Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

Immunoexpression of Steroid Hormone Receptors and Proliferation Markers in Uterine Leiomyoma and Normal Myometrial Tissues from the Miniature Pig, Sus scrofa.

Uterine leiomyomas in miniature pet pigs occur similarly to those in women with regard to frequency, age, parity, and cycling. Clinical signs, gross, and histologic features of the porcine tumors closely resemble uterine leiomyomas (fibroids) in women. Although fibroids are hormonally responsive in women, the roles of estrogen and progesterone have not been fully elucidated. In this study, immunohistochemistry was used to assess the expression of the steroid hormone receptors, estrogen receptor alpha (ER-α), estrogen receptor beta (ER-ß) and progesterone receptor (PR), and cell proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67 in tumor and matched myometrial tissues sampled from miniature pigs. A "quickscore" method was used to determine receptor expression and labeling indices were calculated for the markers. ER-α/ß and PR were localized to the nuclei of smooth muscle cells in both tissues. PR expression was intense and diffuse throughout all tissues, with correlation between tumors and matched myometria. Conversely, ER-α expression was variable between the myometrial and tumor tissues, as well as between animals. ER-ß expression was low. PCNA and Ki-67 were localized to the nucleus and expression varied among tumors; however, normal tissues were overall negative. These findings support further investigation into the use of the miniature pig as a model of fibroids in women.

Development, implementation, and preliminary evaluation of a recovery-based curriculum for community navigation specialists working with individuals with serious mental illnesses and repeated hospitalizations.

A recovery-oriented curriculum for training the Community Navigation Specialists (CNSs) of the new Opening Doors to Recovery in Southeast Georgia program was developed, implemented, and preliminarily evaluated. This new mental health program provides mobile, community-based support services to individuals with serious mental illnesses and a history of psychiatric inpatient recidivism (and commonly past incarcerations and homelessness). Teams of CNSs include a licensed social worker, a family member of an individual with a serious mental illness, and a peer specialist with lived experience. In two courses held in February and June of 2011, 14 newly hired CNSs participated in the new training. A pre-training/post-training evaluation demonstrated statistically significant improvements in pertinent knowledge and self-efficacy for working in a community navigation role. As the recovery paradigm continues to be implemented in diverse real-world mental health treatment settings, recovery-based training curricula should be carefully constructed and evaluated.

Community navigation to reduce institutional recidivism and promote recovery: initial evaluation of opening doors to recovery in Southeast Georgia.

New approaches for preventing repeated inpatient psychiatric stays, detention in jails and prisons, and homelessness among individuals with serious mental illnesses with established histories of such recidivism, while promoting recovery, are direly needed. We present findings from an initial program evaluation of a new community-based, recovery-oriented "community navigation" program in southeast Georgia, called Opening Doors to Recovery. Twenty-three in-depth interviews were conducted with key stakeholders, program participants, community navigation specialist team members, and referring mental health professionals to identify hopes and strengths, challenges and weaknesses, and recommendations pertaining to the new program. Cited strengths included teamwork and pooling of resources from various partners, as well as the novel recovery-based, community navigation team approach. An initial lack of fidelity processes across teams and an ongoing scarcity of safe and affordable housing were identified as weaknesses, with the latter seen as a liability of the overall mental health and social service systems rather than the program itself. Findings from this evaluation highlight strengths and opportunities of this new community navigation approach, including those related to the involvement of certified peer specialists and multiple community partners.

An essential role of p27 downregulation in fenvalerate-induced cell growth in human uterine leiomyoma and smooth muscle cells.

Previously, we reported that fenvalerate (Fen) promotes proliferation of human uterine leiomyoma (UtLM) cells by enhancing progression of cells from G(0)-G(1) to S phase through molecular mechanisms independent of estrogen receptor-α and -ß. The cyclin-dependent kinase (CDK) inhibitor p27, which blocks G(1) to S phase transitions and is an important regulator of CDK2, is often decreased in hormonally regulated diseases, including uterine leiomyomas. Therefore, we were interested in whether Fen could regulate the expression of p27 and whether p27 might play a role in Fen-induced cell proliferation. Expression of p27 in Fen-treated UtLM and uterine smooth muscle cells (UtSMCs) was examined. We found that p27 mRNA was significantly downregulated and that protein levels were decreased in both cell types treated with 10 µM Fen for 24 h compared with respective controls. Overexpression of p27 in UtLM cells and UtSMCs using an adenovirus doxycycline (Dox)-regulated Tet-off system abrogated the proliferative effects of Fen, as evidenced by decreased total cell numbers and BrdU incorporation. Fen treatment increased CDK2 mRNA expression levels; however, overexpression of p27 also abolished this effect. In contrast, Dox treatment dramatically restored the above muted responses. Finally, we utilized siRNA to knock down p27 expression. After transfection, mRNA levels of p27 were downregulated in UtLM cells and UtSMCs and total cell numbers and BrdU incorporation increased significantly compared with nontransfected cells. Fen treatment in the presence of p27 silencing enhanced the increased cell counts and BrdU labeling in UtLM cells and UtSMCs. Taken together, these results indicate that p27 downregulation is critical for Fen-induced cell proliferation.

Effects of hormonally active agents on steroid hormone receptor expression and cell proliferation in the myometrium of ovariectomized macaques.

Hormone replacement therapy and selective estrogen receptor modulators have been controversial treatment options for postmenopausal women because of their potential health benefits and/or risks. In this study, we determine the effects of the hormonally active compounds, conjugated equine estrogens (CEE), medroxyprogesterone acetate (MPA), CEE + MPA, and tamoxifen (TAM) on the myometrium of ovariectomized macaques. Immunoexpression of estrogen receptor-α (ERα), progesterone receptor (PR), and Ki-67 in the myometrium is assessed. We found no significant difference in ERα myometrial expression in the CEE, MPA, and CEE + MPA treatment groups, but there was a significant decrease in expression in animals administered TAM versus controls. Conjugated equine estrogen-, TAM-, and CEE + MPA-treated animals had significantly increased expression of PR in myometrial cells and there was no difference in PR expression in cells from MPA-treated animals versus control animals. Myometrial cell proliferation did not significantly differ between the controls and any of the treatment groups, although normalized Ki-67 values were somewhat higher in the CEE and TAM groups. These data suggest that ERα and PR expression in the myometrium is influenced by treatment with hormonally active agents.

Human uterine leiomyoma-derived fibroblasts stimulate uterine leiomyoma cell proliferation and collagen type I production, and activate RTKs and TGF beta receptor signaling in coculture.

BACKGROUND: Uterine leiomyomas (fibroids) are benign smooth muscle tumors that often contain an excessive extracellular matrix (ECM). In the present study, we investigated the interactions between human uterine leiomyoma (UtLM) cells and uterine leiomyoma-derived fibroblasts (FB), and their importance in cell growth and ECM protein production using a coculture system. RESULTS: We found enhanced cell proliferation, and elevated levels of ECM collagen type I and insulin-like growth factor-binding protein-3 after coculturing. There was also increased secretion of vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor-2, and platelet derived growth factor A and B in the media of UtLM cells cocultured with FB. Protein arrays revealed increased phosphorylated receptor tyrosine kinases (RTKs) of the above growth factor ligands, and immunoblots showed elevated levels of the RTK downstream effector, phospho-mitogen activated protein kinase 44/42 in cocultured UtLM cells. There was also increased secretion of transforming growth factor-beta 1 and 3, and immunoprecipitated transforming growth factor-beta receptor I from cocultured UtLM cells showed elevated phosphoserine expression. The downstream effectors phospho-small mothers against decapentaplegic -2 and -3 protein (SMAD) levels were also increased in cocultured UtLM cells. However, none of the above effects were seen in normal myometrial cells cocultured with FB. The soluble factors released by tumor-derived fibroblasts and/or UtLM cells, and activation of the growth factor receptors and their pathways stimulated the proliferation of UtLM cells and enhanced the production of ECM proteins. CONCLUSIONS: These data support the importance of interactions between fibroid tumor cells and ECM fibroblasts in vivo, and the role of growth factors, and ECM proteins in the pathogenesis of uterine fibroids.

Bisphenol A induces human uterine leiomyoma cell proliferation through membrane-associated ERα36 via nongenomic signaling pathways.

The role of ERα36 in regulating BPA's effects and its potential as a risk factor for human uterine fibroids were evaluated. BPA at low concentrations (10-6 µM - 10 µM) increased proliferation by facilitating progression of hormonally regulated, immortalized human uterine leiomyoma (ht-UtLM; fibroid) cells from G0-G1 into S phase of the cell cycle; whereas, higher concentrations (100 µM-200 µM) decreased growth. BPA upregulated ERα36 gene and protein expression, and induced increased SOS1 and Grb2 protein expression, both of which are mediators of the MAPKp44/42/ERK1/2 pathway. EGFR (pEGFR), Ras, and MAPKp44/42 were phosphorylated with concurrent Src activation in ht-UtLM cells within 10 min of BPA exposure. BPA enhanced colocalization of phosphorylated Src (pSrc) to ERα36 and coimmunoprecipitation of pSrc with pEGFR. Silencing ERα36 with siERα36 abolished the above effects. BPA induced proliferation in ht-UtLM cells through membrane-associated ERα36 with activation of Src, EGFR, Ras, and MAPK nongenomic signaling pathways.

Estrogen receptor alpha (ERalpha) phospho-serine-118 is highly expressed in human uterine leiomyomas compared to matched myometrium.

It is thought that the growth of uterine leiomyomas may be mediated by the interaction of estrogen receptor alpha (ERalpha) and growth factor pathways and that phosphorylation of ERalpha at serine 118 (ERalpha-phospho-Ser118) is important in this interaction. In this study, immunoblotting and immunohistochemistry were used to investigate the expression of ERalpha-phospho-Ser118, phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK), and proliferating cell nuclear antigen (PCNA) in human leiomyoma and myometrial tissues during the proliferative and secretory phases of the menstrual cycle. We found that tumors taken from the proliferative phase expressed significantly higher levels of ERalpha-phospho-Ser118, phospho-p44/42 MAPK, and PCNA compared to patient-matched myometria and had significantly higher ERalpha-phospho-Ser118 and PCNA expression compared to secretory phase tumors. Also, enhanced colocalization and association of phospho-p44/42 MAPK and ERalpha-phospho-Ser118 were observed in proliferative phase tumors by confocal microscopy and immunoprecipitation, respectively. These data suggest that ERalpha-phospho-Ser118 may be important in leiomyoma growth and is possibly phosphorylated by phospho-p44/42 MAPK.

Association of race, age and body mass index with gross pathology of uterine fibroids.

OBJECTIVE: To determine the associations of race, age and body mass index (BMI) with the gross pathology parameters of uterine leiomyomas in premenopausal women undergoing hysterectomy or myomectomy. STUDY DESIGN: Participants (N = 107) were recruited from surgical rosters of the George Washington University (GWU) Medical Center Gynecology Department as part of the National Institute of Environmental Health Sciences Fibroid Study. Tumor data and patient demographics were obtained from clinical reports, pathology forms and interviews. RESULTS: Surgical cases consisted of 78% African Americans, 13% Caucasians and 9% others (non-African American, non-Caucasian or race unknown). This proportion of African Americans was significantly higher than the distribution of GWU health plan participants. Fibroids were localized predominantly within the intramural region. Subserosal tumors were more common in patients with more than 9 tumors. African Americans had the highest mean BMI and mean myomatous uterine weight. CONCLUSION: African Americans were the disproportionate majority coming to surgery for fibroids. The average BMI and uterine weight were greater in African Americans than in Caucasians, although these differences were marginal. Race did not influence the size, location or number of fibroids in these surgical cases. Subserosal tumors were more common in patients with more than 9 tumors.

The Life Cycle of the Uterine Fibroid Myocyte.

PURPOSE OF REVIEW: Uterine fibroids are common benign tumors of women in the USA and worldwide, yet the biological nature and pathogenesis of these tumors remain largely unknown. This review presents our view of the stages in the life cycle of a subset of uterine fibroid myocytes, introduces hypothetical concepts and morphological data to explain these changes, and relates these changes in individual myocytes to the phases of fibroid tumor development. RECENT FINDINGS: The observations gained from light and electron microscopic, immunohistochemical, and morphometric studies in our laboratory have led to the hypothesis that fibroid changes over time may relate to the excessive production of collagen by phenotypically transformed myocytes. This accumulation of collagen results in decreased microvessel density, followed by myocyte injury and atrophy, with eventual senescence and involution through ischemic cellular degeneration and inanition. SUMMARY: Uterine leiomyomas, or fibroids, are characterized by two histologic features-proliferation of myocytes and production of an extracellular collagenous matrix. In the larger tumors, the collagenous matrix is often abundant. Within those regions in which the accumulating collagen is excessive, the myocytes are progressively separated from their blood supply, resulting in myocyte atrophy and eventually cell death. It is within these hypocellular, hyalinized areas that the complete lifecycle of the fibroid myocyte is realized. It begins with the phenotypic transformation of a contractile cell to one characterized by proliferation and collagen synthesis, progresses through an intermediate stage of atrophy related to interstitial ischemia, and eventuates in cell death due to inanition. Lastly, resorption of inanotic cells appears to occur by a non-phagocytic, presumably enzymatic process of degradation and recycling that we refer to as reclamation.

A High Concentration of Genistein Induces Cell Death in Human Uterine Leiomyoma Cells by Autophagy.

Genistein, an estrogenic, soy-derived isoflavone, may play a protective role against hormone-related cancers. We have reported that a high concentration of genistein inhibits cell proliferation and induces apoptosis in human uterine smooth muscle cells, but not in leiomyoma (fibroid) cells. To better understand the differential cell death responses of normal and tumor cells to a high concentration of genistein, we treated uterine smooth muscle cells and uterine leiomyoma cells with 50 µg/ml of genistein for 72 h and 168 h, and assessed for mediators of apoptosis, cytotoxicity and autophagy. We found that leiomyoma cells had increased protection from apoptosis by expressing an increased ratio of Bcl-2: bak at 72 h and 168 h; however, in smooth muscle cells, the Bcl-2: bak ratio was decreased at 72 h, but significantly rebounded by 168 h. The apoptosis extrinsic factors, Fas ligand and Fas receptor, were highly expressed in uterine smooth muscle cells following genistein treatment at both time points as evidenced by confocal microscopy. This was not seen in the uterine leiomyoma cells; however, cytotoxicity as indicated by elevated lactate dehydrogenase levels was significantly enhanced at 168 h. Increased immunoexpression of an autophagy/autophagosome marker was also observed in the leiomyoma cells, although minimally present in smooth muscle cells at 72 h. Ultrastructurally, there was evidence of autophagic vacuoles in the leiomyoma cells; whereas, the normal smooth muscle cells showed nuclear fragmentation indicative of apoptosis. In summary, our data show differential cell death pathways induced by genistein in tumor and normal uterine smooth muscle cells, and suggest novel cell death pathways that can be targeted for preventive and intervention strategies for inhibiting fibroid tumor cell growth in vivo.

Cadmium and proliferation in human uterine leiomyoma cells: evidence of a role for EGFR/MAPK pathways but not classical estrogen receptor pathways.

BACKGROUND: It has been proposed that cadmium (Cd) is an environmental "metalloestrogen" and that its action is mediated via the estrogen receptor (ER). Cd mimics the effects of estrogen in the rat uterus, and blood Cd concentrations positively correlate with ER levels in uteri of women with fibroids. OBJECTIVES: In the present study we explored whether Cd could stimulate proliferation of estrogen-responsive human uterine leiomyoma (ht-UtLM) cells and uterine smooth muscle cells (ht-UtSMCs) through classical interactions with ERα and ERß, or by nongenomic mechanisms. METHODS: We used estrogen response element (ERE) reporters, phosphorylated receptor tyrosine kinase arrays, Western blot analysis, estrogen binding, and cell proliferation assays to evaluate the effects of Cd on ht-UtLM cells and ht-UtSMCs. RESULTS: Cd stimulated growth of both cell types at lower concentrations and inhibited growth at higher concentrations (≥ 50 µM). Cd did not significantly bind to ERα or ERß, nor did it show transactivation in both cell types transiently transfected with ERE reporter genes. However, in both cells types, Cd (0.1 µM and 10 µM) activated p44/42 MAPK (ERK1/2), and a MAPK inhibitor (PD98059) abrogated Cd-induced cell proliferation. Cd in ht-UtLM cells, but not in ht-UtSMCs, activated the growth factor receptors EGFR, HGFR, and VEGF-R1 upstream of MAPK. Additional studies in ht-UtLM cells showed that AG1478, an EGFR inhibitor, abolished Cd-induced phosphorylation of EGFR and MAPK. CONCLUSIONS: Our results show that low concentrations of Cd stimulated cell proliferation in estrogen-responsive uterine cells by nongenomic activation of MAPK, but not through classical ER-mediated pathways.

Cell proliferation and apoptosis in human uterine leiomyomas and myometria.

To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis -- Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) -- and two endogenous markers of cell replication - proliferating cell nuclear antigen (PCNA) and Ki-67 - in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly ( P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.

Perceptions of injustice in family work: the role of psychological distress.

During the transition to parenthood, perceived imbalances in family work typically increase. Little is known, however, about which individuals are especially prone to perceive unfairness in the division of family work during this time. Using data from a longitudinal study of married couples expecting their first child and controlling for marital distress and other relevant variables, we observed that when husbands were psychologically distressed, both they and their wives were subsequently more likely to perceive unfairness to wives in the division of family work. No analogous significant and prospective effects of wives' levels of distress on their own or their husbands' perceptions of unfairness were found. We also found that once wives perceived the amount of child care they did as unfair, both they and their husbands were later more likely to experience psychological distress, controlling for marital distress and other relevant variables.

The natural history of uterine leiomyomas: morphometric concordance with concepts of interstitial ischemia and inanosis.

Based upon our morphologic observations, we hypothesize and also provide morphometric evidence for the occurrence of progressive developmental changes in many uterine fibroids, which can be arbitrarily divided into 4 phases. These developmental phases are related to the ongoing production of extracellular collagenous matrix, which eventually exceeds the degree of angiogenesis, resulting in the progressive separation of myocytes from their blood supply and a condition of interstitial ischemia. The consequence of this process of slow ischemia with nutritional and oxygen deprivation is a progressive myocyte atrophy (or inanition), culminating in cell death, a process that we refer to as inanosis. The studies presented here provide quantitative and semiquantitative evidence to support the concept of the declining proliferative activity as the collagenous matrix increases and the microvascular density decreases.

The natural history of uterine leiomyomas: light and electron microscopic studies of fibroid phases, interstitial ischemia, inanosis, and reclamation.

We propose, and offer evidence to support, the concept that many uterine leiomyomas pursue a self-limited life cycle. This cycle can be arbitrarily divided on the basis of morphologic assessment of the collagen content into 4 phases: (1) proliferation, (2) proliferation and synthesis of collagen, (3) proliferation, synthesis of collagen, and early senescence, and (4) involution. Involution occurs as a result of both vascular and interstitial ischemia. Interstitial ischemia is the consequence of the excessive elaboration of collagen, resulting in reduced microvascular density, increased distance between myocytes and capillaries, nutritional deprivation, and myocyte atrophy. The end stage of this process is an involuted tumor with a predominance of collagen, little to no proliferative activity, myocyte atrophy, and myocyte cell death. Since many of the dying cells exhibit light microscopic and ultrastructural features that appear distinct from either necrosis or apoptosis, we refer to this process as inanosis, because it appears that nutritional deprivation, or inanition, is the underlying cause of cell death. The disposal of myocytes dying by inanosis also differs in that there is no phagocytic reaction, but rather an apparent dissolution of the cell, which might be viewed as a process of reclamation as the molecular contents are reclaimed and recycled.

Estrogen Regulates MAPK-Related Genes through Genomic and Nongenomic Interactions between IGF-I Receptor Tyrosine Kinase and Estrogen Receptor-Alpha Signaling Pathways in Human Uterine Leiomyoma Cells.

Estrogen and growth factors play a major role in uterine leiomyoma (UtLM) growth possibly through interactions of receptor tyrosine kinases (RTKs) and estrogen receptor-alpha (ERα) signaling. We determined the genomic and nongenomic effects of 17ß-estradiol (E(2)) on IGF-IR/MAPKp44/42 signaling and gene expression in human UtLM cells with intact or silenced IGF-IR. Analysis by RT(2) Profiler PCR-array showed genes involved in IGF-IR/MAPK signaling were upregulated in UtLM cells by E(2) including cyclin D kinases, MAPKs, and MAPK kinases; RTK signaling mediator, GRB2; transcriptional factors ELK1 and E2F1; CCNB2 involved in cell cycle progression, proliferation, and survival; and COL1A1 associated with collagen synthesis. Silencing (si)IGF-IR attenuated the above effects and resulted in upregulation of different genes, such as transcriptional factor ETS2; the tyrosine kinase receptor, EGFR; and DLK1 involved in fibrosis. E(2) rapidly activated IGF-IR/MAPKp44/42 signaling nongenomically and induced phosphorylation of ERα at ser118 in cells with a functional IGF-IR versus those without. E(2) also upregulated IGF-I gene and protein expression through a prolonged genomic event. These results suggest a pivotal role of IGF-IR and possibly other RTKs in mediating genomic and nongenomic hormone receptor interactions and signaling in fibroids and provide novel genes and targets for future intervention and prevention strategies.

A high concentration of genistein down-regulates activin A, Smad3 and other TGF-ß pathway genes in human uterine leiomyoma cells.

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-ß signaling pathway genes, activin A, activin B, Smad3, TGF-ß2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real- time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-ß pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.

Histopathologic changes in the uterus, cervix and vagina of immature CD-1 mice exposed to low doses of perfluorooctanoic acid (PFOA) in a uterotrophic assay.

The estrogenic and antiestrogenic potential of perfluorooctanoic acid (PFOA) was assessed using an immature mouse uterotrophic assay and by histologic evaluation of the uterus, cervix and vagina following treatment. Female offspring of CD-1 dams were weaned at 18days old and assigned to groups of equal weight, and received 0, 0.01, 0.1, or 1mg PFOA/kg BW/d by gavage with or without 17-ß estradiol (E(2), 500µg/kg/d) from PND 18-20 (n=8/treatment/block). At 24h after the third dose (PND 21), uteri were removed and weighed. Absolute and relative uterine weights were significantly increased in the 0.01mg/kg PFOA only group. Characteristic estrogenic changes were present in all E(2)-treated mice; however, they were minimally visible in the 0.01 PFOA only mice. These data suggest that at a low dose PFOA produces minimal histopathologic changes in the reproductive tract of immature female mice, and does not antagonize the histopathologic effects of E(2).