.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Division; Chromatography, Affinity; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Leiomyoma; Mice; Mice, Inbred BALB C; Mitogens; Myometrium; Ribonucleases; RNA, Messenger; Uterine Neoplasms

We studied the estrogen-dependent expression of epidermal growth factor (EGF), transforming growth factor (TGF) alpha, and EGF receptor gene transcripts in human fallopian tubes in vivo and in vitro. Competitive polymerase chain reaction (PCR) was performed on the fallopian tube RNA samples from the postmenopausal women with or without estrogen replacement. Amounts of EGF, TGF alpha, and EGF receptors gene transcripts in the estrogen-treated group (n = 3) were significantly (P < 0.01) more than those in the untreated group (n = 3). Competitive PCR also showed that EGF, TGF alpha, and EGF receptor gene transcripts level in tubal cells were increased by estrogen in vitro: messenger RNA levels of these factors were significantly (P < 0.01, n = 3) increased in cells incubated with 10(-8) M estrogen compared with those in cells without estrogen treatment. We studied whether EGF and/or TGF alpha is involved in the estrogen-induced tubal cell growth in vitro. Estrogen enhanced the [3H]-thymidine incorporation into the cell in dose- and time-dependent manners in culture: estrogen treatment for more than 12 h significantly (P < 0.05) enhanced the [3H]-thymidine incorporation into the cell at 10(-8) M. The estrogen-induced cell growth was observed in association with the increase in EGF, TGF alpha, and EGF receptor messenger RNA levels by estrogen. If the EGF and/or TGF alpha is involved in the cell growth, then the estrogen-induced cell growth should be suppressed by blocking the action of EGF and/or TGF alpha. Therefore, we examined the effects of neutralizing monoclonal antibodies against EGF, TGF alpha, and EGF receptors. Anti-EGF antibody significantly reduced the estrogen-induced increase in [3H]-thymidine incorporation, whereas anti-TGF alpha antibody failed to show the effect. Anti-EGF receptor antibody showed a significant suppressive effect on the estrogen-induced increase in [3H]-thymidine incorporation. Moreover, the growth inhibitory effect by 1 microgram/ml anti-EGF was restored by 10(-8) M EGF but not by TGF alpha even at 10(-6) M. All these data suggest that estrogen induces EGF and TGF alpha/EGF receptors in the human fallopian tube and that EGF but not TGF alpha may be involved in the estrogen-induced human tubal cell growth in vitro.

Topics: Antibodies, Monoclonal; Base Sequence; Catechols; Cells, Cultured; DNA; DNA Primers; Epidermal Growth Factor; Epithelium; ErbB Receptors; Estrogens, Conjugated (USP); Fallopian Tubes; Female; Gene Expression; Growth Inhibitors; Humans; Hysterectomy; Leiomyoma; Middle Aged; Molecular Sequence Data; Nitriles; Polymerase Chain Reaction; Postmenopause; Thymidine; Transforming Growth Factor alpha; Tyrphostins; Uterine Neoplasms

Fibroids (leiomyomata) are the commonest tumours in women, but their aetiology is unknown. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF beta) may be important factors involved in fibroid growth. We examined the mRNA expression of these two growth factors in fibroids and corresponding myometrium from 20 women who underwent hysterectomy because of fibroids. We also examined these factors in samples of fibroids from 9 women who underwent myomectomy after pretreatment with luteinizing hormone-releasing hormone agonists. We found that both factors were expressed in the three types of tissue examined. We also found that there was no difference in the relative abundance of either of the two growth factors between the tissues studied. Despite the lack of difference, we postulate that EGF and TGF beta may be important in fibroid growth because of a possible interaction between the two factors in this tissue.

Topics: Autoradiography; Blotting, Northern; Epidermal Growth Factor; Estrogens; Female; Humans; Leiomyoma; Myometrium; RNA, Messenger; Transforming Growth Factor beta; Uterine Neoplasms

Analysis of clinical data has implicated ethanol (EtOH) as an embryotoxic agent and as an agent that disrupts normal placental structure and function. Because epidermal growth factor (EGF) is an important regulator of placental function, we have studied the effects of EtOH on EGF-induced hormone secretion using JEG-3 choriocarcinoma cells that serve as a model for trophoblast cells. EtOH at physiological (5-100 mM) concentrations modulated effects of EGF in a time and dose-dependent manner. EGF-induced P4 secretion was increased by 20-100 mM EtOH after a 2-day pretreatment of cells with EtOH, but not after a 6-day pretreatment. Preincubation with 50 mM EtOH doubled the P4 responses to 50 and 100 ng/ml EGF. Although a 2- or 4-day preincubation of cells with 10-50 mM EtOH increased the secretion of E2 in response to 20 ng/ml EGF, a 6-day preincubation inhibited the secretory response to EGF. Pretreatment of cells with 10-50 mM, but not 100 mM EtOH for 2 to 6 days enhanced the human chorionic gonadotropin (hCG) secretory response to EGF. At 50 mm EtOH, the secretion of hCG in response to EGF was increased 2-fold. EtOH also increased basal hCG secretion in a dose-dependent manner between 10-50 mM EtOH. These results suggest that EtOH may modulate EGF-stimulated hormone secretion from cells of placental origin. Such alterations, if they occur in vivo, may impact on the function of the placenta and could potentially explain the pathophysiology of alcohol toxicity during pregnancy.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Epidermal Growth Factor; Estradiol; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Humans; Placental Hormones; Pregnancy; Progesterone; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

Although there are some reports, including our previous study, indicating the existence and biological action of epidermal growth factor (EGF) in human decidua, the site of its synthesis remains unknown. To clarify the EGF production during decidualization at a molecular level, gene expression of EGF in human endometrium/decidua was examined by Northern blot analysis and in situ hybridization. Northern blot hybridization using 32P-labeled human prepro-EGF complementary DNA was carried out for nonpregnant human endometria from hysterectomized uteri of leiomyoma, decidua from early pregnancy, and in vitro decidualized endometrial cells by medroxyprogesterone acetate (MPA). Although no hybridized band was found in the proliferative and secretory phase endometria, a specific band of 5 kilobases, in agreement with the size of human prepro-EGF messenger ribonucleic acid, was detected in decidua of early pregnancy as well as in in vitro MPA-induced decidual cells. In situ hybridization revealed that prepro-EGF messenger ribonucleic acid was observed in the stromal cells of decidua. These results demonstrate that the EGF gene is expressed in the process of decidualization, suggesting that EGF may play an important role in the decidualization process of human endometrium.

Topics: Blotting, Northern; Decidua; DNA Probes; Endometrium; Epidermal Growth Factor; Female; Gene Expression; Humans; In Situ Hybridization; Leiomyoma; Medroxyprogesterone Acetate; Pregnancy; Protein Precursors; RNA, Messenger; Uterine Neoplasms

Epidermal growth factor (EGF) stimulates the secretion of hCG in choriocarcinoma cells. However, the molecular mechanisms involved in this EGF action have never previously been investigated. The present study investigated them as well as EGF regulation of the hCG/LH (LH) receptor gene in JEG-3 human choriocarcinoma cells. The JEG-3 cells contain multiple EGF receptor messenger RNA (mRNA) transcripts and a single 170-kilodalton immunoreactive receptor protein. The human EGF can bind to the receptor protein and stimulate the receptor autophosphorylation as well as the phosphorylation of four other membrane proteins. Culturing JEG-3 cells with recombinant human EGF resulted in a dose- and time-dependent increase in hCG secretion. The maximal effect was seen at 100 ng/ml EGF, with a time lag of about 5 h. Tyrosine kinase, but not protein kinase-C or protein kinase-A, signaling was involved in the EGF action to increase hCG secretion. The EGF-induced increase in hCG secretion was not due to an increase in cell number or differentiation into multinuclear syncytia. EGF treatment resulted in a dose- and time-dependent increase in steady state levels of hCG alpha and hCG beta mRNAs. This increase was due to the stabilization of subunit mRNA transcripts. The increase in subunit mRNAs preceded the increase in hCG secretion. The EGF treatment resulted in a dose- and time-dependent decrease in steady state levels of the hCG/LH receptor mRNA transcripts. The decrease was due to a transcriptional inhibition of receptor gene. EGF treatment paradoxically stabilized hCG/LH receptor protein. In summary, EGF treatment up-regulates hCG subunits gene expression and down-regulates hCG/LH receptor mRNAs involving transcriptional and posttranscriptional mechanisms in JEG-3 human choriocarcinoma cells.

Topics: Cell Differentiation; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Hydatidiform Mole, Invasive; Mitogens; Pregnancy; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Receptors, LH; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

Epidermal growth factor (EGF) mRNA was quantified in samples of human myometrium, untreated leiomyomata, and leiomyomata from patients treated with a GnRH analog. Quantitative reverse transcriptase-polymerase chain reaction, using a synthetic internal standard, was applied to determine levels of EGF mRNA. In myometrium from uteri with no leiomyomata, levels of EGF mRNA did not differ between the proliferative and secretory phase of the cycle. Leiomyomata from women who had received no drug therapy had significantly higher amounts of EGF mRNA than myometrium from a normal uterus, but only in the secretory phase of the cycle. In the proliferative phase, leiomyomata did not have different amounts of EGF mRNA compared to normal myometrium. Untreated leiomyomata in the secretory phase of the cycle, but not those in the proliferative phase, had significantly more EGF mRNA than leiomyomata from women who had received treatment with a GnRH analog. These findings suggest that EGF is important in leiomyomata development, but imply that its production is only increased during the secretory phase of the cycle. This challenges the hypothesis that EGF production in leiomyomata is mediated by estrogen and raises the possibility that progesterone may be the more important hormone in fibroid growth.

Topics: Adult; Epidermal Growth Factor; Female; Humans; Leiomyoma; Middle Aged; Myometrium; Polymerase Chain Reaction; RNA, Messenger; Uterine Neoplasms

Gestational trophoblastic neoplasms comprise the neoplastic spectrum of nonmalignant hydatidiform mole, invasive hydatidiform mole, and truly malignant choriocarcinoma. Increasing evidence indicates that epidermal growth factor (EGF) acts as an enhancer of trophoblast function to produce human chorionic gonadotropin and that EGF and its receptor may provide a growth advantage to certain carcinoma cells. The current study was undertaken to evaluate a possible link between malignant transformation of trophoblast and expression of EGF and EGF receptor.. Cytologic localization and cellular levels of expression of EGF and EGF receptor in hydatidiform mole, invasive hydatidiform mole, and choriocarcinoma tissue specimens were examined by the avidin-biotin immunoperoxidase techniques with monoclonal antibodies against EGF and EGF receptor.. EGF in hydatidiform mole and invasive mole was localized in syncytiotrophoblasts, whereas cytologic localization of EGF receptor in hydatidiform mole and invasive mole was observed in both cytotrophoblasts and syncytiotrophoblasts. By contrast, EGF and EGF receptor in choriocarcinoma were exhibited in cytotrophoblastic and syncytiotrophoblastic elements. Most (72%) hydatidiform moles immunostained intensely for EGF and EGF receptor, whereas most (78%) choriocarcinomas immunostained slightly for EGF and EGF receptor. Invasive mole occupied the middle position in the staining intensity for EGF and EGF receptor, between hydatidiform mole and choriocarcinoma, with 50% of the cases exhibiting moderate staining.. The simultaneous expression of EGF and EGF receptor in the neoplastic trophoblasts implies that EGF may act in an autocrine-paracrine manner in trophoblastic neoplasms. Furthermore, the results obtained suggest that cytologic expression of EGF and EGF receptor in trophoblastic neoplasms decreases in the malignant transformation of trophoblast.

Topics: Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Hydatidiform Mole; Hydatidiform Mole, Invasive; Immunoenzyme Techniques; Pregnancy; Staining and Labeling; Trophoblasts; Uterine Neoplasms

The cycle of phospholipid turnover has been found to be under the negative control of hormonal cytostatics (progesterone) and under the positive control of proliferation stimulants (17 beta-estradiol, epidermal growth factor). Specific changes in the synthesis of phospholipids are shown when tamoxiphen, an antiestrogen and an inhibitor of protein kinase C, was used. The findings suggest that changes in the turnover rate of phospholipids are one of the key stages of steroid action on target cells and may be regarded as an additional criterion of tumor genetic sensibility.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Protein Kinase C; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

Epidermal growth factor (EGF) elicits estrogen receptor (ER)-dependent physiological sequelae and estrogen-like biochemical effects on the ER in the mouse uterus. These in vivo observations indicate that EGF may elicit some of its actions by activation of the ER. The effect of peptide growth factors on activation of a consensus estrogen-responsive element was assessed in a strain of Ishikawa human endometrial adenocarcinoma cells with negligible levels of ERs, as determined by Western blot and [3H]estradiol binding, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ERs. EGF and transforming growth factor-alpha induced transcriptional activation of a consensus ERE in an ER-dependent manner in both cell types. Transcriptional activation by the growth factors was inhibited by ICI 164,384, an ER receptor antagonist, and neutralizing antibodies to the EGF receptor. Immunodetection of the ER in BG-1 cells demonstrated that receptor levels were not induced by transforming growth factor-alpha vs. untreated cells. ER deletion mutants containing amino acids 1-339 and 121-599 were transfected into Ishikawa cells. The 1-339 mutant was more active in inducing transcription after EGF treatment than the 121-599 mutant. Estrogen only stimulated transcription in the presence of the 121-599 mutant, while 1-339 was inactive. Interestingly, synergism between a physiological dose of estrogen and peptide growth factors was observed. The presence of cross-talk between EGF receptor and ER signaling pathways suggests that interactions between growth factors and steroid receptors may modulate hormonal activity influencing normal and aberrant function in mammalian cells.

Topics: Adenocarcinoma; Animals; Base Sequence; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Humans; Mice; Molecular Sequence Data; Ovarian Neoplasms; Polyunsaturated Alkamides; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Fusion Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Simian virus 40; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Neoplasms; Vitellogenins

Sex steroid hormone dependent cell proliferation and inducing growth factors of endometrial carcinoma cells were investigated using in vitro culture systems. The cell proliferation of Ishikawa cells derived from well-differentiated endometrial adenocarcinoma which possess both estrogen and progesterone receptors were stimulated by either estradiol added to culture media or EGF and TGF-alpha acting through EGF receptors. These stimulatory effects of TGF-alpha were antagonized by the anti TGF-alpha and EGF-receptor antibodies. The cell proliferations of other endometrial cancer cells were also inhibited by those antibodies. All endometrial cancer cells secrete TGF-alpha into their culture media measured by TGF-alpha ELISA methods. The expression of TGF-alpha mRNA and secretion of TGF-alpha of Ishikawa cells were induced by estradiol but not of hormone independent HEC-50 cells. Thus suggest that estradiol dependent growth factor should be TGF-alpha in human endometrial carcinoma cells.

Topics: Carcinoma, Endometrioid; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Humans; Neoplasms, Hormone-Dependent; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Neoplasms

The in vitro effects of the epidermal growth factor (EGF) and progesterone on phospholipid turnover in cells of 19 human adenocarcinomas (postsurgical material) have been studied. In 58% of tumours EGF increased the 32P incorporation into two basic cell phospholipids--phosphatidylcholine and phosphoinositides. In EGF-insensitive cells progesterone induced no noticeable changes in the basal level of phospholipid metabolism. However, in 10 out of 11 positively responding to EGF adenocarcinomas progesterone inhibited the EGF-dependent activation of 32P incorporation into the phospholipids already on the 15th min after its addition to the cells. Analysis of effects of EGF and the anti-estrogen drug tamoxifen on phospholipid turnover in 22 human mammary tumours did not reveal any significant differences in tamoxifen effect on tumour cells differing in their sensitivity to EGF. Independently of cell sensitivity to EGF, tamoxifen caused some decrease in the 32P incorporation into phosphatidylcholine but increased the label incorporation into phosphoinositides. Tamoxifen added to tumour cells prestimulated with EGF or 17 beta-estradiol failed to abrogate the effect of these compounds on phospholipid turnover. At the same time, treatment of cells with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate fully inhibited the effect of tamoxifen on phospholipid metabolism. The results obtained suggest that the EGF-dependent activation of intracellular phospholipid turnover is under the negative control of progesterone. As for tamoxifen, its effect on cells is independent of EGF and consists, apparently, in the inhibition of protein kinase C activity.

Topics: Adenocarcinoma; Breast Neoplasms; Chromatography, Thin Layer; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

The null hypothesis is that partial hydatidiform moles have normal differentiated function (human chorionic gonadotropin and human placental lactogen secretion) in response to epidermal growth factor and 8-bromo-cyclic adenosine monophosphate.. Two complete moles, 10 partial hydatidiform moles, and 19 normal first-trimester placentas in monolayer culture were exposed to 10 ng/ml epidermal growth factor, 1 mmol/L 8-bromo-cyclic adenosine monophosphate plus 1 mmol/L theophylline, or control. Human chorionic gonadotropin and human placental lactogen secretion was measured. Frequency of response to stimuli was compared by chi 2 analysis, and hormone secretion was compared by analysis of variance.. Partial moles demonstrated reduced frequencies of response of human chorionic gonadotropin and human placental lactogen to epidermal growth factor (partial moles 2/8 and 2/8, respectively; normal placentas 16/19 and 7/18, respectively; p less than 0.025) and of human chorionic gonadotropin to 8-bromo-cyclic adenosine monophosphate (partial moles 3/5, normal placentas 13/16; p less than 0.005).. Partial hydatidiform moles demonstrate impaired human chorionic gonadotropin and human placental lactogen secretory responsiveness to epidermal growth factor and cyclic nucleotides in comparison with normal first-trimester trophoblast.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Calcium-Binding Proteins; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Hydatidiform Mole; Immunoenzyme Techniques; Keratins; Placental Lactogen; Pregnancy; Tumor Cells, Cultured; Uterine Neoplasms; Vimentin

Exposure of RL95-2 human endometrial adenosquamous carcinoma cells of early passage (less than 30 passages) and late passage (greater than 250 passages) to epidermal growth factor (EGF) resulted in density- and concentration-dependent effects. At low seeding density, EGF (20 nM) inhibited the growth of early passage cells, whereas at high seeding density, 4.98 nM and 20 nM concentrations of EGF stimulated their growth. Furthermore, the growth of late passage cells was stimulated by 0.0166 nM EGF and inhibited by 4.98 nM and 20 nM EGF at both seeding densities. EGF (20 nM) caused marked morphological changes of both passages at the low seeding density. Inhibition of invasion of both passages through Matrigel-coated filters was seen at low seeding density, while at the high seeding density, EGF enhanced invasiveness. At high seeding density, EGF stimulated an increase in urokinase type plasminogen activator activity, which may have augmented the ability of cells to degrade the extracellular matrix. In addition, the ability of high seeding density cells of both passages to adhere to matrigel after EGF treatment correlated with invasiveness.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Tissue Plasminogen Activator; Tumor Cells, Cultured; Uterine Neoplasms

There is a paucity of data concerning the factors involved in the growth of uterine leiomyomata. The expression of mRNAs encoding EGF and the EGF receptor in myometrial and leiomyoma cultures suggest that EGF may be involved in the autocrine/paracrine regulation of human uterine leiomyomata and myometrial growth.

Topics: Amino Acid Sequence; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Leiomyoma; Molecular Sequence Data; Myometrium; Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

Insulin-like growth factor I (IGF-I) binding sites were characterized in normal and neoplastic endometrium. The characteristics of the endometrial IGF-I receptor are similar to those reported for other tissues. The binding of 125I-IGF-I to the endometrial membranes is saturable and time, temperature, and pH dependent. The 125I-IGF-I binding activity to the membranes obtained from differentiated and undifferentiated adenocarcinoma as well as sarcoma of the endometrium was significantly higher (P less than 0.05) when compared to the binding activity of the membranes obtained from normal endometrium. The Scatchard analysis of the competitive binding data of both normal and neoplastic endometrium revealed linear plots. This indicated a single class binding site for IGF-I with equilibrium dissociation constants (Kd) of 5.0, 6.8, 6.94, and 6.88 nM for normal, differentiated, and undifferentiated adenocarcinoma, and sarcoma of the endometrium, respectively. Therefore, the differences observed in 125I-IGF-I binding between normal and neoplastic endometrial membranes was due to an increase in the number of IGF-I binding sites and not to a change in receptor binding affinity. Autoradiograms from affinity labelling studies revealed a band corresponding to Mr 132,000 subunit of the receptor which is characteristic of the type I receptor reported for other tissues. A dimer of the alpha subunit (Mr 263,000) was also observed in all four categories of endometrial tissue. Additionally, autoradiograms obtained from sarcoma of the endometrium revealed a Mr 40,000 band that was only displaced by IGF-I and IGF-II peptides but not by the monoclonal antibody alpha IR-3 to the type I receptor. These suggest that the band is representative of the IGF-I or IGF-II binding protein. A similar band was not observed in the other tissues. The results show that the human endometrium contains high affinity IGF-I or IGF-II binding sites. The fact that IGF-I binding activity was significantly higher for neoplastic endometrium suggests that IGF-I may play an important role on supporting the growth of this neoplastic tissue.

Topics: Affinity Labels; Cell Membrane; Endometrium; Epidermal Growth Factor; Female; Humans; Hydrogen-Ion Concentration; Insulin; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Molecular Weight; Receptors, Cell Surface; Receptors, Somatomedin; Sarcoma; Uterine Neoplasms

Growth factors, including epidermal growth factor (EGF), have been implicated in the growth of several types of cancer. This study compares EGF receptors in normal and neoplastic endometrium. Membrane fractions were isolated from surgical specimens. Radioreceptor assays demonstrated the presence of receptors with a dissociation constant of 0.64 nmol/l in normal endometrium. Affinity cross-linking revealed receptor molecular weight of 150 to 170 kiloDaltons (KD). A survey of samples (n = 37) revealed progressive decrease of EGF receptors in cancers of increasing grade: Grade 1-2 adenocarcinoma decreased 34% from control (n = 6, P less than 0.01), whereas Grade 3 adenocarcinoma decreased 90% (n = 7, P less than 0.01) and sarcoma decreased by 72% (n = 3, P less than 0.01). The dissociation constant and molecular weight of the receptor in neoplastic endometrium did not differ significantly from normal. The inverse relationship with grade suggests receptor alteration or down regulation by hormones and/or growth factors.

Topics: Adenocarcinoma; Animals; Endometrium; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Molecular Weight; Uterine Neoplasms

Stearic acid and iodo-stearic and inhibited cell growth in a cervical cancer cell line (HOG-1) in a dose-related manner, with a half maximal effect at 50 microM stearic acid. Addition of oleic acid abrogated the effect of stearic acid. EGF-stimulated DNA synthesis and growth of HOG-1 cells was inhibited in the presence of stearic acid without any apparent effect on EGF receptor number or affinity.

Topics: Cell Division; Epidermal Growth Factor; Female; Humans; Stearic Acids; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Uterine Neoplasms

UM-EC-2 was established from a patient with poorly differentiated stage IB endometrial carcinoma. This cell line produces tumors in nude mice that have the same histological features as the patient's tumor. UM-EC-2 cells express b2-microglobulin, the epidermal growth factor receptor (EGF), and the H blood group antigen. This membrane antigen phenotype is consistent with cells of human endometrial origin. The karyotype of UM-EC-2 is fairly complex, with rearrangements affecting all chromosomes except 3, 10, 14, 19, and 20. There were two populations of cells, a hyperdiploid population with a modal number of 53-55 and a hypertetraploid population with a modal number of 109. A postulated sequence of events before and after tetraploidization is suggested based on the number of copies of individual chromosomes and rearrangements. Comparison of the UM-EC-2 karyotype to that of UM-EC-1 (a previously described line from a different patient with endometrial carcinoma) revealed that the two lines share eight very similar chromosome changes, which include loss of most of chromosome 4, breakpoints affecting proximal bands on 8p, loss of most of 9q, a breakpoint at 12q22, loss of 13q, breakpoints in proximal bands on 18q, and a breakpoint at 22p11. These changes may represent nonrandom chromosome abnormalities in poorly differentiated endometrial cancer. Estrogen (ER) and progesterone (PgR) receptors were not detected in either the primary tumor or the cell line. Nevertheless, UM-EC-2 cells were very sensitive to growth inhibition by tamoxifen (TAM) in vitro. One micromolar TAM caused 50% inhibition of cell growth, 2.5 microM caused cytostasis, and 5 microM TAM was cytotoxic, killing all cells after 5-7 days of exposure to the drug. Paradoxically, 100 nM estradiol (E2) caused a moderate increase in the growth of the cells but it did not prevent or reverse growth inhibitory effects of TAM. These findings support the concept that in some tumors TAM causes growth inhibition by an ER-independent mechanism. UM-EC-2 cells were also sensitive to growth regulation by EGF. Thus, these cells provide a new in vitro model of human endometrial cancer in which the roles of both TAM and EGF as growth regulatory substances can be investigated.

Topics: Antigens, Neoplasm; Antigens, Surface; Cell Division; Drug Resistance; Epidermal Growth Factor; Estradiol; Female; Humans; Karyotyping; Middle Aged; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

We examined the presence and characteristics of epidermal growth factor (EGF) receptors in hCG producing tumors (CC-2-JCK) transplanted in female nude mice. We also examined the in vivo effects of EGF on tumor growth. Specific receptors with apparent dissociation constants of 3.89 x 10(-10) and 1.0 x 10(-9) M and binding capacities of 5.96 x 10(-10) and 1.52 x 10(-9) M/mg protein for EGF have been identified in the hCG-producing tumor. [125I]EGF binding to the tumor tissues was time, temperature, and tissue weight dependent and specific. EGF and transforming growth factor-alpha (TGF alpha) competed for [125I]EGF binding, with 50% of the bound [125I]EGF displaced by approximately 0.52 nM EGF and 3.10 nM TGF alpha. TGF beta competed for [125I]EGF binding slightly. Five micrograms of EGF caused an increase in the rate of tumor growth, while 50 micrograms EGF strongly inhibited tumor growth. The concentration of [125I]EGF binding in the tumor treated with low doses of EGF was high, and that in the tumor treated with high doses of EGF was low. These changes in EGF binding were attributable to the changes in the number of high affinity EGF receptors with no significant alteration in binding affinity. In conclusion, the existence of high concentrations of EGF receptors with high affinity and specificity to EGF was demonstrated in an hCG-producing tumor transplanted in nude mice and appeared to be correlated with tumor growth.

Topics: Animals; Binding, Competitive; Cell Division; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Pregnancy; Transplantation, Heterologous; Uterine Neoplasms

The specific binding of epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin were measured in matching cultures of human leiomyoma and myometrial cells, along with the effects of these proteins on DNA and protein syntheses. Scatchard analyses of the binding data revealed that the EGF receptor sites/cell were significantly lower in leiomyoma than myometrial cultures. Two types of PDGF binding were observed when porcine PDGF was used, and one type was seen with human PDGF. By contrast to EGF, more PDGF receptor sites/cell were found in leiomyoma than myometrium but the receptor affinity was higher in the latter. Insulin binding was similar among the myometrial and leiomyoma cells. Protein synthesis was stimulated 3-fold by EGF, PDGF, or insulin in both cell types. DNA synthesis, was higher in myometrial than leiomyoma cells in the basal state and was stimulated by EGF, insulin, or PDGF. A synergistic stimulation (p less than 0.02) of DNA synthesis was observed in both myometrial and leiomyoma cells when EGF was added with insulin. The addition of PDGF with insulin caused only additive stimulation of DNA synthesis. However, the addition of EGF with PDGF caused a synergistic decrease (p less than 0.05) in DNA synthesis by myometrial but no leiomyoma cells. Cultures of human vascular smooth muscle cells obtained from umbilical veins gave results similar to those from myometrium. These findings single out the EGF receptor and EGF, or perhaps an EGF-like growth factor, and to a lesser degree PDGF, as potential regulators of uterine leiomyomata.

Topics: Adult; Analysis of Variance; Cell Division; Culture Techniques; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Insulin; Leiomyoma; Middle Aged; Platelet-Derived Growth Factor; Receptor, Insulin; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Regression Analysis; Tumor Cells, Cultured; Uterine Neoplasms

Topics: DNA, Neoplasm; Epidermal Growth Factor; Female; Humans; Lymphatic Metastasis; Neoplasm Invasiveness; Neoplasm Staging; Ploidies; Prognosis; Receptors, Cell Surface; Uterine Neoplasms

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxygenase; Phorbol Esters; Pregnancy; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

The precursor of cathepsin D, a lysosomal acidic protease, is secreted by human breast cancer cells, where its synthesis is specifically induced by estrogens and growth factors. In this study, we investigated the hormonal regulation of cathepsin D and its mRNA in uterine cells. In the Ishikawa endometrial cancer cell line, epidermal growth factor (EGF) increased the level of cathepsin D and its mRNA 2- to 3-fold. Although expression of the transiently transfected estrogen-responsive recombinant (Vit. tk. CAT) and the endogenous progesterone receptor was markedly increased by estradiol in Ishikawa cells, estradiol did not alter the level of cathepsin D or its mRNA. The progestin R5020 induced the expression of the LTR sp65 CAT, which contains the progesterone-responsive element of the MMTV but it too was without effect on cathepsin D. By contrast, the expression of cathepsin D gene, in normal rat uterus, was increased by R5020 but not by estradiol. We conclude that cathepsin D gene expression is regulated differently by sex steroid hormones in endometrial and breast cancer cell lines, whereas it is similarly induced by EGF in these cells.

Topics: Animals; Blotting, Northern; Breast Neoplasms; Cathepsin D; Cell Line; Epidermal Growth Factor; Estradiol; Female; Gene Expression Regulation; Gonadal Steroid Hormones; Humans; In Vitro Techniques; Promegestone; Rats; RNA, Messenger; Transfection; Uterine Neoplasms

In this study we investigated the presence of epidermal growth factor receptors (EGF-R) and the tissue levels of EGF-like factors (EGF-F) in ovarian, endometrial, cervical and breast carcinomas. EGF-R were found in 33/40 (83%) cervical, 15/26 (58%) endometrial, 64/141 (45%) ovarian, and 19/59 (33%) breast carcinomas. The highest number of EGF-R binding sites was detected in cervical carcinomas followed by endometrial, breast and ovarian carcinomas. The tissue concentrations of EGF-like factors, were investigated in extracts of 63 ovarian, 25 breast, 12 cervical, 14 endometrial carcinomas and in 21 biopsies of nonmalignant tissue such as myometrium and ovaries. The extracts of nonmalignant tissues had a mean EGF-F level of 1.5 +/- 0.7 ng/mg with a concentration range from 0 to 4 ng/mg. The mean EGF-F levels of malignant tissues were: ovarian carcinomas 4.2 +/- 1.5 ng/mg (range 0-15 ng), endometrial carcinomas 4.5 +/- 1.7 ng/mg (range 0-12 ng), cervical carcinomas 4.15 +/- 1.1 ng/mg (range 0-8) and breast carcinomas 3.16 +/- 1.1 ng/mg (range 0-10 ng). About 30% ovarian, endometrial and cervical carcinomas and 16% breast carcinomas, respectively, had enhanced EGF levels from 5 ng/mg to 15 ng/mg compared to nonmalignant tissues. The EGF-F of tissue extracts consists of EGF and transforming growth factor TGF alpha) as shown by the results of EGF and TGF alpha radioimmunoassays. It is assumed that in some tumors the EGF-F tissue levels influence the number of biochemically detectable EGF binding sites.

Topics: Binding Sites; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Prognosis; Radioimmunoassay; Transforming Growth Factors; Uterine Cervical Neoplasms; Uterine Neoplasms

Samples of uterine myometrium and leiomyoma from 11 women were analyzed for the presence of epidermal growth factor receptors and insulin-like growth factor I receptors. In addition, the content of soluble insulin-like growth factor binding protein (IGF-BP/PP12) was measured in the tissue cytosols. Cell membrane preparations of myoma tissue bound significantly more insulin-like growth factor I than did those of adjacent normal myometrium, whereas myoma tissue bound less epidermal growth factor than did the normal myometrium. The differences in both insulin-like growth factor I and epidermal growth factor binding were due to changes in receptor concentration rather than to alterations in receptor affinity. Neither myoma nor myometrial tissue contained detectable levels of insulin-like growth factor binding protein. The changes in epidermal growth factor and insulin-like growth factor I binding to the myometrium may play a role in the pathogenesis of uterine leiomyomata.

Topics: Adult; Binding Sites; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; Iodine Radioisotopes; Leiomyoma; Middle Aged; Myometrium; Somatomedins; Uterine Neoplasms

The aim of the present work was to improve quantitative assessment of angiogenesis by chick chorioallantoic membrane (CAM) assay based on measurement of DNA synthesis. Following incubation of [3H]thymidine (3H-T) with CAM in vivo, incorporation of 3H-T to DNA fraction was expressed as percent of total 3H-T present in the initial homogenate of CAM, regardless of CAM weight and full recovery of applied radioactivity. The assay required simple, partial isolation of DNA to remove traces of free or unspecifically bound 3H-T. These modifications gave a reproducible assay with adequate precision (10-20%). DNA content of CAM did not change between 10-15 days of embryo development and growth of CAM was completed on day 11. 10 to 12-day old CAMs were used for evaluation of the assay. Using a qualitative approach (visual scoring), tissues of several tumors were implanted on CAM. Extracts of tumors that produced the highest score stimulated DNA synthesis in CAM. A similar effect was induced by EGF while cytostatics inhibited DNA synthesis in CAM. Since stimulation of angiogenesis is not a cell type-specific phenomenon, the assay in the present modification should aid the studies on angiogenic factors. With the help of this assay the angiogenic activity of adenocarcinoma of human endometrium was described.

Topics: Allantois; Animals; Chick Embryo; Chorion; DNA; Epidermal Growth Factor; Extraembryonic Membranes; Female; Meningioma; Neovascularization, Pathologic; Thymidine; Tissue Extracts; Uterine Neoplasms

The binding of epidermal growth factor (EGF) to human myometrium and leiomyomata was assessed in a group of women rendered hypo-oestrogenic with the LHRH agonist Zoladex (ICI 118630). The results were compared with those obtained with tissues from women with normal cycles. In normal women, the specific binding of radiolabelled [125I] EGF to both myometrial and fibroid homogenates did not vary during the menstrual cycle, but the specific binding of [125I] EGF to fibroid in women treated with LHRH agonist was significantly less than in the untreated group. Since the hypo-oestrogenic state induced by the agonist is associated with a decrease in fibroid size, the results suggest that the effect of oestrogen on fibroid tissue may partly be mediated by EGF.

Topics: Adult; Buserelin; Epidermal Growth Factor; Estradiol; Female; Goserelin; Humans; Leiomyoma; Menstrual Cycle; Myometrium; Uterine Neoplasms

We determined the concentrations of immunoreactive epidermal growth factor in the urine (U-irEGF) of 97 adult patients with various malignancies, including carcinomas of the urinary bladder, kidney, stomach, colon, rectum, breast, endometrium, uterine cervix, ovary, vagina, prostate, pancreas and thyroid, liposarcoma and skin melanoma. The relative U-irEGF concentrations (ng m-1 creatinine) were higher (P = 0.002) for the whole series of female patients than for healthy controls matched for sex and age. Such difference did not appear for male patients. The only specific group with a statistically supranormal U-irEGF concentration (P = 0.0005) comprised women with endometrial carcinoma of the uterus.

Topics: Adult; Aged; Aged, 80 and over; Creatinine; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Neoplasms; Sex Factors; Uterine Neoplasms

The effects of cholera toxin (CT), which stimulates adenylate cyclase, 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on progesterone (P) and estradiol (E2) secretion by human choriocarcinoma JEG-3 cells were studied. During a 48 h incubation, CT, TPA and EGF stimulated P production in a concentration-dependent manner, whereas IGF-I was without effect. CT (1.0 ng/ml), TPA (10 ng/ml) and EGF (10 ng/ml) stimulated P production maximally 4.3-, 3.3- and 2.3-fold over basal, respectively. When added together with CT, TPA and EGF stimulated P production 10.0- and 5.0-fold over basal production showing that the effects of CT plus TPA were more than additive but those of CT plus EGF less than additive. Time-course studies indicated that the effects were detectable at 12 h, and continued to increase up to 48 h. The conversion of added dehydroepiandrosterone sulfate (DHEAS) to E2 was stimulated by CT and TPA and inhibited by IGF-I in a concentration-dependent manner. By contrast, EGF had no effect. The maximal responses in E2 production were 3.2- and 2.0-fold over unstimulated cells by CT (1.0 ng/ml) and TPA (10 ng/ml), respectively. When both agents were added together, their effects on E2 production were additive with 5.5-fold increase over unstimulated cells. IGF-I (30 ng/ml) inhibited maximally basal and CT-stimulated E2 production by 33% and 42%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cholera Toxin; Choriocarcinoma; Epidermal Growth Factor; Estradiol; Female; Humans; Insulin-Like Growth Factor I; Phorbol Esters; Placental Hormones; Pregnancy; Progesterone; Somatomedins; Tumor Cells, Cultured; Uterine Neoplasms

The effects of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and epidermal growth factor (EGF) on the synthesis and secretion of human chorionic gonadotropin (hCG) and its subunits by normal and malignant trophoblasts as well as by non-trophoblastic cells were investigated in vitro. The explants of normal early placental tissues, choriocarcinoma cell line BeWo and non-trophoblastic tumor cell line CaSki from epidermoid carcinoma of the cervix, respectively, were cultured in the presence or absence of dibutyryl cAMP or EGF. The addition of either dibutyryl cAMP (1 mM) or EGF (100 ng/ml) caused significant increases in the synthesis and secretion of hCG and its subunits in cultures of normal and malignant trophoblasts, but had no stimulatory effect on hCG beta synthesis and secretion in culture of non-trophoblastic cell line CaSki that secretes predominantly hCG beta-like material. The magnitude of the stimulatory effects of dibutyryl cAMP and EGF on hCG (alpha,beta) synthesis and secretion by BeWo cells was much greater than that observed in normal trophoblasts. The time course of these stimulatory effects indicated that EGF-stimulated increase in hCG synthesis and secretion required a lag period longer than that for the dibutyryl cAMP-stimulated increase. These results suggest that there were no differences in normal and malignant trophoblasts in the mechanism for the stimulatory regulation of hCG (alpha, beta) synthesis and secretion, but immunoreactive hCG beta synthesis and secretion in non-trophoblastic tumor cells are regulated by a mechanism different from that in trophoblastic cells.

Topics: Bucladesine; Carcinoma, Squamous Cell; Cell Line; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Pregnancy; Trophoblastic Neoplasms; Uterine Cervical Neoplasms; Uterine Neoplasms

Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF.

Topics: Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Methylamines; Peptides; Transforming Growth Factors; Uterine Neoplasms

Specific epidermal growth factor (EGF) receptors were measured in RL95-2 human endometrial carcinoma cells. At 37 C, binding of 125I-labeled EGF was associated with marked ligand internalization. Maximal cell surface binding occurred within 10 min. Bound [125I]EGF was partially degraded to low mol wt products, and this degradation was blocked by the lysosomotropic compound ammonium chloride. At 4 C, maximal cell surface binding occurred at 3 h. Scatchard analysis of data obtained after 3 h at 4 C revealed a single order of binding sites with a Kd of 1.8 nM and approximately 150,000 surface receptors/cell. EGF, at a concentration of 0.83 nM, inhibited the proliferation of RL95-2 cells. Inhibition was reversible and was associated with morphological changes leading to a fusiform appearance of the growth-arrested cells. These findings indicate that RL95-2 human endometrial carcinoma cells bind, internalize, and degrade EGF in a manner similar to that described for other target cells for EGF action. The ability of EGF to inhibit the growth of RL95-2 cells supports the hypothesis that this type of inhibition is dependent on postreceptor events, rather than on the presence of an overabundance of EGF receptors.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Receptors, Cell Surface; Temperature; Uterine Neoplasms

Epidermal growth factor (EGF) binds specifically (Ka = 4 X 10(9) M-1; 1.3 X 10(11) receptors/mg cellular protein) to JEG-3 cells, which respond in the succeeding 24 h by a 400% increase in hCG secretion without a significant change in cell number. Since JEG-3 cells store less than 2% of the 24-h hCG secretion, a significant increase in hCG in the culture medium reflects increased hCG biosynthesis. It is observed that a tumor promoter, phorbol myristate acetate (PMA), binds specifically to the cells (Ka = 5 X 10(8) M-1; 3.9 X 10(11) receptors/mg), reduces (33%) the affinity but not the number of EGF receptors, and stimulates hCG to the same extent as does EGF. The relative potencies of PMA (100%), phorbol dibutyrate (25%), and nonesterified phorbol (no effect) to stimulate hCG parallel their known tumor-promoting activities. Since phospholipids and fatty acids have been implicated in the mechanism of action of EGF and PMA, the effects of arachidonic acid (AA) and inhibitors of its metabolism were studied. The addition of AA had no effect on basal or PMA-stimulated hCG secretion, but it potentiated the effect of EGF by 200%. Indomethacin (a cyclooxygenase inhibitor) had an effect similar to that of AA. Nordihydroguairetic acid (a cyclooxygenase and lipoxygenase inhibitor) reduced by 90% basal and EGF- or PMA-stimulated hCG secretion. These results indicate that the AA pathway can influence the effects of EGF and PMA on hCG secretion and suggest an intermediary role for the lipoxygenase system.

Topics: Arachidonic Acid; Arachidonic Acids; Caenorhabditis elegans Proteins; Carrier Proteins; Catechols; Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Indomethacin; Masoprocol; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pregnancy; Protein Kinase C; Receptors, Cell Surface; Receptors, Drug; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Uterine Neoplasms

Both 12-O-tetradecanoyl-phorbol-1- acetate and teleocidin B stimulated the secretion of human chorionic gonadotropin by cultured choriocarcinoma cells. These tumor promoters also stimulated production of progesterone in the cells. However, the 2 tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor that also had a stimulatory effect on production of these hormones.

Topics: Alkaloids; Animals; Carcinogens; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Kinetics; Lyngbya Toxins; Male; Mice; Phorbols; Pregnancy; Progesterone; Tetradecanoylphorbol Acetate; Uterine Neoplasms

A new human functional tumor cell line, designated as T3M-3, has been established from a xenotransplanted choriocarcinoma grown in nude mice. One of the biggest problems of the in vitro culture of these tumor cells using the xenotransplanted tumors had been the dense contamination of fibroblasts of host nude mouse origin. In the present study, these fibroblasts were completely removed by incubating the cells with antiserum raised against nude mouse spleen cells. The cell line established from the remaining tumor cells has been successfully propagated in vitro for as long as 4 years. These cells show the morphology of epithelioid cells containing a prominent nucleus with one or two large nucleoli. The cells grow in a monolayered sheet with the population-doubling time of 19 hr. The cells show perfect tumor takes when they are reinoculated into nude mice. Chromosomal analysis revealed that the cell is a human aneuploid one with a hypotriploid mode. These cultured cells maintained well the function of secreting large amounts of human chorionic gonadotropin, progesterone, and estrogen. The secretion of human chorionic gonadotropin and progesterone by these cells is enhanced by stimulation with tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate and teleocidin B, or with epidermal growth factor in a dose-and time-dependent manner. Interestingly, however, the tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor, indicating that the receptors for these reagents in T3M-3 cells are not shared by epidermal growth factor.

Topics: Animals; Carcinogens; Cell Division; Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Pregnancy; Uterine Neoplasms

Using human trophoblastic (SCH) and nontrophoblastic (HeLa S3) tumour cell lines, specific binding sites for epidermal growth factor (EGF), a potent stimulator of growth in many tissues, and its effect on secretion of human chorionic gonadotrophin (hCG) and/or its subunits were compared between these two tumour cells. Both SCH and HeLa S3 cells possessed two populations of specific binding sites for 125I-labelled EGF: the high affinity (Kd approximately 10(-10) M) and the low affinity (Kd approximately 7 x 10(-10) M) system. Tetradecanoyl phorbol acetate (TPA), a tumour promotor, showed a potent competitor of labelled tracer binding to its receptor sites in both cell lines. EGF stimulated both hCG-alpha and hCG and/or hCG-beta secretion in a dose-responsive manner from SCH cells, whereas it had no effect on hCG-alpha secretion from HeLa S3 cells. In contrast, dibutyryl cyclic AMP plus theophylline, a phosphodiesterase inhibitor, enhanced hCG-alpha secretion from both cells, while TPA had no effect in either cells. These data suggest that EGF may play a physiological role in hCG secretion from trophoblastic tissues and that the mechanism by which hCG and/or its subunits are secreted may differ between trophoblastic and non-trophoblastic tumour cells.

Topics: Bucladesine; Cell Line; Chorionic Gonadotropin; Epidermal Growth Factor; ErbB Receptors; Female; HeLa Cells; Humans; Male; Middle Aged; Pregnancy; Receptors, Cell Surface; Theophylline; Trophoblastic Neoplasms; Uterine Neoplasms

The cultured human choriocarcinoma JEG-3 cells secrete biologically active HCG and free HCG alpha-subunit. When compared with the alpha-subunit dissociated from HCG obtained either from pregnancy urine or JEG-3 cells, free alpha-subunit has a larger molecular weight, is more acidic and is non-functional, lacking the property to recombine with the HCG beta-subunit. The understanding of the biochemical differences observed between free alpha-subunit and alpha-subunit found in HCG is important and should help to unravel the biosynthesis of gonadotrophins. Two proteins which bind to the cell membrane, epidermal growth factor and concanavalin A, are capable of stimulating JEG-3 cell secretion. Epidermal growth factor stimulates the secretion of HCG while concanavalin A stimulates both HCG and HCG alpha-subunit secretion. Amphotericin B, an antifungal agent commonly used in tissue cultures, which also affects the cell membrane, was shown to stimulate HCG and HCG alpha-subunit secretions. The use of these agents should contribute to the understanding of membrane-related events which lead to the secretion of HCG and alpha-subunit.

Topics: Amphotericin B; Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Concanavalin A; Epidermal Growth Factor; Female; Humans; Pregnancy; Uterine Neoplasms

The ability of epidermal growth factor (EGF) to modulate the secretion of human chorionic gonadotropin (hCG) in both normal and malignant placental cells was compared. Receptors for EGF were present on the JAr line of choriocarcinoma cells and were localized to the trophoblast cells of normal placental organ cultures as detected by immunofluorescence. Despite the presence of EGF receptors, the normal placenta did not respond to EGF by significantly increasing its levels of hCG production. The JAr line of choriocarcinoma exhibited a 2-fold increase in hCG secretion after the addition of EGF. EGF stimulated growth in the JAr cells, as measured by the protein content of the cultures, but did not elevate the incorporation of [methyl-3H]thymidine in either the JAr cells or placental organ cultures.

Topics: Cell Line; Choriocarcinoma; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Humans; Organ Culture Techniques; Placenta; Pregnancy; Uterine Neoplasms

Epidermal growth factor (EGF) stimulates the production of progesterone by JEG-3, a clonal strain of human choriocarcinoma cells. Stimulation occurs in a time and dose-dependent manner. In addition, EGF increases [14C]-acetate incorporation into [14C]-cholesterol in JEG-3 cells, and this may constitute its mechanism of action in enhancing progesterone synthesis.

Topics: Animals; Cell Line; Choriocarcinoma; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Kinetics; Mice; Peptides; Pregnancy; Progesterone; Uterine Neoplasms

Epidermal growth factor (EGF) binds to JEG-3 cells, a tissue culture line of human choriocarcinoma. EGF also stimulates secretion of human chorionic gonadotropin (hCG) and to a lesser extent the secretion of free hCG-alpha.

Topics: Cells, Cultured; Choriocarcinoma; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Peptide Fragments; Peptides; Pregnancy; Uterine Neoplasms