Granulosa Cell Tumors Cell Research Articles

Anti-Müllerian hormone inhibits growth of AMH type II receptor-positive human ovarian granulosa cell tumor cells by activating apoptosis

Ovarian granulosa cell tumors (GCTs) are sex cord stromal tumors that constitute 3–5% of all ovarian cancers. GCTs usually present with an indolent course but there is a high risk of recurrence, which associates with increased mortality, and targeted treatments would be desirable. Anti-Müllerian hormone (AMH), a key factor regulating sexual differentiation of the reproductive organs, has been implicated as a growth inhibitor in ovarian cancer. GCTs and normal granulosa cells produce AMH, but its expression in large GCTs is usually downregulated. Further, as the lack of specific AMH-signaling pathway components leads to GCT development in mice, we hypothesized that AMH inhibits growth of GCTs. Utilizing a large panel of human GCT tissue samples, we found that AMH type I receptors (ALK2, ALK3 and ALK6) and type II receptor (AMHRII), as well as their downstream effectors Smad1/5, are expressed and active in GCTs. AMHRII expression was detected in the vast majority (96%) of GCTs and correlated with AMH mRNA and protein expression. AMH mRNA level was low in large GCTs, confirming previous findings on low-AMH protein expression in large human as well as mouse GCTs. To study the functional role of AMH in this peculiar ovarian cancer, we utilized a human GCT cell line (KGN) and 10 primary GCT cell cultures. We found that the AMH–Smad1/5-signaling pathway was active in these cells, and that exogenous AMH further activated Smad1/5 in KGN cells. Furthermore, AMH treatment reduced the number of KGN cells and primary GCT cells, with increasing amounts of AMH leading to augmented activation of caspase-3 and subsequent apoptosis. All in all, these data support the premise that AMH is a growth inhibitor of GCTs.

Serum Vascular Endothelial Growth Factor A (VEGF) Is Elevated in Patients with Ovarian Granulosa Cell Tumor (GCT), and VEGF Inhibition by Bevacizumab Induces Apoptosis in GCTin Vitro

Ovarian granulosa cell tumors (GCT) are highly vascularized and express vascular endothelial growth factor A (VEGF) and its functional receptor VEGF receptor 2 (VEGFR-2). Angiogenesis inhibitors have been used in the treatment of ovarian carcinomas, whereas their roles in GCT remain unknown. The aim was to assess serum levels of VEGF and endostatin, an endogenous angiogenesis inhibitor, in GCT patients and to study the effect of bevacizumab (BVZ), a VEGF-binding monoclonal antibody, on human GCT cells in vitro. Using ELISA, we measured soluble VEGF and endostatin in the sera of 54 GCT patients and in conditioned media from cultures of 14 primary GCT and an established GCT cell line (KGN). The expression of activated VEGFR-2 was analyzed in GCT tissues using immunohistochemistry. GCT cells were treated with BVZ and analyzed for cell number and apoptosis. Serum VEGF was elevated in GCT patients, and the levels significantly decreased after tumor removal (P < 0.05), whereas serum endostatin levels changed conversely. Human GCT expressed activated VEGFR-2 protein, and the level of expression was associated with tumor VEGF and vascularization. In addition, the cultured GCT cells produced significant amounts of VEGF but not endostatin. Treatment of KGN cells with BVZ significantly reduced the number of viable cells by 41% and induced a 3.3-fold increase in apoptosis. Furthermore, BVZ induced a mean 2.6-fold increase in apoptosis in six primary GCT cell cultures studied. These data suggest an autocrine role for VEGF in GCT and encourage clinical studies on anti-VEGF treatments in this disease.

TLR4 activates NF-κB in human ovarian granulosa tumor cells

Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-κB) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFα plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-κB does not sensitize GCTs to TRAIL or cisplatin.

Abstract 3193: Valosin containing protein (VCP) is a potential novel serum marker for ovarian cancer

Abstract The measurement of various serum markers (estradiol, inhibin, MIS) is often performed as an ancillary diagnostic technique for ovarian granulosa cell tumor (GCT), and is widely used for postoperative surveillance for disease recurrence. However, both the sensitivity and specificity of these markers has been repeatedly questioned, and a truly reliable serum marker for GCT has yet to be identified. We have recently developed the Ctnnb1tm1Mmt/+;Ptentm1Hwu/tm1Hwu;Amhr2tm3(cre)Bhr/+ (CPA) transgenic mouse model, which features genetic modifications that mimic pathologically relevant genetic lesions and signaling processes that occur in GCT in women. CPA mice develop anaplastic, highly aggressive GCTs that can spread both by forming distant metastases and by seeding into the peritoneal cavity, which are the main modes of GCT dissemination in women. Due to the biological similarities between GCT in women and in CPA mice, we hypothesized that the CPA model could be used in a translational approach to identify novel serum markers for GCT. To test this, granulosa cells from control mice and GCT cells from CPA mice were placed in culture, and differential secretion or shedding of proteins into the culture supernatant was analyzed by SDSPAGE and silver staining. Proteins bands present in the experimental samples but absent (or present at much lower levels) in control media were isolated from the gels and identified by tryptic digestion followed by LC/MS/MS analyses. By this method, VCL, VCP, HSPA4, HSP90A, HSP70, SPARC, WIF1, CTSB, FAM3C, TIMP2, CFL2 and B2M were found to be selectively secreted/shed by CPA GCT cells. An in vivo validation step was then performed to determined if these proteins were overexpressed in the serum of CPA mice relative to controls. Western blotting showed that CPA mice had elevated (P&amp;lt;0.05) serum levels of VCP, HSP4, CTSB, TIMP2 and B2M. The latter proteins were therefore tested as potential serum diagnostic markers for GCT in a small cohort of women with GCT (n=10) and healthy women (n=7), as well as in patients with ovarian epithelial carcinomas (n=8) to provide an initial assessment of marker specificity. Of the proteins tested, mean serum levels of Valosin Containing Protein (VCP) were found to be over 9-fold higher in women with GCT or ovarian carcinoma relative to healthy women (P&amp;lt;0.01) by western blotting. Furthermore, 90% of women with GCT and 100% of women with ovarian carcinomas had serum VCP levels above the highest level measured in the reference group. While these results indicate that VCP is unlikely to serve as a GCT-specific serum marker, it may nonetheless provide a valuable tool for the diagnosis of ovarian cancer. Analyses of larger cohorts will be required to determine the sensitivity, specificity and predictive values of VCP measurement for various forms and stages of ovarian cancer, as well as to determine if non-ovarian cancers also affect serum VCP levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3193. doi:10.1158/1538-7445.AM2011-3193

Negative Regulation of Human Granulosa Tumor Cell Survival by TGF-Beta.

Disruption to the regulatory pathways that govern granulosa cell proliferation, differentiation, and death during ovarian development and folliculogenesis can result in a variety of disorders, including granulosa cell tumor (GCT) formation. Candidate growth regulatory molecules produced within the ovary include several TGF-beta superfamily members, but the specific stimuli and intracellular signalling pathways which determine human granulosa cell proliferation and survival are still poorly understood. We have used a human ovarian cancer cell line of GCT origin, the KGN cells, to study TGF-beta superfamily actions. We have recently reported that this GCT line exhibited very little expression of the TGF-beta accessory receptor betaglycan, a TGF-beta and inhibin co-receptor which determines cellular sensitivity to these ligands. We hypothesized that GCTs would be refractory to the growth-inhibitory effects of TGF-beta and/or inhibin and that overexpression of betaglycan would restore responsiveness to TGF-beta mediated growth inhibition. To test this hypothesis, KGN cells were stably transfected with a wildtype betaglycan expression construct or the empty vector as a control. Overexpression of betaglycan in the KGN cells restored sensitivity to TGF-beta and inhibin in luciferase reporter assays but had little effect on cell proliferation, cell cycle progression, or death in either GCT cell line as determined by flow cytometry. Furthermore, treatment with either exogenous TGF-beta2 or inhibin A, INHA knockdown, or neutralisation of endogenous TGF-betas also did not significantly affect cell survival and proliferation. As the GCT cells are tumour cell lines which exhibit constitutively active NF-kappaB, which is a pro-survival factor, we pre-treated the cells with the chemical NF-kappaB inhibitor, BAY11-7082. Under these conditions, overexpression of betaglycan resulted in a decrease in proliferation (15%; p<0.05) and an increase in apoptosis (300% p<0.05). The addition of TGF-beta1 and TGF-beta2 dose-dependently decreased cell viability, and this effect was enhanced by the presence of betaglycan. However, knockdown of INHA expression did not affect cell viability even in the presence of BAY11-7082. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from betaglycan- and vector-transfected GCT cells. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that overexpression of betaglycan in the GCT cells increased both the basal and TGF-beta2-induced levels of activated SMAD3, with a smaller effect observed on activated SMAD2. Collectively, these data establish the TGF-beta-SMAD2/3-betaglycan signalling pathway as an important regulator of granulosa cell viability and suggest that deficiency of this pathway and/or inhibition by NF-kappaB contributes to dysregulated granulosa cell growth and cancer. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds. (platform)

GATA4 protects granulosa cell tumors from TRAIL-induced apoptosis

Disturbances in granulosa cell apoptosis have been implicated in the pathogenesis of human granulosa cell tumors (GCTs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cytokine that induces apoptosis in a variety of malignancies without toxic effects on benign cells. The aim of this study was to investigate the expression and functionality of the TRAIL receptors DR4 and DR5 in human GCTs. Additionally, we examined the role of GATA4, a transcription factor expressed in normal and malignant granulosa cells, in TRAIL-induced GCT apoptosis. For this purpose, a tissue microarray of 80 primary and 12 recurrent GCTs was subjected to immunohistochemistry for DR4 and DR5, and freshly isolated primary GCT cultures were utilized to evaluate the functional effects of TRAIL on GCT cells. To clarify the role of GATA4 in the regulation of TRAIL-induced apoptosis, a human GCT-derived cell line (KGN) was transduced with lentiviral vectors expressing small hairpin RNAs targeting GATA4 or transfected with adenovirus expressing either wild-type or dominant negative mutant GATA4. We found that receptors DR4 and DR5 are expressed in a vast majority of GCTs as well as in primary GCT cultures, and that TRAIL induces apoptosis in the primary GCT cultures. Moreover, we showed that overexpressing GATA4 protects GCTs from TRAIL-induced apoptosis in vitro, whereas disrupting GATA4 function induces apoptosis and potentiates the apoptotic effect of TRAIL administration. Our results demonstrate that the TRAIL pathway is functional in GCT cells, and suggest that transcription factor GATA4 may function as a survival factor in this ovarian malignancy.

Granulosa cell tumor with activated mTOR‐HIF‐1α‐VEGF pathway

The DNA-binding activity of hypoxia-inducible factor-1 alpha (HIF-1alpha) has been analyzed for various gynecological tumors. Among the tumors that were studied, there was a finding of a high level of DNA-binding HIF-1alpha activity, although it was limited to one case of adult type granulosa cell tumor (GCT). In this case a 60-year-old female had marked immunohistochemical expression of HIF-1alpha. The expressions of the mammalian target of rapamycin (mTOR) and phosphorylated-mTOR (p-mTOR) were also marked, and vascular endothelial growth factor (VEGF) was moderately expressed. To compare the expression profiles, 11 consecutive cases with adult type GCT were used. All cases showed marked expressions of HIF-1alpha and mTOR, but p-mTOR expression was moderately to markedly observed in four of the 12 cases. VEGF was expressed in all cases in varying degrees. Based on the evidence that downregulation of the mTOR pathway due to treatment with rapamycin (everolimus) would suppress tumor cell growth, an experimental study using the GCT cell line was designed to clarify whether HIF-1alpha and VEGF expressions decline. As a result, the expressions of p-mTOR, HIF-1alpha and VEGF were suppressed, but those of mTOR were not. It was concluded that mTOR-targeted therapy may represent a promising strategy for some GCT with an activated mTOR-HIF-1alpha-VEGF pathway.

Betaglycan as a Determinant of TGF-beta Superfamily Action in Human Granulosa Tumor Cells.

Granulosa cell tumors (GCTs) of the ovary belong to a group of ovarian sex cord stromal tumors that account for approximately 5% of total ovarian cancer incidences. The mechanisms by which ovarian granulosa cells undergo malignant transformation are poorly understood but are likely to involve the disruption of regulatory pathways that function during normal folliculogenesis. The type III TGF-beta receptor, betaglycan, and its ligands are essential regulatory factors in reproduction. Within the ovary, these factors regulate multiple processes in normal granulosa cell biology in a species-dependent manner. We have recently reported that human GCTs exhibit low betaglycan expression relative to the normal pre-menopausal ovary and that reduced betaglycan expression may contribute to the malignant behavior of granulosa tumor cells, increasing their ability to migrate, invade extracellular matrix, and metastasize. In two human GCT cell lines, the COV434 and KGN cells, we demonstrated that INHA gene silencing blocked the betaglycan-mediated inhibition of migration and invasion whereas both INHA gene silencing and TGF-beta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. The objective of the current study was to determine which signaling pathways are modulated by the presence of betaglycan on GCT cells. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from KGN and COV434 cells which had been stably transfected with a full-length wildtype betaglycan or a vector control. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that the COV434 and KGN cells exhibit different profiles of activation of each of the SMAD molecules, with COV434 cells exhibiting lower basal and stimulated levels of SMAD2, SMAD3, and SMAD1/5 than KGN cells. The presence of betaglycan did not significantly affect basal SMAD activation, but in both COV434 and KGN cells, the expression of betaglycan significantly increased the activation of SMAD2 in response to TGF-beta2. Furthermore, the knockdown of INHA expression further enhanced TGF-beta2 mediated SMAD2 activation in the betaglycan-expressing KGN cells. Collectively, our data indicate that the presence of betaglycan on GCT cells impacts on both inhibin- and TGF-beta-mediated GCT behaviors. Furthermore, the elimination of autocrine inhibin actions in the GCT cells enhanced TGF-beta2 stimulated SMAD2 activation in a betaglycan-dependent manner, suggesting that the presence of betaglycan on the surface of GCT cells counterbalances inhibin- and TGF-beta mediated signalling. (poster)

Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells

Betaglycan is a type III TGFβ receptor that modulates cellular sensitivity to inhibins and TGFβ. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFβ and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 ...

Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells

Betaglycan is a type III TGFbeta receptor that modulates cellular sensitivity to inhibins and TGFbeta. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFbeta and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells, decreased cellular invasion through Matrigel, and inhibited wound healing. Expression of mutant forms of betaglycan that are defective in TGFbeta and/or inhibin binding in each GCT cell line revealed that the inhibitory effects of betaglycan on wound healing were most strongly linked to the inhibin-binding region of betaglycan. Furthermore, knockdown of INHA mRNA expression abrogated the betaglycan-mediated inhibition of wound healing and invasion, whereas both INHA silencing and TGFbeta neutralization abolished the betaglycan-mediated increase in adhesion to substrate. These data suggest that loss of betaglycan contributes to the pathogenesis of GCTs.

Synergistic effects of Pten loss and WNT/CTNNB1 signaling pathway activation in ovarian granulosa cell tumor development and progression

The mechanisms of granulosa cell tumor (GCT) development may involve the dysregulation of signaling pathways downstream of follicle-stimulating hormone, including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this hypothesis, a genetically engineered mouse model was created to derepress the PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K antagonist gene Pten (Pten(flox/flox);Amhr2(cre/+)). The majority of Pten(flox/flox);Amhr2(cre/+) mice featured no ovarian anomalies, but occasionally ( approximately 7%) developed aggressive, anaplastic GCT with pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1 was abrogated in Pten(flox/flox);Amhr2(cre/+) GCT, indicating a mechanism by which GCT cells may increase proliferation and evade apoptosis. To relate these findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT expression were performed on human and equine tumors. Although PTEN loss was not detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has been reported, we tested whether these pathways could synergize in GCT. Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells of a mouse model (Pten(flox/flox);Ctnnb1(flox(ex3)/+);Amhr2(cre/+)) resulted in the development of GCT similar to those observed in Pten(flox/flox);Amhr2(cre/+) mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the ability to spread by seeding into the abdominal cavity. These data indicate a synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the development and progression of GCT and provide the first animal models for metastatic GCT.

GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors

Excessive cell proliferation and decreased apoptosis have been implicated in the pathogenesis of ovarian granulosa cell tumors (GCTs). We hypothesized that transcription factor GATA-4 controls expression of the antiapoptotic factor Bcl-2 and the cell cycle regulator cyclin D2 in normal and neoplastic granulosa cells. To test this hypothesis, a tissue microarray based on 80 GCTs was subjected to immunohistochemistry for GATA-4, Bcl-2, and cyclin D2, and the data were correlated to clinical and histopathological parameters. In addition, quantitative RT-PCR for GATA-4, Bcl-2, and cyclin D2 was performed on 21 human GCTs. A mouse GCT model was used to complement these studies. The role of GATA-4 in the regulation of Bcl2 and ccdn2 (coding for cyclin D2) was studied by transactivation assays, and by disrupting GATA-4 function with dominant negative approaches in mouse and human GCT cell lines. We found that GATA-4 expression correlated with Bcl-2 and cyclin D2 expression in human and murine GCTs. Moreover, GATA-4 enhanced Bcl-2 and cyclin D2 promoter activity in murine GCT cells. Whereas GATA-4 overexpression up-regulated and dominant negative GATA-4 suppressed Bcl-2 expression in human GCT cells, the effects on cyclin D2 were negligible. Our results reveal a previously unknown relationship between GATA-4 and Bcl-2 in mammalian granulosa cells and GCTs, and suggest that GATA-4 influences granulosa cell fate by transactivating Bcl-2.

Cisplatin-mediated sensitivity to TRAIL-induced cell death in human granulosa tumor cells

Objectives. The goal of the present study was to determine the efficacy of combinatorial treatment using cisplatin and tumor necrosis factor-related apoptosis including ligand (TRAIL) to promote apoptosis in granulosa cell tumor (GCT) lines, in vitro. Methods. Two human GCT lines (COV434 and KGN) were treated with cisplatin or TRAIL, alone or in combination. The cytotoxic effects of each treatment were evaluated using a methyl tetrazolium salt (MTS) assay. Initiation of TRAIL-induced apoptosis was verified by PARP- and FLIP-cleavage. Overexpression and knockdown studies were conducted to evaluate the role of p53 in TRAIL-induced cell death. Real-time PCR was used for gene expression analysis of the TRAIL receptor dr5 and the pro-apoptotic bax following treatment with cisplatin. Results. Treatment with TRAIL (100–200 ng/ml) led to a slight, but significant, loss of cell viability following an 18-h culture. This effect was enhanced following pre-treatment with cisplatin (25 μM) for 2 or 18 h. Moreover, pre-treatment with cisplatin decreased the maximal effective dose of TRAIL from 100 ng/ml to as low as 3 ng/ml in both cell lines. GCT lines overexpressing or deficient in p53 were used to determine the requirement for p53 on TRAIL-induced apoptosis. While the level of p53 expression enhanced both the death-inducing and TRAIL-sensitizing effects of cisplatin, TRAIL-induced cell death was found to occur independent of p53. Conclusions. These data suggest that the efficacy of cisplatin in GCT cells can be enhanced through combinatorial treatment with TRAIL. This result is due to both p53-dependent (cisplatin) and -independent (TRAIL) mechanisms. Combinatorial treatment of GCTs with cisplatin and TRAIL may provide an efficacious addition to cisplatin-based regimens.

CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell tumors (GCTs) of ovarian origin

BackgroundGranulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.MethodsWe examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control.ResultsFrequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.ConclusionsThese results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.

Differential expression of GRK isoforms in nonmalignant and malignant human granulosa cells.

Granulosa cell tumors are serious ovarian neoplasms that can occur in women of all ages. While there have been numerous attempts to understand the cause of these malignancies, the pathogenesis of granulosa cell tumors (GCTs) still remains largely unknown. G-protein coupled receptor kinases (GRKs) are important regulators of signal transduction through the process of receptor desensitization and internalization. Receptors that are regulated by GRKs are members of the large family of seven-transmembrane receptors and include the follicle stimulating hormone receptor (FSHR). In granulosa cells, the FSH signaling system is responsible for cell proliferation, differentiation, and steroidogenesis. In the studies presented, we examined GRK mRNA and protein expression in nonmalignant human granulosa cells, in KGN cells, a human GCT cell line, and in a panel of human GCT samples. The KGN tumor cells express significantly less GRK4 alpha/beta protein and higher levels of GRK2 and GRK4 gamma/delta protein as compared to nonmalignant human granulosa cells. In human GCT samples, GRK4 alpha/beta protein was detected in 3 of the 13 tumor samples, whereas gamma/delta proteins expression was detected in all samples. These findings suggest that GRK protein expression is altered in GCTs and may be involved in the pathogenesis of these tumors.

Epidermal growth factor receptors in spontaneous ovarian granulosa cell tumors of SWR-derived mice.

Epidermal growth factor (EGF) receptor binding properties were examined in spontaneous ovarian granulosa cell (GC) tumors from SWR and SWR-derived strains of mice. EGF binding was measured at room temperature in tissue homogenates from GC tumors and normal ovaries from adult randomly cycling mice. GC tumor tissue displayed significantly increased EGF binding and 2 receptor populations (R1 and R2). Normal ovarian tissue appeared to have only one receptor population with a dissociation constant (KD) similar to the R1 (high-affinity) receptor in GC tumors. In subsequent experiments, GC tumor and normal granulosa cells from immature mice were analyzed in primary cultures for EGF binding, immunofluorescence microscopy for receptors, and cell proliferation. After 24 hr in culture, the GC tumors bound 10-fold more EGF/micrograms protein than did normal granulosa cells. GC tumor cells, but not normal granulosa cells, showed specific immunofluorescence when reacted with a polyclonal antibody to mouse EGFR. During 96 hr in culture, GC tumor cells, but not normal cells, showed a significant proliferative response to EGF. In conclusion, the EGF binding capacity is markedly increased in GC tumor cells and the proliferation data suggest that this growth factor supports tumor growth in the SWR model system.