Mark W. Nachtigal

Anti-Müllerian Hormone Inhibits Initiation of Primordial Follicle Growth in the Mouse Ovary

Recruitment of primordial follicles is essential for female fer- tility; however, the exact mechanisms regulating this process are largely unknown. Earlier studies using anti-Mullerian hormone (AMH)-deficient mice suggested that AMH is in- volved in the regulation of primordial follicle recruitment. We tested this hypothesis in a neonatal ovary culture system, in which ovaries from 2-d-old C57Bl/6J mice were cultured for 2 or 4di n theabsence or presence of AMH. Ovaries from 2-d-old mice contain multiple primordial follicles, some naked oo- cytes, and no follicles at later stages of development. We ob- served that in the cultured ovaries, either nontreated or AMH- treated, follicular development progressed to the same extent as in in vivo ovaries of comparable age, confirming the validity of our culture system. However, in the presence of AMH, cul- tured ovaries contained 40% fewer growing follicles compared with control ovaries. A similar reduction was found after 4 d of culture. Consistent with these findings, we noted lower inhibin -subunit expression in AMH-treated ovaries com- pared with untreated ovaries. In contrast, expression of AMH ligand type II receptor and the expression of oocyte markers growth and differentiation factor 9 and zona pellucida protein 3 were not influenced by AMH. Based on the results, we suggest that AMH inhibits initia- tion of primordial follicle growth and therefore functions as an inhibitory growth factor in the ovary during these early stages of folliculogenesis. (Endocrinology 143: 1076 -1084, 2002)

Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.

Autocrine BMP4 signalling regulates ID3 proto-oncogene expression in human ovarian cancer cells.

Bone morphogenetic protein (BMP)-4 signalling leads to the direct upregulation of ID3 proto-oncogene expression in human ovarian cancer cells. An upstream BMP4-responsive enhancer element consisting of a palindromic BMP response element (BRE) site and CAGA box was identified ~3.0 kb upstream of the human ID3 gene, and a nearly-identical element exists in the second intron of the ID3 gene. BMP4 stimulation leads to the direct binding of Smads 1/5 and Smad4 to the upstream and intronic enhancers, and together both enhancers cooperate to yield heightened BMP4-mediated ID3 promoter activity. We further demonstrate that ID3 is overexpressed in human ovarian cancer cells when compared to normal ovarian surface epithelial cells, and treatment of ovarian cancer cells with the BMP4 antagonist Noggin abrogates endogenous ID3 gene expression. Our findings define the mechanism of BMP4-mediated ID3 gene expression, and support the notion that ovarian cancer cells possess autocrine BMP4 signalling required to sustain ID3 overexpression which may contribute to human ovarian cancer pathogenesis.

TGFβ-Induced Smad Signaling Remains Intact in Primary Human Ovarian Cancer Cells

Disruptions in TGFβ signaling have been implicated in various human cancers, including ovarian cancer. Our goal was to determine whether ovarian cancer cells isolated from patient ascites fluid were growth inhibited by TGFβ1 treatment and further characterize the expression and activity profile of TGFβ/Smad signaling components in human ovarian cancer cells. We found that 9 of 10 primary cultures of ovarian cancer cells (OC2–10) were growth inhibited by 16 pm TGFβ1. One primary ovarian cancer sample (OC1) and the established ovarian cancer cell lines CaOV3 and SkOV3 continued to grow in the presence of TGFβ1. All cells expressed components of the TGFβ/Smad signaling pathway including TGFβ1, TβRI, TβRII, Smad2, -3, -4, and Smad anchor for receptor activation. Although OC1, CaOV3, and SkOV3 are not growth inhibited by TGFβ1, they can transmit the TGFβ1 signal to turn on a transfected TGFβ/Smad reporter gene, p3TP.lux. In addition, all cells up-regulate the endogenous TGFβ target genes Smad7 and PAI-1. p15In...

Valproic acid: how it works. Or not

Abstract Bipolar disorder (BPD) affects approximately 1% of the population worldwide (Weissman MM, Bland RC, Canino GJ, et al. Cross-national epidemiology of major depression and bipolar disorder. J Am Med Assoc 1996;276:293–99, [1] ) and is a debilitating illness associated with high morbidity and mortality (10% lifetime risk of suicide). Valproic acid (VPA) is a widely used alternative to lithium salts for the treatment of BPD, and has been in clinical use for epilepsy for years, but its mechanism of action has not been defined in any setting. A large number of indirect targets as well as a more limited number of direct in vitro targets for VPA have been described, but strong evidence that these targets play a role in the therapeutic response is lacking. VPA was recently shown to inhibit histone deacetylases (HDACs), key regulators of chromatin structure and transcription. In this review we summarize the history and pharmacology of VPA and address the potential role of HDACs in the response to VPA. Review of these studies suggests that inhibition of HDACs is a highly plausible mechanism for VPA-mediated cellular differentiation and teratogenesis, but cannot account for the anticonvulsant and mood regulating actions of VPA that more likely involve different or additional targets.

Primary culture and mRNA analysis of human ovarian cells.

Established cell lines are invaluable for studying cell and molecular biological questions. A variety of human ovarian cancer (OC) cell lines exist, however, most have acquired significant genetic alterations from their cells of origin, including deletion of important cell cycle regulatory genes. In order to analyze signaling events related to cell cycle control in human OC, we have modified existing protocols for isolating and culturing OC cells from patient ascites fluid and normal ovarian surface epithelial (OSE) cells from benign ovarian tissue sections. These cells maintain an epithelial phenotype and can be manipulated experimentally for several passages before cellular senescence. An example using TGF1 treatment of OC cells to examine signaling and target gene activation is presented.

Differential binding of rat pituitary-specific nuclear factors to the 5′-flanking region of pituitary and placental members of the human growth hormone gene family

SummaryPlacental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5′-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (−140/−107) and proximal site (−97/−66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (−140/−116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.

Inhibition of the antiproliferative effect of TGFβ by EGF in primary human ovarian cancer cells

The majority of ovarian cancers (OCs) arise from the ovarian surface epithelium (OSE). Proliferation of the OSE can be regulated by a number of autocrine and paracrine factors, including transforming growth factor beta (TGFβ). Defects in the TGFβ signaling pathway have been implicated in a number of cancers, including ovarian. We previously found that the TGFβ signaling pathway is intact and functional in primary human OC cells, and that these cells stop growing in response to TGFβ. Ovarian cancer cells in vivo are exposed to TGFβ, yet continue to proliferate, therefore, mechanisms must exist to inhibit TGFβ signaling contributing to uncontrolled cellular proliferation. Numerous signaling pathways converge with the TGFβ pathway to modulate its effects, including signaling induced by epidermal growth factor (EGF). We hypothesized that EGF can modulate TGFβ signaling and contribute to uncontrolled cellular proliferation of OC cells. Our results show that EGF abrogates the antiproliferative effect of TGFβ. EGF does not modulate TGFβ signaling by inhibiting receptor-activated Smad (R-Smad) phosphorylation or nuclear translocation. Rather, EGF decreases TGFβ-induced mRNA expression of the cell cycle regulator, p15INK4B, contributing to decreased sensitivity of OC cells to the antiproliferative effect of TGFβ.

Constitutive activation of BMP signalling abrogates experimental metastasis of OVCA429 cells via reduced cell adhesion

BackgroundActivation of bone morphogenetic protein (BMP)4 signalling in human ovarian cancer cells induces a number of phenotypic changes in vitro, including altered cell morphology, adhesion, motility and invasion, relative to normal human ovarian surface epithelial cells. From these in vitro analyses, we had hypothesized that active BMP signalling promotes the metastatic potential of ovarian cancer.MethodsTo test this directly, we engineered OVCA429 human ovarian cancer cells possessing doxycycline-inducible expression of a constitutively-active mutant BMP receptor, ALK3QD, and administered these cells to immunocompromised mice. Further characterization was performed in vitro to address the role of activated BMP signalling on the EOC phenotype, with particular emphasis on epithelial-mesenchymal transition (EMT) and cell adhesion.ResultsUnexpectedly, doxycycline-induced ALK3QD expression in OVCA429 cells reduced tumour implantation on peritoneal surfaces and ascites formation when xenografted into immunocompromised mice by intraperitoneal injection. To determine the potential mechanisms controlling this in vivo observation, we followed with several cell culture experiments. Doxycycline-induced ALK3QD expression enhanced the refractile, spindle-shaped morphology of cultured OVCA429 cells eliciting an EMT-like response. Using in vitro wound healing assays, we observed that ALK3QD-expressing cells migrated with long, cytoplasmic projections extending into the wound space. The phenotypic alterations of ALK3QD-expressing cells correlated with changes in specific gene expression patterns of EMT, including increased Snail and Slug and reduced E-cadherin mRNA expression. In addition, ALK3QD signalling reduced β1- and β3-integrin expression, critical molecules involved in ovarian cancer cell adhesion. The combination of reduced E-cadherin and β-integrin expression correlates directly with the reduced EOC cell cohesion in spheroids and reduced cell adhesion to the extracellular matrix substrates fibronectin and vitronectin that was observed.ConclusionsWe propose that the key steps of ovarian cancer metastasis, specifically cell cohesion of multicellular aggregates in ascites and cell adhesion for reattachment to secondary sites, may be inhibited by overactive BMP signalling, thereby decreasing the ultimate malignant potential of ovarian cancer in this model system.

FGF-2 protects cardiomyocytes from doxorubicin damage via protein kinase C-dependent effects on efflux transporters

AIMS The anti-cancer anthracycline doxorubicin (DOX) increases the risk of cardiac damage, indicating a need to protect the heart and still allow the benefits of drug treatment. Fibroblast growth factor-2 (FGF-2) is cardioprotective against ischaemia-reperfusion injury. Our aim is to investigate: (i) the ability of FGF-2 to protect against DOX-induced cardiomyocyte damage and (ii) the contribution of efflux drug transport to any increase in injury-resistance. METHODS AND RESULTS Neonatal rat cardiomyocyte damage was assessed by measuring cell death markers and lactate dehydrogenase (LDH) activity in the culture medium. LDH activity was increased significantly after incubation with 0.5 μM DOX for 24 h in the absence but not presence of 10 nM FGF-2; this beneficial effect of FGF-2 was blocked by tyrosine kinase (FGF) receptor inhibition. An increase in efflux drug transporter RNA levels was also detected after FGF-2 treatment in the presence of DOX. The beneficial effect of FGF-2 against cell damage and increased transporter RNA levels were blunted with protein kinase C (PKC) inhibition. Finally, FGF-2 stimulated efflux transport of calcein and DOX, and treatment with efflux transporter inhibitors significantly attenuated the protective effect of FGF-2 from DOX-induced injury. CONCLUSION Administered FGF-2 increases resistance to DOX-induced cardiomyocyte damage, by a mechanism dependent on PKC as well as regulation of efflux transporter production and/or function.