Urinary angiostatin levels are elevated in patients with epithelial ovarian cancer.

OBJECTIVE: The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC. METHODS: VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. RESULTS: AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements. CONCLUSIONS: Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted.

VEGF- and LPA-induced telomerase in human ovarian cancer cells is Sp1-dependent.

OBJECTIVE: Both vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) are secreted by ovarian cancer cells and are known to promote cancer cell growth though the exact mechanism(s) are not completely understood. Since telomerase, a ribonucleprotein expressed in 95% of ovarian cancers, plays an important role in cellular immortalization, growth, and tumor progression, we examined whether telomerase is a molecular target of LPA and VEGF in ovarian cancer. METHODS: Telomerase-positive ovarian carcinoma cell lines PA-1, SW 626, and one telomerase-negative, non-tumorigenic SV40 large-T antigen-transfected human ovarian surface epithelial (IOSE) cell line, FHIOSE 118, derived from normal ovarian surface epithelium were cultured with and without VEGF and LPA for 4 h and 24 h, respectively. Telomerase PCR-ELISA, RT-PCR, VEGF ELISA and luciferase assays were performed to determine the effect of VEGF and LPA on telomerase activity in ovarian cancer cells. Western blot analyses were used to examine the signaling pathway involved in telomerase regulation by VEGF and LPA. RESULTS: We report that: (1) both VEGF and LPA upregulate telomerase activity; (2) LPA induction of telomerase activity is VEGF-dependent; (3) VEGF and LPA induction of telomerase activity is ERK 1/2-dependent; and (4) Sp1 binding sites within the proximal 976- to 378-bp regions of the hTERT promoter are essential for VEGF- and LPA-induced hTERT promoter activity. CONCLUSION: Consequently, these data show the novel finding that VEGF can regulate telomerase activity in non-endothelial cells and that telomerase appears to be a novel molecular target of LPA.

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells.

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.