cyclin-d1 and Leiomyoma

To investigate the link between EZH2 and Wnt/β-catenin signaling and its role in uterine fibroids (UFs) pathogenesis and explore the potential effect of natural compound methyl jasmonate (MJ) against UFs.. EZH2 overexpression or inhibition was achieved in human uterine leiomyoma (HuLM) cells using EZH2-expressing adenovirus or chemical EZH2 inhibitor (DZNep), respectively. The HuLM and normal uterine smooth muscle cells were treated with 0.1-3 mM of MJ, and several experiments were employed.. Laboratory study.. None.. Methyl jasmonate.. Protein expression of EZH2, β-catenin, and proliferating cell nuclear antigen (PCNA) was measured by Western blot as well as gene expression alterations of Wnt ligands (Wnt5A, Wnt5b, and Wnt9A), WISP1, CTNNB1, and its responsive gene PITX2 using quantitative real-time polymerase chain reaction. The protein and ribonucleic acid (RNA) levels of several markers were measured in MJ-treated or untreated HuLM cells, including EZH2 and β-catenin, extracellular matrix markers collagen type 1 (COL1A1) and fibronectin (FN), proliferation markers cyclin D1 (CCND1) and PCNA, tumor suppressor marker p21, and apoptotic markers (BAX, cytochrome c, and cleaved caspase 3).. EZH2 overexpression significantly increased the gene expression of several Wnt ligands (PITX2, WISP1, WNT5A, WNT5B, and WNT9A), which increased nuclear translocation of β-catenin and PCNA and eventually HuLM cell proliferation. EZH2 inhibition blocked Wnt/β-catenin signaling activation where the aforementioned genes significantly decreased as well as PCNA, cyclin D1, and PITX2 protein expression compared with those in untreated HuLM. Methyl jasmonate showed a potent antiproliferative effect on HuLM cells in a dose- and time-dependent manner. Interestingly, the dose range (0.1-0.5 mM) showed a selective growth inhibitory effect on HuLM cells, not on normal uterine smooth muscle cells. Methyl jasmonate treatment at 0.5 mM for 24 hours significantly decreased both protein and RNA levels of EZH2, β-catenin, COL1A1, FN, CCND1, PCNA, WISP1, and PITX2 but increased the protein levels of p21, BAX, cytochrome, c and cleaved caspase 3 compared with untreated HuLM. Methyl jasmonate-treated cells exhibited down-regulation in the RNA expression of 36 genes, including CTNNB1, CCND1, Wnt5A, Wnt5B, and Wnt9A, and up-regulation in the expression of 34 genes, including Wnt antagonist genes WIF1, PRICKlE1, and DKK1 compared with control, confirming the quantitative real-time polymerase chain reaction results.. Our studies provide a novel link between EZH2 and the Wnt/β-catenin signaling pathway in UFs. Targeting EZH2 with MJ interferes with the activation of wnt/β-catenin signaling in our model. Methyl jasmonate may offer a promising therapeutic option as a nonhormonal and cost-effective treatment against UFs with favorable clinical utility, pending proven safe and efficient in human clinical trials.

Topics: bcl-2-Associated X Protein; beta Catenin; Caspase 3; Cyclin D1; Enhancer of Zeste Homolog 2 Protein; Female; Humans; Leiomyoma; Ligands; Proliferating Cell Nuclear Antigen; RNA; Uterine Neoplasms; Wnt Signaling Pathway

Diagnostic criteria, biological behavior, and treatment approaches of leiomyosarcomas (LMS) may differ according to the origin of the tumor. This is important in terms of patient's management, especially in tumors located in the peritoneum and retroperitoneal sites. In our study, we aimed to demonstrate the immunophenotypic characteristics of uterine and extra-uterine LMS using a large antibody panel, and to determine whether they potentially play a role in the differences among these tumor groups. Between 2006 and 2018, 29 uterine and 42 extra-uterine primary LMS were included in this study. Using tissue samples taken from the areas that best represented the tumor, an immunohistochemical study was performed on the blocks prepared by tissue micro-array method with estrogen and progesterone receptor (PR), WT-1, SMA, desmin, caldesmon, calponin, p16, p53, MDM2, CDK4, bcl-2, cyclin D1, fascin, EMMPRIN, FOXM1, c-erb-B2, c-Myc, PAX8, and CD117. Staining results of uterine and extra-uterine LMS were evaluated with these 20 antibodies. In uterine LMS compared with extra-uterine LMS, estrogen receptor (48% vs. 12%), PR (62% vs. 21%), desmin (79% vs. 50%), and EMMPRIN (69% vs. 45%) staining rate was detected higher. In extra-uterine LMS, caldesmon (88% vs. 69%), c-Myc (33% vs. 10%), and cyclin D1 (52% vs. 28%) were stained higher than uterine LMS (p < 0.05). No significant staining difference was detected with other antibodies. We concluded that estrogen receptor, PR, desmin, EMMPRIN, caldesmon, c-Myc, and cyclin D1 antibodies may help to determine primary origin of the tumor in LMS cases.

Topics: Basigin; Biomarkers, Tumor; Calmodulin-Binding Proteins; Cyclin D1; Desmin; Female; Humans; Leiomyoma; Leiomyosarcoma; Receptors, Estrogen; Uterine Neoplasms

Cadmium (Cd) is a toxic metal reported to act as an estrogen "mimic" in the rat uterus and in vitro. We have reported that Cd stimulates proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM; fibroid) cells through nongenomic signaling involving the G protein-coupled estrogen receptor (GPER), with activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (pMAPK44/42). In this study, we explored Cd-induced mechanisms downstream of MAPK and whether Cd could stimulate phosphorylation of Histone H3 at serine 10 (H3Ser10ph) through activated Aurora B kinase (pAurora B), a kinase important in activation of histone H3 at serine 10 during mitosis, and if this occurs via Fork head box M1 (FOXM1) and cyclin D1 immediately downstream of MAPK. We found that Cd increased proliferating cell nuclear antigen (PCNA) and H3Ser10ph expression by immunofluorescence, and that H3ser10ph and pAurora B were coexpressed along the metaphase plate in ht-UtLM cells. In addition, Cd-exposed cells showed higher expression of pMAPK44/42, FOXM1, pAurora B, H3ser10ph, and Cyclin D1 by western blotting. Immunoprecipitation and proximity ligation assays further indicated an association between FOXM1 and Cyclin D1 in Cd-exposed cells. These effects were attenuated by MAPK kinase (MEK1/2) inhibitor. In summary, Cd-induced proliferation of ht-UtLM cells occurred through activation of Histone H3 and Aurora B via FOXM1/Cyclin D1 interactions downstream of MAPK. This provides a molecular mechanism of how Cd acts as an "estrogen mimic" resulting in mitosis in hormonally responsive cells.

Topics: Aurora Kinase B; Cadmium; Cell Proliferation; Cells, Cultured; Cyclin D1; Female; Forkhead Box Protein M1; Histones; Humans; Leiomyoma; Mitogen-Activated Protein Kinases; Mitosis; Receptors, Estrogen; Receptors, G-Protein-Coupled; Signal Transduction; Uterine Neoplasms

Uterine leiomyoma (UL) is the most common type of benign tumor in the women's reproductive system. A number of genes has been found to play an important role in the initiation and progression of UL, including miRNAs. In this study, our results exhibited that miR-93, a member of mir-106b-25 cluster, significantly reduced the cell viability, promoted cell cycle arrest, caused apoptosis, and inhibited migration in UL cells (p < .01). Moreover, our results have provided experimental evidence that miR-93 regulated the biological functions of UL cells by targeting CCND1.

Topics: Adult; Apoptosis; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; MicroRNAs; Middle Aged; Uterine Neoplasms

Uterine fibroids (UF) are the most common benign tumor of the myometrium (MM) in women of reproductive age. However, the mechanism underlying the pathogenesis of UF is largely unknown.. To explore the link between nuclear β-catenin and UF phenotype and β-catenin crosstalk with estrogen and histone deacetylases (HDACs).. Protein/RNA levels of β-catenin (CTNNB1 gene), its responsive markers cyclin D1 and c-Myc, androgen receptor (AR), p27, and class-I HDACs were measured in matched UF/MM tissues or cell populations. The effects of chemical inhibition/activation and genetic knockdown of CTNNB1 on UF phenotype were measured. The anti-UF effect of 2 HDAC inhibitors was evaluated.. β-catenin nuclear translocation in response to β-catenin inhibition/activation, estrogen, and HDAC inhibitors in UF cells.. UF tissues/cells showed significantly higher expression of nuclear β-catenin, cyclin D1, c-Myc, and HDACs 1, 2, 3, and 8 than MM. Estradiol induced β-catenin nuclear translocation and consequently its responsive genes in both MM and UF cells, while an estrogen receptor antagonist reversed this induction effect. Treatment with β-catenin or HDAC inhibitors led to dose-dependent growth inhibition, while Wnt3a treatment increased proliferation compared with control. Chemical inhibition of β-catenin decreased cyclin D1 and c-Myc expression levels, while β-catenin activation increased expression of the same markers. Genetic knockdown of CTNNB1 resulted in a marked decrease in β-catenin, cyclin D1, c-Myc, and AR expression. Treatment of UF cells with HDAC inhibitors decreased nuclear β-catenin, cyclin D1, and c-Myc expression. Moreover, HDAC inhibitors induced apoptosis of UF cells and cell cycle arrest.. β-catenin nuclear translocation contributes to UF phenotype, and β-catenin signaling is modulated by estradiol and HDAC activity.

Topics: beta Catenin; Biomarkers; Cyclin D1; Estrogens; Female; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Humans; Leiomyoma; Receptors, Androgen; Receptors, Estrogen; Signal Transduction; Tumor Cells, Cultured

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/β-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and β-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/β-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

Topics: Autophagy; beta Catenin; Cell Cycle; Cyclin D1; Female; Humans; Leiomyoma; Mediator Complex; Mutation; Myometrium; TOR Serine-Threonine Kinases; Wnt Signaling Pathway; Wnt4 Protein

Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress β1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies.. This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells.. This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 μM, or control) for 48 hours. Protein and mRNA levels of β1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction.. We found that simvastatin significantly reduced the protein expression of β1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in β1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1.. The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.

Topics: A Kinase Anchor Proteins; Collagen Type I; Cyclin D1; Extracellular Matrix; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Integrin beta1; Leiomyoma; Mechanotransduction, Cellular; Minor Histocompatibility Antigens; Myosin-Light-Chain Kinase; Phosphorylation; Primary Cell Culture; Proto-Oncogene Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; RNA, Messenger; Simvastatin; Uterine Neoplasms

The objective of this study was to determine whether miR-93, miR-29c, and miR-200c, which we previously reported to be downregulated in leiomyomas, target cell cycle regulatory proteins that influence cell proliferation. Based on TargetScan algorithm 3 cell cycle regulatory proteins namely, E2F transcription factor 1 (E2F1), Cyclin D1 (CCND1) and CDK2 which were predicted to be targets of these miRNAs were  further analyzed. In 30 hysterectomy specimens, we found the expression of E2F1 and CCND1 messenger RNA (mRNA) was increased in leiomyoma as compared to matched myometrium, with no significant changes in CDK2 mRNA levels. There was a significant increase in the abundance of all 3 proteins in leiomyoma in comparison with matched myometrium. Using luciferase reporter assay, we demonstrated E2F1 and CCND1 are targets of miR-93 and CDK2 is a target of miR-29c and miR-200c. We confirmed these findings through transfection studies in which transfection of primary leiomyoma cells with miR-93 resulted in a significant decrease in the expression of E2F1 and CCND1 mRNA and protein levels, whereas knockdown of miR-93 had the opposite effect. Similarly, overexpression of miR-29c and miR-200c in leiomyoma cells inhibited the expression of CDK2 protein and mRNA, whereas knockdown of this microRNAs (miRNA) had the opposite effect. Transfection of miR-29c, miR-200c, and miR-93 in primary leiomyoma cells resulted in a time-dependent inhibition of cell proliferation and cell motility. These results collectively indicate that the 3 miRNAs known to be downregulated in fibroid tumors are critical in regulation of cell proliferation because of their effects on 3 key cell cycle regulatory proteins, which are overexpressed in uterine leiomyomas.

Topics: Adult; Cell Cycle Proteins; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Female; Gene Expression Regulation, Neoplastic; Humans; Leiomyoma; MicroRNAs; Middle Aged; Uterine Neoplasms

Aspirin has been confirmed as an effective antitumor drug in various cancers. However, the relationship between aspirin and uterine leiomyoma is still underexplored. Here, we explored the effects of aspirin on human uterine leiomyoma cells and provide insights into the underlying mechanisms. Cell Counting Kit-8 (CCK-8) and flow cytometry analysis showed that aspirin treatment inhibited cell proliferation and promoted cell cycle arrest at G0/G1 phase in a dose- and time‑dependent manner of human uterine leiomyoma cells. Further studies revealed that aspirin blocked the interaction between K-Ras and p110α by co-immunoprecipitation and immunofluorescence. Western blotting demonstrated K‑Ras‑p110α interaction was required for the effects of aspirin‑induced inhibition on cell growth and cell cycle transition via cell cycle regulators, including cyclin D1 and cyclin-dependent kinase 2 (CDK2). PI3K/Akt/caspase signaling pathway was involved in human uterine leiomyoma cell growth under aspirin treatment. Taken together, these results suggest that aspirin inhibited human uterine leiomyoma cell growth by regulating K‑Ras‑p110α interaction. Aspirin which targeting on interaction between K-Ras and p110α may serve as a new therapeutic drug for uterine leiomyoma treatment.

Topics: Apoptosis; Aspirin; Caspases; Cell Cycle Checkpoints; Cell Proliferation; Class I Phosphatidylinositol 3-Kinases; Cyclin D1; Cyclin-Dependent Kinase 2; Flow Cytometry; Humans; Leiomyoma; Proto-Oncogene Proteins p21(ras); Signal Transduction

To analyse the effect of dydrogesterone use during pregnancy on uterine fibroids, pregnancy complications, and pregnancy outcome.. In all, 372 pregnant women with uterine fibroids who were treated at the Affiliated Provincial Hospital of Shandong University were included in this study. Thirty-three of these women received dydrogesterone and constituted the treatment group, and the 27 women who were found to have uterine fibroids during the first trimester but did not receive intervention to prevent miscarriage composed the control group. The changes in uterine fibroids before and after pregnancy and the pregnancy complications were recorded; immunohistochemistry was used to detect the expression of progesterone receptor (PR) and proliferation- and apoptosis-related proteins in the uterine fibroid tissue.. No significant difference was observed in the change in uterine fibroid volume during pregnancy between the treatment group and the control group (p > 0.05). The percentage of uterine fibroids with red degeneration was lower in the treatment group than in the control group, but the difference was not statistically significant. No significant difference was observed in newborn weight, height, Apgar score, threatened miscarriage, or premature birth, among other characteristics, between the two groups (p > 0.05). Immunohistochemistry showed no significant difference in the expression of PR, cyclinD1, insulin-like growth factor (IGF1), or B-cell lymphoma 2 (Bcl2) between the two groups.. The use of dydrogesterone during pregnancy has no significant effect on uterine fibroids, pregnancy progression, or pregnancy outcomes in pregnant patients with uterine fibroids.

Topics: Abortion, Spontaneous; Apoptosis; Cell Proliferation; Cyclin D1; Dydrogesterone; Female; Humans; Infant, Newborn; Insulin-Like Growth Factor I; Leiomyoma; Pregnancy; Pregnancy Complications, Neoplastic; Pregnancy Outcome; Progestins; Proto-Oncogene Proteins c-bcl-2; Receptors, Progesterone; Uterine Neoplasms

Uterine leiomyomas are benign tumors that develop from smooth muscle cells (SMCs). The reactive oxygen species (ROS) have been shown to be involved in the signaling pathways that stimulate proliferation of a variety of cell types. Thioredoxin-1 (TRX-1) is a redox-regulating protein, which is overexpressed in various tumors. In the present study, we investigated the expressions of TRX-1 and its related molecules in uterine leiomyomas and matched adjacent myometrium. Our results showed the expression of TRX-1 was increased in leiomyomas compared with the matched adjacent myometrium by quantitative RT-PCR and western blotting. FOXO3A expression was increased in leiomyomas compared with myometrium by western blotting. The mRNA levels of hypoxia-inducible factor-1α, cyclooxygenase-2 and cyclin D1 were increased in leiomyomas compared with the adjacent myometrium. The mRNA level of (thioredoxin-1-binding protein) TBP-2 in leiomyomas was not altered when compared with the matched adjacent myometrium. These results suggest that TRX-1 and some of its related molecules are associated with the pathogenesis of uterine leiomyomas. The identification of TRX-1 signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.

Topics: Adult; Blotting, Western; Cyclin D1; Cyclooxygenase 2; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leiomyoma; Middle Aged; Myometrium; Nuclear Proteins; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Signal Transduction; TATA Box Binding Protein-Like Proteins; Thioredoxins; Uterine Neoplasms

Uterine leiomyoma, the most common tumors found in the women of the reproductive age, may cause abnormal uterine bleeding and be life threatening. Compared with myometrium, leiomyoma contains excessive extracellular matrix (ECM). However, the pathological roles of ECM in the development of leiomyoma remain largely unknown. Integrins are the major adhesion molecules on cell surface to interact with ECM. The interactions of ECM with integrins regulate cell adhesion and initiate signals for cell growth, differentiation, and migration.. The aim of this study was to investigate the expression and functional role of integrin-β1 in leiomyoma pathogenesis.. Levels of integrin-β1 protein were determined by Western blotting in paired normal and leiomyomal tissues (n = 15). Knockdown of integrin-β1 and inhibition of ECM-integrin interaction by disintegrin were used to evaluate the impact of integrin-β1 in cell adhesion, spreading, and proliferation.. Levels of integrin-β1 were significantly up-regulated in leiomyomal cells compared with their normal counterparts. Knockdown of integrin-β1 did not affect cell adhesion on fibronectin or laminin matrix but significantly inhibits cell spreading ability. Consistent with this notion, the phosphorylation of focal adhesion kinase and the recruitment of paxillin to the focal contact were decreased in integrin-β1 knockdown cells, which attenuates contraction force. The inability of cell spreading leads to inhibition of cyclin D1 expression and impedes cell cycle progression. More importantly, disruption of ECM-integrin interaction by the small protein, disintegrin inhibited cyclin D1 expression and cell proliferation.. These data demonstrate that integrin-β1 is a critical ligand to enhance cell-ECM contact force and thus promotes cell proliferation. Disruption of ECM-integrin-β1 signaling may serve as an option to inhibit the progression of leiomyoma.

Topics: Antigens, Neoplasm; Cell Adhesion; Cell Movement; Cell Proliferation; Cell Shape; Cyclin D1; Disease Progression; Disintegrins; Extracellular Matrix Proteins; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin beta1; Leiomyoma; Paxillin; Phosphorylation; Protein Processing, Post-Translational; RNA Interference; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

Obesity is a well-documented risk factor for uterine leiomyoma with a major impact on women health and health care system of the nation. Obesity is associated with increased secretion of adipokines that significantly influence growth and proliferation of tumor stroma and malignant cells. Adipokines, such as tumor necrosis factor α (TNF-α), are produced in the adipose tissue with concomitant expression in other organs and tissues. Increased and sustained cytokine production is associated with alterations in cell growth and differentiation. We, therefore, explored the influence of human adipocytes (SW872 cells)-mediated biological humoral factors on human uterine leiomyoma (HuLM) cells.. We measured cell proliferation and expression of cell-proliferating proteins (proliferating cell nuclear antigen [PCNA], cyclin D1, and B-cell lymphoma 2 [BCL-2]) in human leiomyoma cells cocultured with SW872 cells. SW872-conditioned media was neutralized for TNF-α and proliferation of HuLM cells was observed along with antiapoptotic marker, BCL-2, using Western immunoblot.. We found that both SW872-conditioned media and coculture with SW872 cells increased HuLM cell proliferation significantly (P < .05). We determined that this effect was associated with the upregulation of specific markers for proliferation, such as PCNA, cyclin D1, and BCL-2 (P < .05). Furthermore, the addition of neutralizing antibodies, anti-TNF-α, to SW872-conditioned media reversed the proliferation of leiomyoma cells and induced apoptosis as indicated by the reduced expression of antiapoptotic marker BCL-2.. SW872 cells secrete TNF-α, which is associated with a proliferative gene profile in HuLM cells and may play a role in initiation and/or progression of uterine leiomyoma.

Topics: Adipocytes; Antibodies, Neutralizing; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cyclin D1; Female; Humans; Leiomyoma; Paracrine Communication; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Necrosis Factor-alpha; Uterine Neoplasms

Sphingosine 1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a family of cognate G protein-coupled receptors or as an intracellular second messenger for agonists acting through their membrane receptors. In view of the general growth promoting and developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in adaptation of the uterus to pregnancy. We analyzed its potential role and that of the related lysophospholipid lysophosphatidic acid in the pregnant rat uterus by examining changes in mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only sphingosine kinase-1 (SphK1) was markedly changed ( approximately 30-fold increase), being localized in the glandular epithelium, vasculature, and the myometrium. Uterine SphK1 mRNA and protein levels paralleled those of serum progesterone, and treatment with progesterone or an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also increased SphK1 mRNA steady-state levels in a rat myometrial/leiomyoma cell line (ELT3). Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle regulator cyclin D1 and increased myosin light-chain phosphorylation. Ectopic expression of SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates processes involved in growth and differentiation of uterine tissues during pregnancy.

Topics: Animals; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Enzymes; Female; Genes, Dominant; Humans; Immunologic Techniques; Leiomyoma; Lysophospholipids; Mutation; Myometrium; Myosin Light Chains; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pregnancy; Pregnancy, Animal; Progesterone; Progestins; Rats; Rats, Sprague-Dawley; Receptors, Lysophospholipid; RNA, Messenger; Signal Transduction; Sphingosine; Uterus

Desmoid tumors arising in the lung and pleura are extremely rare and can resemble other, more common neoplasms native to these sites. Alterations of the adenomatous polyposis coli/beta-catenin pathway have been detected in sporadic desmoid tumors and have been associated with nuclear accumulation of beta-catenin and overexpression of cyclin D1.. To analyze the expression of beta-catenin and cyclin D1 in desmoid tumors and solitary fibrous tumors (SFTs), and to compare the utilities of these substances for distinguishing between these entities with those of other, more commonly used stains.. Formalin-fixed, paraffin-embedded sections of 4 desmoid tumors (1 pulmonary, 1 pleural, 2 pleural/chest wall), and 5 benign and 6 malignant SFTs of the pleura were immunostained for beta-catenin, cyclin D1, ALK1, CD34, vimentin, desmin, smooth muscle actin, muscle-specific actin, S100, and pancytokeratin. Staining intensity and the percentage of stained tumor cells were assessed semiquantitatively.. Diffuse moderate or strong nuclear staining for beta-catenin was found in all desmoid tumors, 4 of 5 benign SFTs, and 2 of 6 malignant SFTs. All cases except 1 benign SFT showed concurrent cytoplasmic staining. Nuclear and cytoplasmic cyclin D1 staining was increased in all groups. The best distinction between desmoid tumors and SFTs was provided by CD34 (desmoid tumors, 0/4; SFTs, 8/11) and smooth muscle actin (desmoid tumors, 4/4; SFTs, 0/11).. Our findings suggest that alterations in the adenomatous polyposis coli/beta-catenin pathway and cyclin D1 dysregulation may contribute to the pathogenesis of pleuropulmonary desmoid tumors and SFTs. CD34 and smooth muscle actin stains are particularly useful for differentiating between pleuropulmonary desmoid tumors and SFTs.

Topics: Actins; Adult; Aged; Antigens, CD34; beta Catenin; Cell Nucleus; Child, Preschool; Cyclin D1; Cytoplasm; Diagnosis, Differential; Female; Fibromatosis, Aggressive; Humans; Immunohistochemistry; Leiomyoma; Lung Neoplasms; Male; Middle Aged; Muscle, Smooth; Pleural Neoplasms; Staining and Labeling

The purpose of this study was to detect the expression of cyclin G1 in leiomyoma and to investigate the alteration of its expression compared with normal myometrial tissue that was obtained from the same patient.. With the use of Northern blot analysis, Western blot analysis, and immunohistochemistry, we analyzed the expression of cyclin G1 in 24 patients who underwent hysterectomies.. We found that messenger RNA levels of cyclin G1 were elevated in human leiomyomas compared with their adjacent normal myometrial tissues. Consistent with elevated messenger RNA levels, high levels of cyclin G1 protein expression were detected by immunoblot analysis in all leiomyoma samples. Immunohistochemistry revealed that cyclin G1 is located mainly in the nucleus in both normal myometrium and leiomyoma. However, higher levels of cyclin G1 were apparent in tumor regions compared with adjacent normal myometrial regions. In addition, we found the expression levels of other cyclins (A and E) and CDK2 were elevated in leiomyomas compared with normal myometrium. Because cyclin G1 is a transcriptional target of the p53 tumor suppressor, we examined the p53 status of all eight leiomyoma samples and found no p53 mutations.. These results suggest that cyclin G1 is frequently overexpressed in uterine leiomyoma in a p53-independent manner and that this abnormality could be attributed to the severe proliferation of human uterine leiomyomas.

Topics: Adult; Blotting, Northern; Blotting, Western; Cyclin D1; Female; Genes, p53; Humans; Immunohistochemistry; Leiomyoma; Middle Aged; Mutation; Myometrium; Reference Values; RNA, Messenger; Uterine Neoplasms

The aim of these experiments was to investigate the expression of cyclin D1 and of oestradiol receptors as well as the level of [(3)H]oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases and at an early stage of menopause. [(3)H]oestradiol binding was determined by saturation analysis, while the oestradiol receptor (ER) alpha and beta and cyclin D1 levels were determined by Western blot analysis of 16 samples of human leiomyomas and corresponding myometria at different hormonal stages. In leiomyomas during all phases of the menstrual cycle, ERalpha expression, high affinity oestradiol binding and cyclin D1 expression were all elevated in comparison with adjacent myometrium. ERbeta expression and low affinity oestradiol binding were enhanced in leiomyomas only during the proliferative phase. During menopause, ERbeta expression and low affinity binding were enhanced in leiomyomas, while the ERalpha expression was not significantly enhanced and cyclin D1 levels were similar to that in myometrium. Only the oestradiol binding exhibited any menstrual cycle-related changes. Our data suggest the involvement of cyclin D1 in the growth of leiomyomas during the menstrual cycle. In menopause, there appears to be a switch from ERalpha to ERbeta expression in leiomyomas, and the induction of cyclin D1 is decreased. The regression of tumour may ensue from these changes at menopause.

Topics: Adult; Cyclin D1; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Leiomyoma; Menopause; Menstrual Cycle; Middle Aged; Myometrium; Receptors, Estrogen; Uterine Neoplasms