Ovarian Surface Epithelial Cell Lines Research Articles

Differential Expression of Claudin Family Proteins in Mouse Ovarian Serous Papillary Epithelial Adenoma in Aging FSH Receptor-Deficient Mutants

Ovarian cancer is a deadly disease with long latency. To understand the consequences of loss of folliclestimulating hormone receptor (FSH-R) signaling and to explore why the atrophic and anovulatory ovaries of follitropin receptor knockout (FORKO) mice develop different types of ovarian tumors, including serous papillary epithelial adenoma later in life, we used mRNA expression profiling to gain a comprehensive view of misregulated genes. Using real-time quantitative reverse transcription-polymerase chain reaction, protein analysis, and cellular localization, we show, for the first time, in vivo evidence that, in the absence of FSH-R signaling, claudin-3, claudin-4, and claudin-11 are selectively upregulated, whereas claudin-1 decreases in ovarian surface epithelium and tumors in comparison to wild type. In vitro experiments using a mouse ovarian surface epithelial cell line derived from wild-type females reveal direct hormonal influence on claudin proteins. Although recent studies suggest that cell junction proteins are differentially expressed in ovarian tumors in women, the etiology of such changes remains unclear. Our results suggest an altered hormonal environment resulting from FSH-R loss as a cause of early changes in tight junction proteins that predispose the ovary to late-onset tumors that occur with aging. More importantly, this study identifies claudin-11 overexpression in mouse ovarian serous cystadenoma.

A Mouse Model for the Molecular Characterization of Brca1-Associated Ovarian Carcinoma

Little is known about the mechanisms that underlie Brca1-associated ovarian tumorigenesis, mainly due to the lack of an appropriate experimental model. We developed genetically defined primary mouse ovarian surface epithelial (OSE) cell lines in which the loss of functional Brca1 and p53 recapitulates the events that are thought to occur in early ovarian cancer development in patients with Brca1 mutations. This system allows for the introduction of additional oncogenes that are thought to cooperate with the loss of Brca1 and p53 to induce tumorigenesis. We showed that Myc is sufficient to induce transformation of ovarian cells that are deficient for both Brca1 and p53 but not sufficient for the transformation of cells that are deficient for either Brca1 or p53. The transformed Brca1-deficient OSE cells display an increased number of centrosomes, acquire complex chromosome aberrations, and lack Rad51 nuclear foci in the presence of DNA-damaging agents, such as mitomycin C and cisplatin. Immunocompetent mice injected with transformed OSE cells develop tumors that resemble human metastatic serous ovarian carcinoma, the most common type of ovarian cancer in women. Consistent with the reported platinum chemosensitivity in patients with Brca1-associated ovarian cancer, the Brca1-deficient OSE cells have increased sensitivity to the DNA-damaging agent cisplatin, whereas sensitivity to the microtubule poison paclitaxel is similar between Brca1 wild-type and Brca1-deficient cells. The Brca1 wild-type and Brca1-deficient mouse ovarian tumors and cell lines provide a new experimental system for the evaluation of therapies that target the Brca1 pathway.

Nonphotochemical Hole-Burning Study of Selectively Stained Normal and Cancerous Human Ovarian Tissues

Results are presented of nonphotochemical hole-burning (HB) experiments on cancerous ovarian and analogous normal peritoneal in vitro tissues stained with the mitochondrial-selective dye rhodamine 800. A comparison of fluorescence excitation spectra, hole-growth kinetics data, and external electric field (Stark) effects on the shape of spectral holes burned in cancerous and normal tissues stained with rhodamine 800 revealed significant differences only in the dipole moment change (fDeltamu) measured by a combination of HB and Stark spectroscopies. It is shown that the permanent dipole moment change for the S0--> S1 transition of the rhodamine 800 molecules in cancerous tissue is higher than that of normal tissue by a factor of about 1.4. The finding is similar to the HB results obtained earlier for human ovarian surface epithelial cell lines, i.e., OV167 carcinoma and OSE(tsT)-14 normal cells stained with the same mitochondria-specific dye (Walsh et al. Biophys. J. 2003, 84, 1299). We propose that the observed difference in the permanent dipole moment change in cancerous ovarian tissue is related to a modification in mitochondrial membrane potential.

CD80 in Immune Suppression by Mouse Ovarian Carcinoma–Associated Gr-1+CD11b+ Myeloid Cells

An elevated number of Gr-1+CD11b+ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1+CD11b+ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1+CD11b+ cells from naïve mice. However, the expression of CD80 by Gr-1+CD11b+ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1+CD11b+ cells, this phenomenon likely requires direct interaction. Gr-1+CD11b+ cells derived from 1D8 tumor-bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1+CD11b+ cells from naïve mice did not. Both short interfering RNA-mediated knockdown and genetic knockout of CD80 expression by Gr-1+CD11b+ cells of 1D8 tumor-bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1+CD11b+ myeloid cells was mediated by CD4+CD25+ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1+CD11b+ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4+CD25+ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.

Defining the mechanisms by which leptin stimulates proliferation of ovarian cancer cells

15017 Background: Ovarian cancer (OvCa) ranks fourth as the cause of death related to cancer in women in the U.S. The vast majority (>90%) of OvCa originates from the ovarian surface epithelium. There is sufficient evidence to suggest that hormones, especially estrogen, may be involved in the etiopathogenesis of epithelial OvCa. Recent studies indicate that leptin participates either directly or indirectly to promote carcinogenesis in both breast and endometrial cancers. Furthermore it has been proposed that leptin may elicit its action via an estrogen related pathway. Leptin can stimulate proliferation of some OvCa cell lines and has been implicated as a potential biomarker for OvCa. However the mechanism(s) by which leptin contributes to the growth of OvCa has yet to be defined. We hypothesize that leptin’s effect will be mediated in part by estrogen receptor (ER) pathways. Methods: Three epithelial OvCa cell lines (IGROV1, OVCAR5 and TOV21G) and one benign human ovarian surface epithelial cell line (HOSE) were evaluated. Enzyme linked immunosorbent assay (ELISA) and Western blotting were used to assess leptin and leptin receptor (ObR), respectively. Leptin (0.06 nM–6.25 nM) induced effects on cell proliferation were assessed in the presence or absence of an aromatase enzyme inhibitor (Anastrozole) or the ER antagonist (ICI182780). Further, we explored leptin-induced effects on ERα promoter activity as evidenced by change in fluorescence via a dual luciferase promoter reporter. All experiments were conducted in triplicate. All data were subjected to ANOVA followed by Tukey’s post hoc test (p < 0.05). Results: All ovarian cell lines expressed ObR; whereas, no measurable amounts of leptin were detected in conditioned media. Leptin stimulated cell proliferation in both the benign and malignant lines. Leptin-induced cell proliferation was inhibited by Anastrozole and ICI182780. Furthermore, leptin stimulated luciferase activity of the ERα promoter/reporter. Conclusions: Leptin promotes proliferation of benign and malignant ovarian epithelial cells and appears to be mediated, at least in part, via aromatase and ER which may have therapeutic implications. This work was supported by the Vincent Memorial Hospital, SG Komen Foundation and the Advanced Medical Research Foundation. No significant financial relationships to disclose.

Eukaryotic elongation factor 1 alpha 2 alone is not sufficient to cause tumorigenicity in immortalized and neoplastic ovarian cell lines

15051 Background: EEF1a2 is amplified and overexpressed in 25% of ovarian tumors. Previous studies have shown EEF1a2 to have oncogenic and tumorigenic properties in rodent fibroblasts. However, its role in the tumorigenesis and behavior of human ovarian carcinomas is not known. Methods: Stable expression of EEF1a2 cDNA in both T80 immortalized human ovarian surface epithelial cells and SKOV-3 cell line was followed by standard proliferation assays, soft agar assays for anchorage independent growth, cell cycle analysis, mouse xenograft injections, evaluation for changes in factors such as VEGF and ras and susceptibility to CDDP. Transient transfections with 3 different siRNA’s to EEF1a2 were performed in both cell lines. Results: Stable expression of EEF1a2 was detected by Western Blot and IP. Compared to vector only control, transfection with pCMVTag2B EEF1a2 vector did not change proliferation in either cell line. Anchorage independent growth was slightly higher in the T80 transfected cells. Cell cycle and expression of VEGF and ras were not different than control. IC50 of CDDP was similar between the transfected and control cell lines. No size difference was seen in SKOV transfected and control xenografts. T80 controls grew larger and more frequent tumors than transfected controls. Transient transfection of T80 with siRNA did not result in changes in anchorage independent growth. Conclusion: EEF1a2 alone is not sufficient to act induce tumorigenicity, affect tumor growth rate or drug susceptibility in human ovarian epithelial cells. Experiments utilizing stable siRNA vectors specific to EEF1a2 are underway, as well the combination of EEF1a2 with recognized ovarian tumor oncogenes. No significant financial relationships to disclose.

Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells

Although gonadotropin-releasing hormone (GnRH) has been shown to play a role as an autocrine/ paracrine regulator of cell growth in ovarian surface epithelium and ovarian cancer, the factors which regulate the expression of GnRH and its receptor in these cells are not well characterized. In the present study, we employed real-time PCR to determine the potential regulatory effect of gonadotropins on the expression levels of GnRH I (the mammalian GnRH), GnRH II (a second form of GnRH) and their common receptor (GnRHR) in immortalized ovarian surface epithelial (IOSE-80 and IOSE-80PC) cells and ovarian cancer cell lines (A2780, BG-1, CaOV-3, OVCAR-3 and SKOV-3). The cells were treated with increasing concentrations (100 and 1000 ng/ml) of recombinant follicle-stimulating hormone (FSH) or luteinizing hormone (LH) for 24 h. Treatment with FSH or LH reduced GnRH II mRNA levels in both IOSE cell lines and in three out of five ovarian cancer cell lines (A2780, BG-1 and OVCAR-3). A significant decrease in GnRHR mRNA levels was observed in IOSE and ovarian cancer cells, except CaOV-3 cells, following treatment with FSH or LH. In contrast, treatment with either FSH or LH had no effect on GnRH I mRNA levels in these cells, suggesting that gonadotropins regulate the two forms of GnRH and its receptor differentially. In separate experiments, the effect of gonadotropins on the anti-proliferative action of GnRH I and GnRH II agonists in IOSE-80, OVCAR-3 and SKOV-3 cells was investigated. The cells were pretreated with FSH or LH (100 ng/ml) for 24 h after which they were treated with either GnRH I or GnRH II (100 ng/ml) for 2 days, and cell growth was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Pretreatment of the cells with FSH or LH significantly reversed the growth inhibitory effect of GnRH I and GnRH II agonists in these cell types. These results provide the first demonstration of a potential interaction between gonadotropins and the GnRH system in the growth regulation of normal ovarian surface epithelium and its neoplastic counterparts.

Activin Receptor-Like Kinase 7 Induces Apoptosis through Up-Regulation of Bax and Down-Regulation of Xiap in Normal and Malignant Ovarian Epithelial Cell Lines

Transforming growth factor-beta superfamily has been implicated in tumorigenesis. We have recently shown that Nodal, a member of transforming growth factor-beta superfamily, and its receptor, activin receptor-like kinase 7 (ALK7), inhibit proliferation and induce apoptosis in human epithelial ovarian cancer cell lines. In this study, we further investigated the cellular mechanisms underlying the apoptotic action of ALK7 using an immortalized ovarian surface epithelial cell line, IOSE397, and an epithelial ovarian cancer cell line, OV2008. Infection of these cells with an adenoviral construct carrying constitutively active ALK7 (Ad-ALK7-ca) potently induced cell death; all cells died after 3 and 5 days of Ad-ALK7-ca infection in IOSE397 and OV2008 cells, respectively. ALK7-ca induced the expression of proapoptotic factor Bax but suppressed the expression of antiapoptotic factors Bcl-2, Bcl-XL, and Xiap. Silencing of Bax by small interfering RNA in IOSE397 cells significantly reduced ALK7-ca-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay but partially blocked ALK7-ca-induced caspase-3 activation and did not affect the down-regulation of Xiap by ALK7-ca. Dominant-negative Smad2, Smad3, and Smad4 blocked ALK7-ca-regulated Xiap and Bax expression and caspase-3 activation. Thus, ALK7-induced apoptosis is at least in part through two Smad-dependent pathways, Bax/Bcl-2 and Xiap.

Luteinizing hormone inhibits Fas-induced apoptosis in ovarian surface epithelial cell lines

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.

Gonadotropins mediate DNA synthesis and protection from spontaneous cell death in human ovarian surface epithelium

Gonadotropins have been implicated in the development of epithelial ovarian cancers. These tumors are derived from ovarian surface epithelium (OSE). The purpose of this study was to determine the effects of these hormones on DNA synthesis and spontaneous cell death in primary cultures of OSE and three immortalized OSE cultures. Primary cultures of OSE cells were generated from the ovaries of women with benign disease. The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on DNA synthesis and cell death were determined using [(3)H]thymidine incorporation and JAM assays. Significant inductions of DNA synthesis were demonstrated with LH in 4/12 (33%) primary cultures of OSE and 2/3 OSE cell lines and with FSH in 4/11 (36%) primary cultures of OSE and 2/3 OSE cell lines. A significant protection from cell death was also observed in the presence of FSH in 2/4 primary cultures of OSE and 1/3 OSE cell lines and in the presence of LH in 1/4 primary cultures of OSE and 2/3 OSE cell lines. The results indicate that while gonadotropins have the potential to induce cell proliferation and protect from cell death in OSE cells in vitro, their effects are variable in OSE cells from different women.

Classification of Cancer Cell Lines Using an Automated Two-dimensional Liquid Mapping Method with Hierarchical Clustering Techniques

A two-dimensional liquid mapping method was used to map the protein expression of eight ovarian serous carcinoma cell lines and three immortalized ovarian surface epithelial cell lines. Maps were produced using pI as the separation parameter in the first dimension and hydrophobicity based upon reversed-phase HPLC separation in the second dimension. The method can be reproducibly used to produce protein expression maps over a pH range from 4.0 to 8.5. A dynamic programming method was used to correct for minor shifts in peaks during the HPLC gradient between sample runs. The resulting corrected maps can then be compared using hierarchical clustering to produce dendrograms indicating the relationship between different cell lines. It was found that several of the ovarian surface epithelial cell lines clustered together, whereas specific groups of serous carcinoma cell lines clustered with each other. Although there is limited information on the current biology of these cell lines, it was shown that the protein expression of certain cell lines is closely related to each other. Other cell lines, including one ovarian clear cell carcinoma cell line, two endometrioid carcinoma cell lines, and three breast epithelial cell lines, were also mapped for comparison to show that their protein profiles cluster differently than the serous samples and to study how they cluster relative to each other. In addition, comparisons can be made between proteins differentially expressed between cell lines that may serve as markers of ovarian serous carcinomas. The automation of the method allows reproducible comparison of many samples, and the use of differential analysis limits the number of proteins that might require further analysis by mass spectrometry techniques.

772. Inhibition of Ovarian Tumor Growth Following Treatment with an Oncolytic Vaccinia Virus

Background: Recombinant vaccinia virus is currently being explored as a potential replicating oncolytic virus for cancer virotherapy due to its exceptional ability to replicate in tumor cells. Vaccinia tropism is not limited to tumor tissues, but also other tissues including ovarian follicles, possibly due to the presence of angiogenic factors such as vascular endothelial growth factor (VEGF). We therefore investigated the utility of vaccinia virus as an oncolytic therapy in a murine model of ovarian cancer. Methods: A murine ovarian surface epithelial cell (MOSEC) line was employed for this model. Intraperitoneal inoculation of the MOSEC cell line leads to a highly malignant neoplasm containing both carcinomatous and sarcomatous components as well as production of hemorrhagic ascitic fluid. These mice were treated with a thymidine kinase (TK) and vaccinia growth factor (VGF) double-deleted virus with the cytokine deaminase (CD) gene inserted into the TK locus. This recombinant virus (vvddCD) was found to be equivalent to the native WR strain in tumor tropism with minimal systemic growth. Results: Following intraperitoneal (i.p.) inoculation with 7.5x106 cells, mice were treated with 109 pfu of the vvddCD strain of vaccinia virus i.p. on the same day (concurrent treatment). In vivo studies appeared to show complete inhibition of tumor growth as compared to the control group (median mouse weight with ascites at 88 days: 40.7+/-1.49 (control [n=10]) vs 27.43+/-1.12 (virus [n=10])) (p<0.01). Hemorrhagic ascites formation was completely abrogated through treatment with the virus. All untreated mice either died or were sacrificed due to overwhelming ascitic fluid burden by 94 days following tumor inoculation. Treatment with virus was able to prevent or delay establishment of tumor and/or ascitic fluid with all treated mice viable and healthy at 161 days. Delayed treatment strategies with injections of 109 pfu of vvddCD at thirty (30) or sixty (60) days post tumor cell inoculation again appeared to show significant inhibition of tumor growth compared to control. For the 30 day treatment group the median mouse weight with ascites at day 75 was 39.3+/-1.65 (control [n=10]) vs 29.87+/- 1.54 (virus [n=10])) (p<0.05). All control mice either died or were sacrificed by 88 days; for animals treated at 30 days, 90% were viable at 88 days. By day 105, 50% of the 30 day treatment group were still viable. For the 60 day treatment group, 50% of treated animals were viable at day 88. All animals in the 60 day treatment group died or were euthanized by day 125. Conclusions: Treatment with a double-deleted recombinant vaccinia virus has been demonstrated to either completely abrogate (concurrent treatment) or significantly inhibit (delayed treatment) tumor progression in a murine ovarian model. The therapeutic application of vaccinia vectors expressing recombinant gene products in ovarian cancer represents a potential avenue of use given its biological safety, tumor selectivity and oncolytic potential.

Transcriptomic analysis of an in vitro murine model of ovarian carcinoma: Functional similarity to the human disease and identification of prospective tumoral markers and targets

Ovarian cancer is an aggressive disease of poor prognostic when detected at advanced stage. It is widely accepted that the ovarian surface epithelium plays a central role in disease etiology, but little is known about disease progression at the molecular level. To identify genes involved in ovarian tumorigenesis, we carried out a genome-wide transcriptomic analysis of six spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines, an in vitro model for human ovarian carcinoma. Loess normalization followed by statistical analysis with control of multiple testing resulted in 509 differentially expressed genes using an adjusted P-value < or = 0.05 as cut-off. The top 20 differentially expressed genes included 10 genes (Spp1, Cyp1b1, Btg1, Cfh, Mt1, Mt2, Igfbp5, Gstm1, Gstm2, and Esr1) implicated in various aspects of ovarian carcinomas, and other 3 genes (Gsto1, Lcn7, and Alcam) associated to breast cancer. Upon functional analysis, the majority of alterations affected genes involved in glutathione metabolism and MAPK signaling pathways. Interestingly, over 20% of the aberrantly expressed genes were related to extracellular components, suggestive of potential markers of disease progression. In addition, we identified the genes Pura, Cnn3, Arpc1b, Map4k4, Tgfb1i4, and Crsp2 correlated to in vivo tumorigenic parameters previously reported for these cells. Taken together, our findings support the utility of MOSE cells in studying ovarian cancer biology and as a source of novel diagnostic and therapeutic targets.

Inhibition of TRAIL-induced apoptosis by IL-8 is mediated by the p38-MAPK pathway in OVCAR3 cells

TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of the TNF superfamily of cell death inducing ligands. Interestingly, while malignant cells are responsive to TRAIL-induced cell death when used alone or in combination with other agents, normal cells do not appear to be sensitive to this ligand, making it a desirable therapeutic compound against many cancers, including many ovarian carcinomas. Interleukin-8 (IL-8), a member of the C-X-C chemokine family, has been found to be at significantly higher level in the ascites from patients with ovarian cancer. We have previously demonstrated a role for IL-8 in blocking TRAIL's ability to induce apoptosis in the ovarian cancer cell line, OVCAR3, possibly by repressing the DR4 TRAIL receptor expression and blocking caspase-8 cleavage. In addition, we showed a member of the mitogen-activated protein kinase (MAPK) superfamily, p38gamma, is among the genes regulated in OVCAR3 cells by TRAIL and IL-8. The present study further investigates involvement of the p38 MAPK pathway in IL-8's ability to block TRAIL-induced apoptosis in the ovarian surface epithelial cancer cell line, OVCAR3. In this study we demonstrate that p38gamma as well as p38alpha play a significant role in IL-8's ability to block TRAIL-induced apoptosis. Through array analysis, as well as confirmation with other methods, we detected regulation of p38gamma and p38alpha following treatment of the cancer cell line with IL-8 or TRAIL. We also tested two other isoforms of p38 MAPK, p38beta and p38delta, but did not find significant regulation by IL-8 or TRAIL. We also examined activation of the p38 MAPK pathway, up-stream as well as down-stream, and noticed activation of the pathway following treatment with TRAIL and decreased activity when IL-8 was introduced. With the use of specific inhibitors, we were able to further confirm the role of this pathway in TRAIL-induced apoptosis, and IL-8's ability to block this apoptosis, in ovarian cancer cell lines. Taken together, these results further solidify the role of IL-8 in blocking the TRAIL-induced apoptosis in these ovarian carcinoma cells and provide new molecular insight into this potentially important therapeutic target.

Microarray Comparative Genomic Hybridization Profile of a Murine Model for Epithelial Ovarian Cancer Reveals Genomic Imbalances Resembling Human Ovarian Carcinomas

Microarray comparative genomic hybridization (mCGH) is emerging as a high-resolution technology to detect gene dosage alterations in numerous pathologies, including cancer. We optimized cDNA microarrays to identify genome-wide imbalances in spontaneously transformed mouse ovarian surface epithelial cell lines, an in vitro murine model for ovarian cancer. Amplification of chromosome 19 and a more variable gain pattern of chromosomes 15 and 5 were detected and independently validated using conventional metaphase CGH. In addition, cryptic aberrations in segments of chromosomes 4, 7, 8, 9, 11, 17, and X, allowed identification of 2 related genomic variants among six cell lines studied. Mouse-human synteny revealed an overall early transformation stage with approximately 80% conservation relative to human ovarian malignancies of epithelial origin including low malignant potential tumors, serous carcinoma, and carcinoma cell lines. Importantly, three of the cells bear gained segments 13 and 41 Mbp length of chromosomes 5 and 15, respectively, which are syntenic to human 22q11–13, 8q24 and 12p11–q24, the two latter chromosomal regions thought to define one pathway of karyotypic changes in the development of human ovarian tumors. Our findings support the utility of mouse ovarian surface epithelial (MOSE) cells in studying initiation and progression of human ovarian cancer and as a suitable model to evaluate therapeutic approaches.

Profiling estrogen-regulated gene expression changes in normal and malignant human ovarian surface epithelial cells

Estrogens regulate normal ovarian surface epithelium (OSE) cell functions but also affect epithelial ovarian cancer (OCa) development. Little is known about how estrogens play such opposing roles. Transcriptional profiling using a cDNA microarray containing 2400 named genes identified 155 genes whose expression was altered by estradiol-17beta (E2) in three immortalized normal human ovarian surface epithelial (HOSE) cell lines and 315 genes whose expression was affected by the hormone in three established OCa (OVCA) cell lines. All but 19 of the genes in these two sets were different. Among the 19 overlapping genes, five were found to show discordant responses between HOSE and OVCA cell lines. The five genes are those that encode clone 5.1 RNA-binding protein (RNPS1), erythrocyte adducin alpha subunit (ADD1), plexin A3 (PLXNA3 or the SEX gene), nuclear protein SkiP (SKIIP), and Rap-2 (rap-2). RNPS1, ADD1, rap-2, and SKIIP were upregulated by E2 in HOSE cells but downregulated by estrogen in OVCA cells, whereas PLXNA3 showed the reverse pattern of regulation. The estrogen effects was observed within 6-18 h of treatment. In silicon analyses revealed presence of estrogen response elements in the proximal promoters of all five genes. RNPS1, ADD1, and PLXNA3 were underexpressed in OVCA cell lines compared to HOSE cell lines, while the opposite was true for rap-2 and SKIIP. Functional studies showed that RNPS1 and ADD1 exerted multiple antitumor actions in OVCA cells, while PLXNA3 only inhibited cell invasiveness. In contrast, rap-2 was found to cause significant oncogenic effects in OVCA cells, while SKIIP promotes only anchorage-independent growth. In sum, gene profiling data reveal that (1) E2 exerts different actions on HOSE cells than on OVCA cells by affecting two distinct transcriptomes with few overlapping genes and (2) among the overlapping genes, a set of putative oncogenes/tumor suppressors have been identified due to their differential responses to E2 between the two cell types. These findings may explain the paradoxical roles of estrogens in regulating normal and malignant OSE cell functions.

Erythropoeitin receptor function and expression in epithelial ovarian carcinoma

Our objectives were to determine if the erythropoietin receptor (EpoR) has increased expression in epithelial ovarian carcinoma, and if erythropoietin (Epo) confers malignant properties to ovarian cancer cell lines. A Western blot analysis of protein lysates from normal ovarian surface epithelial cells and ovarian cancer cell lines was performed. In addition, immunohistochemical (IHC) staining for EpoR in tissue specimens of normal, low malignant potential tumor, and epithelial ovarian carcinoma was performed. Epo effect on ovarian cancer cell lines was investigated by a cytotoxicity assay using a cell line with high (OVCAR3) and low (SKOV3) EpoR expression. Western blot analysis revealed increased expression of EpoR in multiple ovarian cancer cell lines. IHC staining revealed limited EpoR expression in benign ovarian tissue and increased levels in ovarian low malignant potential (LMP) tumor and carcinoma. This difference between benign ovarian tissue and carcinoma was found to be statistically significant using a quantitative scoring system. In addition, a cytotoxicity assay with paclitaxel and cisplatin revealed an attenuation of cytotoxic effects. Increased EpoR expression in ovarian LMP tumors and carcinoma is demonstrated by Western blot analysis and IHC staining. Furthermore, adversely effects sensitivity to cisplatin in the ovarian cancer cell lines. These data add to the growing body of evidence suggesting possible detrimental effects of erythropoietin.

The effects of Omega-3 fatty acids on growth regulation of epithelial ovarian cancer cell lines

Objective. Omega-3 fatty acids (OM-3FAs) have been shown to possess anti-carcinogenic properties. We investigated the effect of OM-3FAs on epithelial ovarian cancer cell lines to determine if a growth suppressive effect existed and to gain a better insight on the potential molecular mechanisms that may be involved. Methods. Three epithelial ovarian cancer cell lines (SKOV-3 [p53 null], TOV-21G [wt p53] and OVCAR-3 [mutant p53]) and one immortalized ovarian surface epithelial cell line (IOSE-29 [wt p53]) were treated with OM-3FAs and evaluated for cellular proliferation (WST-1 assay), apoptosis (Annexin V-FITC/PI method) and VEGF expression (VEGF ELISA assay). Results. A statistically significant inhibitory effect under the influence of OM-3FAs was detected in all four cell lines. Apoptosis and VEGF down-regulation were either limited or not detected in the p53 null and mutant cell lines, SKOV-3 and OVCAR-3 respectively. Apoptosis and/or VEGF down-regulation was strongly evident in the wt p53 cell lines TOV-21G and IOSE-29. Conclusion. These data suggest that, under the influence of OM-3FAs, there are definitive growth suppressive mechanisms at work and that the biologic effects of OM-3FAs may in part be mediated by the p53 status.

Establishment of an immortalised human ovarian surface epithelial cell line without chromosomal instability

Epithelial ovarian carcinoma is thought to derive from ovarian surface epithelium (OSE). The black box of the early molecular changes in ovarian carcinogenesis is being interpreted by the development of experimental systems employing immortalised human OSE cells. However, the existing cell lines of the OSE cells have limited utility due to chromosomal instability. Our goal was to establish new immortalised human OSE cells that retain the original characteristics of the primary cells without chromosomal alterations. Using primary human OSE cells obtained from a postmenopausal patient with endometrial cancer, five cell lines (‘HOSE1’ lines) were newly established by infection with retroviral expression vectors containing type 16 human papillomavirus (HPV-16) E6, E7, a variant E6 (E6Δ151), and Bmi1 polycomb gene, in combination with telomerase reverse transcriptase (hTERT). Consequently, five HOSE1s cell lines, HOSE1s-E6/hTERT, -E7/hTERT, -E6/E7/hTERT, -E6Δ151/E7/hTERT, and -E6Δ151/Bmi1/hTERT, grew beyond the population doubling number of 200. These cell lines, except for HOSE1-E6/hTERT, essentially showed the original features of the primary human OSE cells. Of them, HOSE1-E7/hTERT preserved diploidy in a kariotype analysis, and did not show transformed phenotypes in anchorage-independent growth and tumour formation. Thus, HOSE1-E7/hTERT may provide a novel model system with which to investigate the mechanisms of early molecular changes.

BORIS, a novel cancer-testis antigen, is a potential target for immunotherapy in epithelial ovarian cancer

9673 Background: Cancer-testis antigens (CTA), are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTA makes them promising targets for cancer-specific immunotherapy. Brother of Regulator Imprinted Sites (BORIS), a novel CTA located on chromosome 20q13.2, is involved in epigenetic re-programming. The aims of this study were to determine the frequency of aberrant expression of BORIS in epithelial ovarian cancer (EOC) and examine its correlation with clinical outcome. Methods: One step RT-PCR was performed with RNA from various normal and 94 EOC tumor tissues obtained from 1995 to 2002. A 270bp BORIS PCR product was amplified using specific sense 5’-CAGGCCCTACAAGTGTAACGACTGCAA and antisense 5’-GCATTCGTAAGGCTTCTCACCTGAGTG primers. Glyceraldehyde-3-phosphodehydrogenase (GAPDH) specific PCR amplification was used as control The χ2 test was used to analyze the distribution of BORIS expression and clinical outcome. Survival distributions were calculated by the Kaplan-Meier method and statistical significance was determined with the log-rank test. Results: Aberrant expression of BORIS was present in (63/ 94) 67% of EOC primary tumors, and undetectable in a panel of 16 normal tissues. The normal ovarian surface epithelial cell lines, IOSE and HOSE were negative for BORIS while 3/4 (75%) ovarian cancer cell lines (OVCA43, OVCA432, SKOV3) were positive. The median follow up of the patient population was 25 months (range 0.5–119). Tumor expression of BORIS did not correlate with stage, grade or histology. The median survival for BORIS positive patients was 49 months (CI:35,64) compared with 40 months (CI:26,54) for BORIS negative patients (not significant). Conclusion: BORIS is a novel CTA with high frequency expression in EOC. Although the expression of this antigen does not appear to influence survival, the tissue restricted expression pattern, role in epigenetic re-programming, and potential immunogenicity makes BORIS an attractive target for antigen-specific immunotherapy in EOC. The characterization of additional CTA with a frequent expression in EOC is warranted to allow for the development of polyvalent vaccines. No significant financial relationships to disclose.