Procaspase-Activating Compound-1 Synergizes with TRAIL to Induce Apoptosis in Established Granulosa Cell Tumor Cell Line (KGN) and Explanted Patient Granulosa Cell Tumor Cells In Vitro.

Granulosa cell tumors (GCT) constitute only ~5% of ovarian neoplasms yet have significant consequences, as up to 80% of women with recurrent GCT will die of the disease. This study investigated the effectiveness of procaspase-activating compound 1 (PAC-1), an activator of procaspase-3, in treating adult GCT (AGCT) in combination with selected apoptosis-inducing agents. Sensitivity of the AGCT cell line KGN to these drugs, alone or in combination with PAC-1, was tested using a viability assay. Our results show a wide range in cytotoxic activity among the agents tested. Synergy with PAC-1 was most pronounced, both empirically and by mathematical modelling, when combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This combination showed rapid kinetics of apoptosis induction as determined by caspase-3 activity, and strongly synergistic killing of both KGN as well as patient samples of primary and recurrent AGCT. We have demonstrated that the novel combination of two pro-apoptotic agents, TRAIL and PAC-1, significantly amplified the induction of apoptosis in AGCT cells, warranting further investigation of this combination as a potential therapy for AGCT.

New small-molecule compound Hu-17 inhibits estrogen biosynthesis by aromatase in human ovarian granulosa cancer cells.

Estrogen-dependent cancers (breast, endometrial, and ovarian) are among the leading causes of morbidity and mortality in women worldwide. Aromatase is the main enzyme that catalyzes the biosynthesis of estrogen, which drives proliferation, and antiestrogens can inhibit the growth of these estrogen-dependent cancers. Hu-17, an aromatase inhibitor, is a novel small-molecule compound that suppresses viability of and promotes apoptosis in ovarian cancer cells. Therefore, this study aimed to predict targets of Hu-17 and assess its intracellular signaling in ovarian cancer cells. Using the Similarity Ensemble Approach software to predict the potential mechanism of Hu-17 and combining phospho-proteome arrays with western blot analysis, we observed that Hu-17 could inhibit the ERK pathway, resulting in reduced estrogen synthesis in KGN cells, a cell line derived from a patient with invasive ovarian granulosa cell carcinoma. Hu-17 reduced the expression of CYP19A1 mRNA, responsible for producing aromatase, by suppressing the phosphorylation of cAMP response element binding-1. Hu-17 also accelerated aromatase protein degradation but had no effect on aromatase activity. Therefore, Hu-17 could serve as a potential treatment for estrogen-dependent cancers albeit further investigation is warranted.

Effects of human blood levels of two PAH mixtures on the AHR signalling activation pathway and CYP1A1 and COMT target genes in granulosa non-tumor and granulosa tumor cell lines.

Epidemiological studies have shown a link between problems with offspring of couples living in a contaminated environment in comparison to those who live in an uncontaminated environment. We measured the concentrations of 16 priority polycyclic aromatic hydrocarbons (PAHs) in maternal and cord blood. To explore the mechanism of the effects of PAH mixtures on nonluteinized granulosa cells (HGrC1) and granulosa tumor cells (COV434), as well as cell proliferation and apoptosis, we investigated the effect of PAH mixtures on the expression of the aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and aryl hydrocarbon receptor repressor (AHRR) genes, as well as the expression and activity of target genes cytochrome P450 1A1 (CYP1A1) and catechol-O-methyltransferase (COMT). The cells were exposed to mixture 1 (M1), composed of all 16 priority PAHs, and mixture 2 (M2), composed of five PAHs which are not classified as human carcinogens, and which are observed in the highest amounts both in maternal and cord blood. All 16 priority PAHs were bioavailable in maternal and cord plasma, suggesting that perinatal exposure should be considered. In HGrC1 cells, M1 increased AHR and ARNT, but decreased AHRR expression, in parallel with increased CYP1A1 and COMT expression and activity. M2 decreased AHR and AHRR, and increased ARNT, with no effect on CYP1A1 expression and activity; however, it did increase COMT expression and activity. In tumor cells, M1 lowered AHR and up-regulated AHRR and ARNT expression, consequently decreasing CYP1A1 expression and COMT activity. M2 up-regulated AHR and ARNT, down-regulated AHRR, and had no effect on CYP1A1 and COMT expression, but decreased COMT activity. We hypothesise that, dependent on composition, mixtures of PAHs activate the AHR differently through varying transcription responses: in HGrC1, a canonical AHR mechanism of M1, with activation of CYP1A1 important for detoxication, while in COV434, a noncanonical AHR mechanism, probably by activation the nuclear factor NFkB.

Adult granulosa cell tumors of the ovary: a retrospective study of 30 cases with respect to the expression of steroid synthesis enzymes.

OBJECTIVE: Some, but not all, granulosa cell tumors are characterized by estrogen production. This study was designed to determine whether there are clinical or pathological variations in granulosa cell tumors in relation to the expression of sex steroid synthesis enzymes. METHODS: Clinical symptoms, serum hormonal values, and histology of 30 granulosa cell tumor patients who underwent surgery between 2002 and 2014 were retrospectively reviewed. RESULTS: Most patients presented with abnormal genital bleeding including abnormal menstrual cycles. Eight of 16 patients older than 50 years had endometrial hyperplasia and one had endometrial cancer. Serum 17ß-estradiol (E2) levels tended to be higher in patients over 50 years of age (p=0.081). Serum follicle-stimulating hormone (FSH) levels were low in all patients irrespective of serum E2 levels. Magnetic resonance imaging revealed a thicker endometrium in older as compared to younger patients (p<0.05). Tumor cells in the majority of cases were positive for inhibin α and P450 aromatase, irrespective of age and serum E2 levels. P450 17α-hydroxylase (P450c17) expression varied among cases. P450c17 was strongly positive in luteinized tumor cells and weakly positive in theca cells and fibroblasts. High E2 levels were associated with P450c17-positive cells in the tumor (p<0.05). CONCLUSION: The expression of hormone-synthesizing enzymes divides granulosa cell tumors into 2 distinct types; tumors with P450c17-positive cells show elevated serum E2 and related clinical symptoms, while tumors without these cells show symptoms related to FSH suppression by inhibin.

Expression of P450 Aromatase in Granulosa Cell Tumors and Sertoli-Stromal Cell Tumors of the Ovary: Which Cells Are Responsible for Estrogenesis?

Granulosa cell tumors are representative of estrogenic ovarian tumors, and some Sertoli-stromal cell tumors are also estrogenic. The exact cells that are responsible for estrogenesis, however, have yet to be identified. In the present study, 25 sex cord-stromal tumors (20 granulosa cell tumors, 4 Sertoli-Leydig cell tumors, and a Sertoli cell tumor) were immunohistochemically examined for expression of P450 aromatase, which is critical for estrogenesis. All of the tumors had been evaluated for estrogenic function, including contemporaneous endometrial hyperplasia and/or elevation of serum estradiol. Eleven of 14 estrogenic granulosa cell tumors showed sparse or aggregated immunoreactivity for aromatase, whereas 5 of 6 nonestrogenic tumors did not. Aromatase was selectively expressed by plump granulosa cells with eosinophilic or vacuolated cytoplasm, resembling luteinized granulosa cells. Such a localization of aromatase is analogous to that in normal ovaries. Aromatase expression in primary tumors was recapitulated by recurrent tumors. In Sertoli-stromal cell tumors, either undifferentiated plump cells or well-differentiated Sertoli cells expressed aromatase. In conclusion, the expression of P450 aromatase corresponds to specific cell morphology in sex cord-stromal tumors, including recurrent tumors. Aromatase status in granulosa cell tumors provides helpful information on whether serum estradiol could be a marker for recurrence.

A Hot-spot of In-frame Duplications Activates the Oncoprotein AKT1 in Juvenile Granulosa Cell Tumors.

BACKGROUND: Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of Gαs, which transduces mitogenic signals, in 30% of the analyzed JGCTs. METHODS: We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K-AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays. FINDINGS: We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays. INTERPRETATION: Our study incriminates somatic mutations of AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols.

Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition.

Epidermal growth factor receptor (EGFR) is implicated in the progression of many human cancers, but its significance in ovarian granulosa cell tumor (GCT) pathobiology remains poorly understood. We assessed the EGFR gene copy number, surveyed the mRNA and protein expression patterns of EGFR in 90 adult GCTs, and assessed the in vitro sensitivity of GCT cells to EGFR inhibition. Low-level amplification of EGFR gene was observed in five GCTs and high-level amplification in one sample. EGFR mRNA was robustly expressed in GCTs. Most tumors expressed both unphosphorylated and phosphorylated EGFR protein, but the protein expression did not correlate with clinical parameters, including the risk of recurrence. Small-molecule EGFR inhibitors reduced the EGF-induced activation of EGFR and its downstream signaling molecules at nanomolar doses, but cell viability was reduced, and caspase-3/7 was activated in GCT cells only at micromolar doses. Based on the present results, EGFR is active and abundantly expressed in the majority of GCTs, but probably has only minor contribution to GCT cell growth. Given the high doses of EGFR inhibitors required to reduce GCT cell viability in vitro, they are not likely to be effective for GCT treatment as single agents; they should rather be tested as part of combination therapies for these malignancies.

Characterization of the inhibitor of kappaB kinase (IKK) complex in granulosa cell tumors of the ovary and granulosa cell tumor-derived cell lines.

Granulosa cell tumors of the ovary (GCT) are a distinct, hormonally active subset of ovarian cancers. Although it has recently been shown that ∼97 % of all adult GCT harbor a novel somatic missense mutation in the FOXL2 gene, given its almost universal presence, it does not explain differences in tumor stage and/or recurrence. The nuclear factor kappaB (NFκB) transcription factor is constitutively active in two human GCT-derived cell lines, COV434 and KGN, which are useful in vitro models to investigate juvenile and adult GCT, respectively. This study aimed to determine the molecular basis and pathogenetic significance of this aberrant NFκB activity. Selective chemical inhibitors were used to target candidate components of the pathway. The constitutive activity was blocked by two independent inhibitors of IκBα phosphorylation, suggesting that aberrant activation occurs upstream of this point. NFκB inhibition resulted in a dose-dependent decrease in cell proliferation and viability and a dose-dependent increase in apoptosis. Inhibitors of earlier components of the pathway were without effect. Two independent inhibitors of inhibitor of kappaB kinase (IKK)ß, a catalytic subunit of the NFκB activation complex, were unable to inhibit the constitutive activity, but surprisingly also ligand-induced activity. These findings suggest a central role for IKKß; however, no mutations or altered expression of the IKKß, IKKα, or IKKγ genes was observed in the cell lines or in a panel of human GCT samples. This study highlights unresolved issues in understanding the pathogenesis of GCT and in the use of the COV434 and KGN cells lines as model systems.

Betaglycan alters NFκB-TGFß2 cross talk to reduce survival of human granulosa tumor cells.

The molecular pathways controlling granulosa cell tumor (GCT) survival are poorly understood. In many cell types, nuclear factor-κB (NFκB) and TGFß coordinately regulate cell survival to maintain tissue homeostasis. Because GCT cell lines exhibit constitutively activated NFκB, we hypothesized that NFκB blocks TGFß-mediated cell death in GCT cells. To test this hypothesis, we used the human GCT cell line KGN, which exhibits loss of betaglycan, a TGFß co-receptor. After inhibition of NFκB in KGN cells, re-expression of betaglycan resulted in a decrease in cell viability, which was further decreased by TGFß2. Intriguingly, TGFß2 increased NFκB reporter activity in control cells, but betaglycan expression suppressed both basal and TGFß2-stimulated NFκB activity. Chemical inhibition of Mothers against decapentaplegic homolog 2/3 (SMAD2/3) signaling or SMAD2/3 gene silencing revealed that both SMADs contributed to cell survival. Furthermore, inhibiting NFκB activity resulted in a specific reduction in SMAD3 expression. Conversely, overexpression of SMAD3 increased basal NFκB activity and countered betaglycan-mediated suppression of NFκB activity. Finally, ERK1/2 activation emerged as the point of convergence of NFκB, SMAD3, and TGFß2/betaglycan governance of GCT cell viability. Key findings in KGN cells were reproduced in a second GCT cell line, COV434. Collectively, our data establish that both SMAD2/3 and NFκB signaling pathways support GCT cell viability and suggest the existence of a positive feedback loop between NFκB and SMAD3 signaling in late-stage GCT. Furthermore, our data suggest that loss of betaglycan during tumor progression in GCT alters the functional outcomes generated by NFκB and TGFß pathway cross talk.

Unusual virilization in girls with juvenile granulosa cell tumors of the ovary is related to intratumoral aromatase deficiency.

BACKGROUND: Hyperandrogenism is a rare symptom of juvenile ovarian granulosa cell- tumors (JGCTO). This study aimed to determine whether hyperandrogenism was related to overexpression of SOX9, decreased expression of FOXL2 or absent aromatase expression in tumor with particular scheme of expression of P450scc and P450c17alpha. METHODS: Through a nationwide study including the French Society of Pediatric Oncology, 6/30 patients with JGCTO presented with clinical hyperandrogenism and high plasma testosterone. Tumor specimens underwent immunofluorescence (SOX9, FOXL2) and immunochemistry (aromatase, P450scc, P450c17alpha). Results were compared with patients without hyperandrogenism. RESULTS: SOX9 was expressed in the granulosa cell nucleus in 2/6 cases but also in 9/24 tumors without hyperandrogenism (p=n.s.). FOXL2 was absent or decreased in 3/6 cases of JGCTO with hyperandrogenism with no statistical difference from the group without this symptom. In 6/6 patients, the intratumoral expression of aromatase was absent (n=5) or dramatically reduced (n=1). In contrast, 15/24 patients without virilization exhibited conserved aromatase expression in their tumor (p<0.05). A variable number of tumoral cells expressed P450scc while some interstitial cells were focally immunopositive for P450c17alpha. CONCLUSION: Unusual virilization in girls with JGCTO is not explained by a dysregulation in SOX9 or FOXL2 expression, but is related to a localized defect of aromatase expression in granulosa cells and to the ability of interstitial cells to produce testosterone.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: immunohistochemical expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450.

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries.

Dysregulation of WNT/CTNNB1 and PI3K/AKT signaling in testicular stromal cells causes granulosa cell tumor of the testis.

Synergistic effects of dysregulation of the WNT/CTNNB1 and phosphatidylinositol 3-kinase (PI3K)/AKT pathways are thought to be important for the development and progression of many forms of cancer, including the granulosa cell tumor of the ovary. Sustained WNT/CTNNB1 signaling in Sertoli cells causes testicular degeneration and the formation of foci of poorly differentiated stromal cells in the seminiferous tubules in mice. To test if concomitant dysregulation of the WNT/CTNNB1 and PI3K/AKT pathways could synergize to cause testicular cancer, Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) mice that express a dominant, stable CTNNB1 mutant and lack the expression of phosphatase and tensin homolog (PTEN) in their Sertoli cells were generated. These mice developed aggressive testicular cancer with 100% penetrance by 5 weeks of age, and 44% of animals developed pulmonary metastases by 4 months, whereas Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) controls were phenotypically normal. Surprisingly, the tumors could not be classified as Sertoli cell tumors, but rather bore histologic and ultrastructural characteristics of granulosa cell tumors of the testis (GCTT). Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testicular tumors did not express CYP17, CYP19, germ cell nuclear antigen, estrogen receptor 1 or progesterone receptor, but expressed the early granulosa cell markers WNT4 and FOXL2, confirming the diagnosis of GCTT. Immunohistochemical analyses of Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) GCTT demonstrated a tumor marker profile similar to that reported in human GCTT. Immunoblotting analyses revealed high levels of phosphorylation of AKT and the PI3K/AKT signaling effector FOXO1A in Pten(tm1Hwu/tm1Hwu);Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) GCTT, suggesting the involvement of FOXO1A in the mechanism of GCTT development. Together, these data provide the first insights into the molecular etiology of GCTT and the first animal model for the study of GCTT biology.

Expression of c-kit and platelet-derived growth factor receptors in ovarian granulosa cell tumors.

OBJECTIVE: This study aimed at evaluating the expression of tyrosine kinase receptors c-kit, (platelet-derived growth factor receptor-alpha (PDGFR-alpha), and PDGFR-beta in ovarian granulosa cell tumors (GCTs). STUDY DESIGN: Primary ovarian GCT specimens were obtained for immunohistochemical staining.The expressions of c-kit, PDGFR-alpha, and PDGFR-beta were analyzed and scored by a semiquantitative (SQ) method. Normal ovarian tissue from the same patients' specimens served as internal controls. RESULTS: A total of 21 specimens were available for evaluation. C-kit was expressed in only 2 samples, whereas both PDGFR-alpha and PDGFR-beta stained positive in 100% of tumors. PDGFR targets demonstrated strong positive expression in intensity and amount of tissue stained. Normal ovarian tissue demonstrated complete absence of staining for all 3 antibodies evaluated. CONCLUSIONS: The data demonstrated significant expression of PDGFR targets of imatinib mesylate in GCTs, whereas normal ovarian tissues had a complete absence of staining.This expression profile provides the rationale to investigate the role of imatinib mesylate in PDGFR-positive GCTs.

Inhibition of proteasome activity sensitizes human granulosa tumor cells to TRAIL-induced cell death.

Human granulosa tumor cell (GCT) lines (KGN and COV434) were utilized to establish the combinatorial effects of TRAIL treatment and a proteasome inhibitor on cell viability, in vitro. TRAIL induced a slight, but consistent, decrease in viability for both cell lines, and pharmacologic inhibition of proteasome activity, using Z-LLF-CHO (Z-LLF), synergistically enhanced TRAIL-induced loss of viability. This enhanced sensitization was associated with the up-regulation of a TRAIL receptor, DR5, and pro-apoptotic Bax. Targeted reduction of p53 expression revealed that the ability of Z-LLF to enhance DR5 and Bax expression occurs independent of p53 activity. These studies underscore the potential to develop targeted treatments for GCTs using established cell lines.

Expression status and mutational analysis of the ras and B-raf genes in ovarian granulosa cell and epithelial tumors.

OBJECTIVES: The molecular pathogenesis of granulosa cell tumors of the ovary (GCT) is not understood. Activating mutations in the K-, N-, and H-ras protooncogenes have been identified in a wide range of human cancers, including ovarian epithelial tumors. Furthermore, an apparent association has recently been reported between the presence of ras and B-raf mutations in the same cancer types. Activation of the ras/raf pathway would be predicted to be tumorigenic in granulosa cells. METHODS: Gene expression patterns of the three ras and B-raf genes were determined in a panel of GCT and ovarian epithelial tumors, and in normal premenopausal ovaries. Expression was determined by RT-PCR using gene-specific primers combined with Southern blot analysis of the PCR products using gene-specific (32)P-labeled probes. Direct sequence analysis was used to screen for known activating mutations. RESULTS: Widespread expression of the four genes was observed in all tumor types examined. Compared to the normal ovaries, none of the genes was expressed at significantly higher levels in any of the tumor types examined. A heterozygous point mutation in codon 12 of the K-ras gene was found in five of the 10 mucinous tumors. No B-raf mutations were detected in the mucinous tumors. No mutations were detected in any of the genes in the cohort of GCT. CONCLUSION: These results suggest that neither overexpression nor activating mutations of the ras or B-raf genes are associated with the development of GCT.

A novel nonradioactive method for measuring aromatase activity using a human ovarian granulosa-like tumor cell line and an estrone ELISA.

Aromatase is a key enzyme in steroidogenesis and plays an important role in sexual differentiation, fertility, and carcinogenesis. Importantly, a variety of chemicals in the environment may influence its activity and thereby disrupt endocrine function. In the current studies, we developed a novel nonradioactive method for measuring aromatase activity that uses a specific ELISA for estrone along with KGN human ovary granulosa-like carcinoma cells. This cell line has relatively high aromatase activity, and because it lacks 17alpha-hydroxylase, it secretes little or no androstenedione, 17beta-estradiol, or estrone. Therefore, aromatase activity can be assayed simply by measuring the production of estrone in the culture medium after addition of the substrate, androstenedione. Furthermore, by making a slight change in the commercial ELISA kit and optimizing the experimental conditions, we developed a sensitive aromatase assay that could measure a wide range of estrone concentrations with very low interference by androgens. We used this assay to investigate the effects of 23 chemicals that have been previously reported to affect aromatase activity in vitro. We confirmed that 17 of 23 test chemicals had inhibitory or inducible effects, although the specific effects of some were different than previously reported. In conclusion, we have developed a simple, sensitive, and nonradioactive assay that can be used for large-scale screening of compounds that can disrupt endocrine function by influencing aromatase activity.

Evidence of a role for the INK4 family of cyclin-dependent kinase inhibitors in ovarian granulosa cell tumors.

Granulosa cell tumors (GCTs) of the ovary are relatively rare and account for <5% of all ovarian cancers. The molecular pathogenesis of these tumors is not well understood. We tested the hypothesis that cyclin-dependent kinase inhibitors, specifically the inhibitors of the cyclin-dependent kinase 4 (INK4) family, are targets for altered gene expression in GCTs. The status of RB1, INK4A, INK4B, INK4C, INK4D, and ARF in 13 adult and 2 juvenile ovarian GCTs was determined by reverse transcription-polymerase chain reaction of total RNA and exon-specific sequencing of genomic DNA. Tumors showing loss of INK4A expression were assayed further by exon-deletion analysis and methylation-specific PCR. None of the juvenile tumors demonstrated altered expression, but 7/12 (58%) adult GCTs lacked expression of INK4A, INK4B, or both. In one of these cases, we noted a homozygous deletion of the INK4A locus, and in the remaining tumors we found hypermethylation of the promoter region, a mechanism that can lead to gene inactivation. These data support a role for the INK4 family of CDK inhibitors in the biology of GCTs.

Combined treatment with specific ligands for PPARgamma:RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells.

Our previous study demonstrated that PPARgamma specific ligand troglitazone (TGZ) or RXR specific ligand LG100268 (LG) alone decreased the aromatase activity in cultured human ovarian granulosa cells from pre-ovulatory follicles, and combined treatment caused an even greater reduction in this activity. Since similar manners of effects of TGZ or/and LG on the aromatase activity in human ovarian granulosa cancer cell line were observed, we performed the detailed analysis of the mechanisms of these effects using this cell line. The changes in the aromatase activity were associated with comparable changes in the P450arom mRNA levels based on a RNase protection assay. A nuclear run-on assay indicated the P450arom transcript to decrease by 40 and 66% at 24 and 48 h, respectively, after TGZ plus LG treatment. An RNA stability analysis showed the half-life of P450arom mRNA to decrease from 13 to 9 h after the TGZ plus LG treatment. The inhibitory effect of TGZ plus LG on the aromatase activity and P450arom mRNA may not be mediated by the cAMP-PKA pathway that is usually implicated in the regulation of aromatase activity in granulosa cells: because (1) the aromatase activity stimulated by forskolin was not inhibited by TGZ plus LG; (2) the specific PKA inhibitor H89 could not block the inhibitory effect of TGZ plus LG on the aromatase activity; and (3) the luciferase activity of P450arom promoter II did not decrease by the addition of TGZ and LG in transfected cells either at a basic state or in the states stimulated by forskolin or PGE2, respectively. Taken together, these results indicate that TGZ plus LG inhibited the aromatase activity and also decreased the P450arom mRNA level in granulosa cancer cells, and the loss of P450arom mRNA expression was considered to be due to both the decreased transcription and rapid degradation of its RNA.

Immunolocalization of aromatase P-450 in ovarian tissue from pregnant and nonpregnant mares and in ovarian tumours.

Aromatase P-450 (P-450arom) is a crucial regulatory enzyme that is necessary for conversion of androgens to oestrogens. Corpora lutea and follicles were obtained from the ovaries of cyclic mares and from mares at day 20 and days 40-70 of pregnancy. The presence of P-450arom within specific cell types was investigated by immunostaining to determine potential sites of oestrogen synthesis. Immunoreactivity for P-450arom was confined to the granulosa layer of non-atretic follicles > 5 mm in diameter and to corpora lutea at all stages of the oestrous cycle and during pregnancy. These findings confirm that aromatization of androgens occurs within the granulosa cells of the preovulatory follicle of the mare and that the corpus luteum of the mare has the capacity for oestrogen production if adequate androgen substrate is available. Granulosa cells in ovarian tissue from three mares with granulosa cell tumours showed little staining for P-450arom which suggests that these tumours have little aromatizing capacity.