Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.     

Effects of progesterone on uterine leiomyoma growth and apoptosis

Uterine leiomyomas appear during the reproductive years and regress after menopause, indicating the ovarian steroid-dependent growth potential. Recently we have found that the use of levonorgestrel-releasing intrauterine system (IUS) is effective in the long-term contraception and management of menorrhagic women with uterine myomas because of a striking reduction in menorrhagia. These clinical experiences prompted us to characterize the effects of progestin on the proliferation and apoptosis of leiomyoma cells cultured in vitro. 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 E(2) (10 ng/ml) or 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. In contrast, E(2) treatment augmented EGF-R expression in cultured leiomyoma cells, but P(4) did not. These results indicate that P(4) up-regulates the expression of PCNA and EGF in leiomyoma cells, whereas E(2) up-regulates 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, suggesting that the abundant expression of Bcl-2 protein in leiomyoma cells may be one of the molecular bases for the enhanced growth of leiomyoma relative to that of normal myometrium in the uterus. Furthermore, Bcl-2 protein expression in leiomyoma cells was up-regulated by P(4), but down-regulated by E(2). Therefore, it seems likely that P(4) may also participate in leiomyoma growth through the induction of Bcl-2 protein in leiomyoma cells.     

Preoperative administration of GnRH-a plus tibolone to premenopausal women with uterine fibroids: evaluation of the clinical response, the immunohistochemical expression of PDGF, bFGF and VEGF and the vascular pattern

Aim of the study is to compare the effects of preoperative therapy with tibolone plus gonadotropin-releasing hormone analogue (GnRH-a) in premenopausal women with those of GnRH-a alone on clinical response, uterine volume, immunohistochemical expression of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and vascular features of myomas. Seventy women with symptomatic uterine fibromatosis were treated for four months with leuprorelin acetate alone or plus tibolone. Untreated patients were submitted to uterine surgery directly. Uterine volume, hematological data, BMD, myoma-related symptoms and hot flushes were evaluated at the admission and before surgery. Immunohistochemical expression of PDGF, bFGF and VEGF, vascular changes and CD105 expression, as a marker of angiogenesis, were evaluated in myomas obtained after surgery. Uterine volume and myoma-related symptoms reduced and hematological variables increased in treated patients. BMD decreased in patients treated with GnRH-a alone. Hot flushes were less in GnRH-a plus tibolone group than in GnRH-a group. Immunohistochemical expression of PDGF, bFGF and VEGF, vascularization and angiogenesis reduced in treated patients in comparison with untreated ones. In conclusion, the administration of tibolone plus GnRH-a before uterine surgery does not change the clinical and immunohistochemical effects of GnRH-a alone.     

An accumulation of insulin-like growth factor I (IGF-I) in human myometrium and uterine leiomyomas in various stages of tumour growth

It is commonly thought that uterine leiomyomas result from hyperstimulation of myometrium by ovarian hormones. Some observations suggest that cytokines and growth factors are intermediate elements through which the ovarian hormones may exert their growth-stimulatory effects on leiomyomas. Human myometrium and uterine leiomyomas of various weights were homogenised and extracted with 1 M acetic acid or with 0.05 M Tris/HCl, pH 7.6. The extracts were assayed for IGF-I using the ELISA technique. It was found that 0.05 M Tris/HCl extracts contained several times more IGF-I than the 1 M acetic acid extracts. Nanogram amounts of IGF-I were found in both control myometrium and in leiomyomas. It was found that the amounts of IGF-I extracted from leiomyomas were distinctly higher in comparison to control myometrium and they increased as a function of tumour growth. Polyacrylamide gel electrophoresis, followed by Western immunoblotting, demonstrated that IGF-I in acidic and alkaline extracts exists as stable complexes, probably with extracellular matrix components. No free IGF-I was detected. Furthermore, it was found that some components of both the acidic and alkaline extracts were able to bind exogenous (125)I-labeled IGF-I. It is suggested that IGF-I plays an important role both in myometrium biology and in the growth of uterine leiomyomas.     

Fibroblast growth factors (FGF) in human myometrium and uterine leiomyomas in various stages of tumour growth

We previously described that the growth of human uterine leiomyomas was associated with a significant remodelling of the extracellular matrix of these tumours. Significant weight-related increase of collagen and heparan sulphate contents was detected. The latter was known as a component, which bound some peptide growth factors, mainly FGFs, therefore it was decided to evaluate the amounts of acidic FGF (aFGF) and basic FGF (bFGF) in human myometrium and in leiomyomas of various weight and FGF-binding to tissue components. It was found that myometrium and uterine leiomyomas contain picogram amount of aFGF and nanogram amounts of bFGF. No free aFGF was found. Slight amounts of free bFGF were detected both in myometrium and in the tumours. The aFGF and most of bFGF existed in a form of complex with a high molecular component(s). These complexes were very stable and they did not dissociate in denaturation conditions. In comparison to myometrium the tumours contained several times more FGFs and their amounts distinctly increased during the tumour growth. The expression of FGF-receptor I (FGF RI) in the tumours was more distinct in comparison to myometrium. The extracts from myometrium did not bind exogenous 125I-bFGF. In contrast to that the tumours of different weights contained at least two high molecular weight FGF-binding components. One of them (150 kDa) corresponded to FGF-receptor. The other one (190-200 kDa) might be a heparan sulphate-proteoglycan. It seems that aFGF and bFGF play an important role in transformation of normal myometrium into leiomyoma and further growth of this tumour. The action of FGFs on tumour cells enhances biosynthesis of collagen and sulphated glycosaminoglycans, especially heparan sulphate which binds FGFs in the vicinity of cells and facilitates their interaction with membrane receptors. The effect of these processes may be further stimulation of tumour growth and remodelling of tumour extracellular matrix.     

Altered hormonal responsiveness of proliferation and apoptosis during myometrial maturation and the development of uterine leiomyomas in the rat

Uterine leiomyomas are responsive to the ovarian steroids, estrogen and progesterone; however, a mechanistic understanding of the role of these hormones in the development of this common gynecologic lesion remains to be elucidated. We have used the Eker rat uterine leiomyoma model to investigate how ovarian hormones regulate or promote the growth of these tumors. Proliferative and apoptotic rates were quantitated in normal uterine tissues and leiomyomas in response to endogenous ovarian steroids. In 2- to 4-mo-old animals, cell proliferation in the normal uterus corresponded with high serum levels of steroid hormones during the estrous cycle, and apoptosis occurred in the rat uterus in all cell types following sharp, cyclical declines in serum hormone levels. It is interesting that the responsiveness of uterine mesenchymal cells changed between 4 and 6 mo of age, with significant decreases in both proliferative and apoptotic rates observed in myometrial and stromal cells of cycling animals. Leiomyomas displayed much higher levels of proliferation than did age-matched myometrium; however, their apoptotic index was significantly decreased in comparison with normal myometrium. This disregulation between proliferative and apoptotic responses, which were tightly regulated during ovarian cycling in the normal myometrium, may contribute to the disruption of tissue homeostasis and underlie neoplastic growth of these tumors.     

Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma

The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of alpha(s), alphai1/2, alphai3 and alphai0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in alphai3 and alphaio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues.     

Elastin distribution in the normal uterus, uterine leiomyomas, adenomyosis and adenomyomas: a comparison

OBJECTIVE: To describe the histologic distribution of elastin in the nonpregnant human uterus, uterine leiomyomas, adenomyosis and adenomyomas. STUDY DESIGN: Uteri were obtained from women undergoing hysterectomy for benign conditions, including 26 cases of uterine leiomyomas, 24 cases of adenomyosis, 18 adenomyomas and 6 cases of autopsy specimens. Specific histochemical staining techniques were employed in order to demonstrate the distribution of elastin. RESULTS: The distribution of elastin components in the uterus was markedly uneven and showed a decreasing gradient from outer to inner myometrium. No elastin was present within leiomyomas, adenomyomas or adenomyosis. CONCLUSION: The distribution of elastin may help explain the normal function of the myometrium in labor. It implies that the uneven distribution of elastin components and absence of elastin within leiomyomas, adenomyomas and adenomyosis could be of some clinical significance. The altered elastin distribution in disease states may help explain such symptoms as dysmenorrhea in uterine endometriosis.     

miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.     

Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells

When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.     

Binding of 125I-epidermal growth factor in human uterus

Summary Quantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with ¹²⁵I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Effects of tumor necrosis factor and epidermal growth factor on cell morphology, cell surface receptors, and the production of tissue inhibitor of metalloproteinases and IL-6 in human microvascular endothelial cells

The effect of human TNF on cultured human microvascular endothelial (HME) cells was examined. Incubation with TNF alone transformed the morphology of HME cells from a cobblestone-like appearance into a disordered array of criss-crossed, elongated, spindle-shaped cells. Coadministration of epidermal growth factor (EGF) and TNF caused even more dramatic morphologic changes than TNF alone. Addition of basic fibroblast growth factor or insulin-like growth factor-I showed rather weak effects on cell morphology than EGF. Cell growth of HME cells was stimulated up to two-fold by TNF whereas addition of EGF additively enhanced the growth rate. Treatment of HME cells with 10 ng/ml EGF increased the binding of 125I-TNF, and Scatchard analysis showed increased TNF-R number by EGF treatment. Cellular response to TNF in the absence or presence of EGF was assessed by analyzing SDS-PAGE patterns of secreted proteins from HME cells. TNF enhanced the secretion of a protein of molecular weight 25,000 Da (25 kDa) which was found to be IL-6. In contrast, secretion of a polypeptide of 29 kDa was significantly increased when HME cells were treated with EGF, but not with TNF. Coadministration of TNF and EGF synergistically increased the secretion of the 29-kDa protein. This 29-kDa protein was found to be tissue inhibitor of metalloproteinases when assayed with antitissue inhibitor of metalloproteinases antibody. TNF and EGF also enhanced secretion of collagenase with Mr of approximately 55 kDa. Increased steady state levels of the inhibitor mRNA were observed when HME cells were treated with EGF, and coadministration of TNF further increased the levels. The morphologic transformation of HME cells by TNF and/or EGF is discussed in relation to their expression of the secreted proteins.     

Epidermal growth factor-induced alterations in proliferating mouse epithelial cells

The effects of epidermal growth factor (EGF) on the growth and morphology of mouse embryo epithelial cells (MMC-E) were studied in culture. Growing cultures of epithelial cells were incubated in the media containing EGF or certain other mitogenic peptides. It was found that nanogram (ng) quantities of EGF stimulated growth in these cells and caused reversible phenotypic changes in these cells. These changes were not observed in cultures treated with the other mitogens. The compact growing islands of MMC-E cells were surrounded by elongated border cells [12]. EGF induced the elongated border cells to flatten and spread. The change of the elongated border cells into polygonal, flattened cells was dependent on the dose of EGF. After treatment with higher concentrations of EGF all cells appeared more flattened and their cytoplasm was more granular than that of the controls. Scanning electron microscopic studies (SEM) showed that the elongated border cells in the control cultures were distinctly higher than the cells inside the islands, while after exposure to EGF they flattened and had fewer surface microvilli than control cells. When EGF was removed and the cells were further cultivated in media without EGF, the border cells became smaller and elongated, eventually resembling those in the control cultures. These results show that EGF may act as a regulatory factor in the control of the proliferation and differentiation of mouse epithelial cells.     

Epithelial growth factor-induced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8-10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90-95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.     

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor

The capacity of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr = 125,000 amino-terminally truncated degradation product from the mature EGF receptor (EGF-dependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGF-alpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [125I]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [125I]-EGF binding after TGF-alpha treatment did not appear to require protein synthesis. Tyrosine phosphorylation of EGF receptor from cells treated with TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpha allows receptor recycling prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing.