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Abstract Background Ovarian cancer is the most lethal gynecologic malignancy. The ovarian tumor microenvironment is comprised of tumor cells, surrounding stroma, and circulating lymphocytes, an important component of the immune response, in tumors. Previous reports have shown that the anti-apoptotic protein Bcl-2 is overexpressed in many solid neoplasms, including ovarian cancers, and contributes to neoplastic transformation and drug-resistant disease, resulting in poor clinical outcome. Likewise, studies indicate improved clinical outcome with increased presence of lymphocytes. Therefore, we sought to examine Bcl-2 expression in normal, benign, and cancerous ovarian tissues to determine the potential relationship between epithelial and stromal Bcl-2 expression in conjunction with the presence of lymphocytes for epithelial ovarian tumor progression. Methods Ovarian tissue sections were classified as normal (n = 2), benign (n = 17) or cancerous (n = 28) and immunohistochemically stained for Bcl-2. Bcl-2 expression was assessed according to cellular localization, extent, and intensity of staining. The number of lymphocyte nests as well as the number of lymphocytes within these nests was counted. Results While Bcl-2 staining remained cytoplasmic, both percent and intensity of epithelial and stromal Bcl-2 staining decreased with tumor progression. Further, the number of lymphocyte nests dramatically increased with tumor progression. Conclusion The data suggest alterations in Bcl-2 expression and lymphocyte infiltration correlate with epithelial ovarian cancer progression. Consequently, Bcl-2 expression and lymphocyte status may be important for prognostic outcome or useful targets for therapeutic intervention.

Abstract Background The frequently occurring 185delAG mutation occurs in the amino-terminal zinc RING domain of the breast and ovarian cancer susceptibility gene, BRCA1. We sought to determine differential cell viability and apoptotic response of human ovarian surface epithelial cells with and without the 185delAG mutation. Results BRCA1wt and BRCA1+ cells were treated with staurosporine. Cell proliferation assays showed BRCA1wt cells grew to a greater extent compared to BRCA1+ cells. Trypan blue exclusion assays confirmed this observation. Western immunoblot analysis revealed that caspase 3 levels were higher after staurosporine treatment in BRCA1+ cells than in wild type cells, while full length DNA Fragmentation Factor 45 levels were lower in BRCA1+ cells. While there was no significant difference in levels of excision repair cross complementing protein1 (ERCC1) with BRCA1 status, BRCA1+ cells demonstrated cleavage of polyribose ADP polymerase (PARP) before wild type cells. Conclusions Disruption of the BRCA1 RING domain caused altered cell viability and caspase-dependent apoptotic response after chemotoxic stress.

Background: Elevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM's role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC., Results: Despite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells., Conclusion: This pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.

Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (-1800) to (-900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Background: Early detection of epithelial ovarian cancer (OC) is necessary to overcome the high mortality rate of late stage diagnosis; and, examining the molecular changes that occur at early disease onset may provide new strategies for OC detection. Since the deregulation of inflammatory mediators can contribute to OC development, the purpose of this pilot study was to determine whether elevated urinary levels of Interleukin-1beta (IL-1 beta) are associated with OC and associated clinical parameters., Methods: Urinary and serum levels of IL-1 beta were analyzed by ELISA from a patient cohort consisting of healthy women (N = 10), women with ovarian benign disease (N = 23), women with OC (N = 32), women with other benign gynecological conditions (N = 22), and women with other gynecological cancers (N = 6)., Results: Average urinary IL-1 beta levels tended to be elevated in ovarian benign (1.26 pg/ml) and OC (1.57 pg/ml) patient samples compared to healthy individuals (0.36 pg/ml). Among patients with benign disease, urinary IL-1β levels were statistically higher in patients with benign inflammatory gynecologic disease compared to patients with non-inflammatory benign disease. Interestingly, urinary IL-1 beta levels tended to be 3-6x greater in patients with benign ovarian disease or OC as well as with a concomitant family history of ovarian and/or breast cancer compared to similar patients without a family history of ovarian and/or breast cancer. Lastly, there was a pattern of increased urinary IL-1 beta with increasing body mass index (BMI); patients with a normal BMI averaged urinary IL-1 beta levels of 0.92 pg/ml, overweight BMI averaged urinary IL-1 beta levels of 1.72 pg/ml, and obese BMI averaged urinary IL-1 beta levels of 5.26 pg/ml., Conclusions: This pilot study revealed that urinary levels of IL-1 beta are elevated in patients with epithelial OC supporting the thought that inflammation might be associated with cancer progression. Consequently, further studies of urinary IL-1 beta and the identification of an inflammatory profile specific to OC development may be beneficial to reduce the mortality associated with this disease.

Introduction: Fallopian tube intraepithelial cancer is a postulated precursor of epithelial ovarian carcinomas. As research continues on epithelial ovarian carcinomas' developmental pathways, representative tubal tissue must be procured for diagnostic, biological, and molecular studies without compromising pathological diagnosis., Materials and Methods: Fallopian tube fimbrial epithelia were harvested from postmenopausal women undergoing surgery for non-neoplastic gynecologic lesions (n = 16) and epithelial ovarian carcinomas (n = 6). Cytological imprints and washings were obtained from each fimbria and stained by Diff-Quik and rapid Papanicolaou for general cytomorphology; by Trypan blue for cell viability; and by rapid immunohistochemistry for evaluation of low molecular weight cytokeratin, MIB-1, p53, and high-mobility group A (HMGA2) expression., Results: Benign and malignant tubal imprints harvests yielded means of 3.5 × 10 5 and 1.2 × 10 6 cells/fimbria, respectively, with viabilities higher than 85%. A mean of 2.5 × 10 5 cells/fimbria was obtained from fimbrial washings. The mean DNA, RNA, and protein contents of benign imprints were 2.4, 1.5, and 67 μg/fimbria, respectively. Benign cell populations contained nearly 97% cytokeratin-positive and p53/HMGA2-negative cells, which were dispersed within a watery to proteinaceous material and rare microcalcifications. Fimbrial imprints from serous carcinomas involving the fimbriae exhibited abnormal p53 and HMGA2 expression, high proliferation, and diagnostic criteria of malignancy, including prominent nucleoli and cell crowding., Conclusions: Ex vivo harvest from operative specimens allows for collection of cell populations representative of native fimbrial epithelium and free of significant contaminants. Tubal harvest facilitates triaging of cellular material for basic, clinical, and translational studies on cancer pathobiology and also represents a potential diagnostic adjunct to emerging in vivo high-resolution optical technologies., (Copyright © 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.

Ovarian epithelial cancer is the most lethal gynecologic malignancy. The high mortality is attributed to the fact that most cases typically present in late stage when ovarian cancer (OC) has already spread beyond the ovary. Ovarian epithelial cancer cells are shed into intraperitoneal ascites and easily disseminate throughout the peritoneal cavity with preferential metastasis to the omentum, peritoneum, and local organs. Understanding how ovarian epithelial cells interact with and modulate their microenvironment can provide insight into the molecular mechanism(s) involved with malignant transformation and progression which may eventually identify novel diagnostic, prognostic, and therapeutic targets. The objective of this paper is to provide a brief consideration of ovarian surface epithelial-stromal interactions in regard to normal physiological function and tumor progression as influenced by two potentially key interleukins, interleukins-1 (IL-1) and -6 (IL-6), present in the microenvironment. Lastly, we will consider the clinical implications of IL-1 and IL-6 for OC patients.

Mutations in the tumor suppressor breast cancer susceptibility gene 1 (BRCA1), an important player in the DNA damage response, apoptosis, cell cycle regulation and transcription, confer a significantly elevated lifetime risk for breast and ovarian cancer. Although the loss of wild-type BRCA1 function is an important mechanism by which mutations confer increased cancer risk, multiple studies suggest mutant BRCA1 proteins may confer functions independent of the loss of wild-type BRCA1 through dominant negative inhibition of remaining wild-type BRCA1, or through novel interactions and pathways. These functions impact various cellular processes and have the potential to significantly influence cancer initiation and progression. In this review, we discuss the functional classifications of risk-associated BRCA1 mutations and their molecular, cellular and clinical impact for mutation carriers.

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., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objective: Aggressive clinical course and difficult detection of ovarian cancer are major challenges to improving patient survival and necessitate avid investigation into more effective therapeutic approaches. Understanding early molecular and pathological changes in high risk patients, such as BRCA1 mutation carriers, can provide candidates for molecular profiling and novel targets for effective therapies., Methods: Using a culture model system for normal human ovarian surface epithelial cells with and without the BRCA1 185delAG frameshift mutation for the truncated protein product, BRAt, we investigated the role of BRAt in enhanced chemosensitivity. We used MTS, Western immunoblot, semi-quantitative RT-PCR, luciferase reporter and siRNA assays, to identify novel downstream targets of BRAt that promote apoptosis following chemotherapeutic treatment., Results: We identified maspin as a novel downstream target of BRAt. BRAt increases maspin expression with preferential nuclear localization of maspin. Further, Brat-mediated maspin expression is transcriptionally regulated through an AP1 site within the (-520) to (-297) region of the promoter. Lastly, BRAt, enhances chemosensitivity in normal ovarian surface epithelial cells through c-Jun by a mechanism that may involve maspin., Conclusions: BRAt-mediated enhanced chemosensitivity correlates clinically with enhanced chemotherapeutic response in BRCA1 mutation carriers. BRAt-mediated maspin expression also correlates with improved prognostic outlook for ovarian tumors with high levels of nuclear maspin. Consequently, understanding early genotypic and phenotypic changes in the context of high risk disease may provide a better understanding of the mechanism of mutation-associated ovarian cancer and provide new targets for therapeutic intervention., (Copyright 2009 Elsevier Inc. All rights reserved.)

I-kappa-B kinase e (IKBKE; IKKepsilon) has been recently identified as a breast cancer oncogene, and its alteration appears to be an early event in breast cancer development. In this study, we demonstrated that IKKepsilon is frequently overexpressed and activated in human ovarian cancer cell lines and primary tumors. Of 96 ovarian cancer specimens examined, 63 exhibited elevated levels of IKKepsilon. Furthermore, alterations of IKKepsilon were associated with late-stage and high-grade tumors, suggesting a role of IKKepsilon in ovarian tumor progression rather than in tumor initiation. Overall survival in patients with elevated levels of IKKepsilon was significantly lower than patients whose tumors expressed normal levels of IKKepsilon. Moreover, both early and late-stage tumors that overexpressed IKKepsilon conferred a poor prognosis, as compared with those that did not possess elevated IKKepsilon levels. Notably, overexpression of IKKepsilon rendered cells resistant to cisplatin, whereas knockdown of IKKepsilon overcame cisplatin resistance in both A2780CP and C13 cells, which express high levels of endogenous IKKepsilon. Therefore, these data demonstrate for the first time that deregulation of IKKepsilon is a highly recurrent event in human ovarian cancer and could play a pivotal role in tumor progression and cisplatin resistance. IKKepsilon could also serve as a prognostic marker and potential therapeutic target for this malignancy.

In 1932, Bidder postulated that senescence results from "continued action of a (genetic) regulator (of development) after growth ceases (maturation occurs)." A 16-year-old girl who physically appears to be an infant has not been diagnosed with any known genetic syndrome or chromosomal abnormality. The subject's anthropometric measurements are that of an 11-month-old. Coordinated development of structures for swallowing/breathing has not occurred resulting in dysfunctional digestive and respiratory systems. Brain structure, proprioception and neuroendocrine functions are infantile. Dental and bone ages are pre-teen, while telomere length and telomerase inactivity suggest a cellular age at least comparable to her chronological age. Sub-telomeric microdeletions known to be responsible for developmental delay and chromosomal imbalances are not present. Findings suggest that the subject suffers from "developmental disorganization" resulting from spontaneous mutation of Bidder's putative "regulator" of development, thereby providing an opportunity to locate and identify developmental gene(s) responsible for ensuring integrated and coordinated change in form and function from conception to adulthood. If their continued expression beyond maturation erodes internal order to promote senescence then further study of her DNA and testing of homologous genes in animal models may provide clues to genetic determinants of aging and human life span.

Objective(s): The poor prognosis associated with ovarian cancer is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors overexpress anti-apoptotic proteins, the purpose of this study was to determine whether elevated levels of the anti-apoptotic protein Bcl-2 were present in urine from patients with ovarian cancer., Methods: Bcl-2 was assayed by ELISA in urine samples from two cohorts consisting of a total of 77 healthy women, 161 women with benign gynecologic disease and 150 women with ovarian cancer, 13 with early and 137 with late stage disease, respectively. Wherever possible, parallel serum samples were measured for CA125 levels by ELISA., Results: Urinary levels of Bcl-2 from healthy individuals or women with benign disease averaged 0.59 ng/ml+/-0.61 and 1.12 ng/ml+/-0.79, respectively. In contrast, urinary levels of Bcl-2 averaged 2.60 ng/ml+/-2.23 and 3.58 ng/ml+/-1.55 from women with early (N=13) and late (N=137) stage ovarian cancer. Further, urinary levels of Bcl-2 were elevated in ovarian cancer patients regardless of tumor grade, stage, size, histologic subtype, creatinine levels or patient age, but appeared to complement CA125 measurements., Conclusion(s): Levels of Bcl-2 are elevated in the urine of patients with ovarian cancer and may be of diagnostic and/or prognostic clinical importance. Further studies of urinary Bcl-2 as a biomarker for ovarian cancer alone or in combination with other markers are warranted.

While semaphorins and their receptors appear to play a role in tumor carcinogenesis, little is known about the role of semaphorin 3F (S3F) in epithelial ovarian cancer (EOC) development. Therefore, we sought to determine the clinical relationship between S3F and its receptors, neuropilin-2 (NP-2) and neuropilin-1 (NP-1) with EOC progression. We analyzed the immunohistological expression of S3F, NP-2, and NP-1 in clinical specimens of normal ovaries (N), benign cystadenomas (Cy), well-differentiated adenocarcinomas (WD), poorly-differentiated adenocarcinomas (PD), inclusion cysts (IC), paraovarian cysts (PC), and fallopian tubes (FT). Tissue sections were evaluated for staining intensity and percentage of immunoreactive epithelia. We found that expression of S3F and NP-2 decreased while NP-1 expression increased with EOC progression. Interestingly, we also found elevated expression of S3F, NP-2, and NP-1 in epithelia of ICs, PCs, and FT. Our findings indicate that loss or deregulation of semaphorin signaling may play an important role in EOC development.

Breast and ovarian cancer patients with germline mutations in BRCA1 respond more favorably to initial chemotherapy. We previously reported that cells from women carrying the BRCA1 185delAG founder mutation undergo an enhanced caspase-3-mediated apoptotic response. Here, we report on the transient and stable transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human ovarian surface epithelial cells and ovarian cancer cells, resulting in cells with a heterozygous background containing two BRCA1 wild-type alleles and the BRCA1 185delAG transcript. The BRCA1 185delAG truncation (BRAt) protein did not alter epithelial cell morphology or induce tumorigenesis. However, upon treatment with staurosporine, BRAt cells showed increased levels of active caspase-3 and increased cleavage of caspase-3 substrates, PARP and DFF45. Additionally, XIAP and cIAP-1 protein are at reduced levels in untreated BRAt cells as compared to control cells. BRAt also reduced levels of phosphorylated Akt and overexpression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild-type cells. Further, BRAt expression increased chemosensitivity in platinum-resistant ovarian cancer cells. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis, suggesting a possible mechanism by which ovarian cancer patients with this germline BRCA1 mutation may respond better to initial chemotherapy.

Epidermal growth factor (EGF) promotes growth of normal ovarian surface as well as malignant ovarian epithelial cells. Further, EGF receptors are present on both normal and malignant ovarian surface epithelial cells and they are often constitutively activated in many cancers. Since telomerase confers cellular immortalization and survival through increased cellular proliferation, we sought to investigate the potential role of EGF to regulate telomerase activity in normal and ovarian cancer cells. While exogenous EGF failed to activate telomerase in normal ovarian surface epithelial cells, in cancer cells we herein report that: exogenous EGF activates telomerase activity and human telomerase reverse transcriptase gene (hTERT) transcription; EGF-induced telomerase activity is ERK 1/2-dependent; EGF targets Sp1 and c-Myc binding sites within the core region of the hTERT promoter; and proline-rich tyrosine kinase 2 (Pyk2) is a key mediator of EGF-mediated telomerase activity. Together, these data show that dysregulation of EGF signaling may promote cancer cell survival through up-regulation of telomerase activity.

MicroRNAs (miRNA) represent a novel class of genes that function as negative regulators of gene expression. Recently, miRNAs have been implicated in several cancers. However, aberrant miRNA expression and its clinicopathologic significance in human ovarian cancer have not been well documented. Here, we show that several miRNAs are altered in human ovarian cancer, with the most significantly deregulated miRNAs being miR-214, miR-199a*, miR-200a, miR-100, miR-125b, and let-7 cluster. Further, we show the frequent deregulation of miR-214, miR-199a*, miR-200a, and miR-100 in ovarian cancers. Significantly, miR-214 induces cell survival and cisplatin resistance through targeting the 3'-untranslated region (UTR) of the PTEN, which leads to down-regulation of PTEN protein and activation of Akt pathway. Inhibition of Akt using Akt inhibitor, API-2/triciribine, or introduction of PTEN cDNA lacking 3'-UTR largely abrogates miR-214-induced cell survival. These findings indicate that deregulation of miRNAs is a recurrent event in human ovarian cancer and that miR-214 induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway.

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.

Background: Dietary factors influence tumor formation and progression. Vitamin E is a dietary anti-oxidant capable of eliminating free radical damage, inducing apoptosis and decreasing oncogene expression. Therefore, Vitamin E may be a strong candidate for cancer prevention and/or chemotherapeutic intervention. Since telomerase, a ribonucleoprotein uniquely expressed in over 95% of cancers, plays an important role in cellular immortalization, cell growth and tumor progression, the present study investigated the effects of Vitamin E on telomerase activity in human ovarian cancer., Methods: Normal and malignant ovarian surface epithelial (OSE) cells were cultured with and without D-alpha tocopheryl acetate (Vitamin E). MTS and Western immunoblot assays were used to examine the effect of Vitamin E on cell growth, survival and cytotoxicity. PCR-ELISA, RT-PCR and luciferase reporter assays were performed to determine the effect of Vitamin E on telomerase activity., Results: Vitamin E suppressed endogenous telomerase activity in ovarian cancer cells, but had no similar effects in telomerase-negative normal OSE cells. Vitamin E also reduced hTERT-mRNA transcript levels and reduced hTERT promoter activity maximally targeting the -976 to -578bp promoter regions. In addition, Vitamin E improved cisplatin-mediated cytotoxicity as evidenced by reduced cancer cell growth and increased cleaved caspase 3 activity. In contrast, Vitamin E protected telomerase-negative OSE cells from cisplatin-mediated cytotoxicity as evidenced by decreased cleaved caspase 3 activity., Conclusion: Our data suggest that, by suppressing telomerase activity, Vitamin E may be an important protective agent against ovarian cancer cell growth as well as a potentially effective therapeutic adjuvant.

There is growing evidence that accelerated telomeric attrition and/or aberrant telomerase activity contributes to pathogenesis in a number of diseases. Likewise, there is increasing interest to develop new therapies to restore or replace dysfunctional cells characterized by short telomeric length using telomerase-positive counterparts or stem cells. While telomerase adds telomeric repeats de novo contributing to enhanced proliferative capacity and lifespan, it may also increase cellular survival by conferring resistance to apoptosis. Consequently, we sought to determine the involvement of telomerase for reduced apoptosis using ovarian surface epithelial cells. We found that expression of hTERT, the catalytic component of telomerase, was sufficient and specific to reduce caspase-mediated cellular apoptosis. Further, hTERT expression reduced activation of caspases 3, 8, and 9, reduced expression of pro-apoptotic mitochondrial proteins t-BID, BAD, and BAX and increased expression of the anti-apoptotic mitochondrial protein, Bcl-2. The ability of telomerase to suppress caspase-mediated apoptosis was p-jnk dependent since abrogation of jnk expression with jip abolished resistance to apoptosis. Consequently, these findings indicate that telomerase may promote cellular survival in epithelial cells by suppressing jnk-dependent caspase-mediated apoptosis.

Objective: This review will discuss the discovery and development of RNA interference (RNAi) technology, small interfering RNA (siRNA) design and delivery, and the implications of RNAi on gynecologic cancers., Methods: Systematic review of English language literature using searches for RNAi and gynecologic cancers in established databases, including Pubmed and Ovid, was employed., Results: The high degrees of efficiency and specificity are the main advantages of RNAi. Consequently, RNAi is used in functional genomics and developing therapies for the treatment of viral infection, dominant disorders, neurological disorders, and cancers, including gynecologic cancers., Conclusion: RNAi represents an exciting technology for functional genomics by selective targeting of genes. While issues regarding delivery remain, the therapeutic advantages of siRNA in cancer treatment warrant further investigation.

BRCA1 mutations have long been associated with altered apoptosis. We have recently reported that caspase 3 activation is increased in human ovarian surface epithelial (OSE) cells expressing a germline N-terminal BRCA1 185delAG mutation. Here, we report increased caspase 3 activity in 185delAG OSE cells associated with decreased expression of cIAP-1 and X-linked inhibitor of apoptosis (XIAP), and decreased ubiquitination of caspase 3. Overexpression of XIAP restored active caspase 3 ubiquitination and lowered levels of caspase 3 activity. Further, the BRCA1 185delAG mutation was associated with reduced levels of phosphorylated Akt1. Transfection with activated Akt1 led to increased cIAP-1 and XIAP levels similar to that seen in BRCA1 185delAG cell lines. Taken together, these data suggest a direct link between the BRCA1 185delAG mutation and alterations in the caspase-mediated apoptotic pathway.

Background: The human ovarian surface epithelium (HOSE) is the putative source of ovarian epithelial cancer, the most lethal gynecologic malignancy that affects women in the United States. The current study was designed to provide a database of normal HOSE cell features for diagnostic and research applications., Methods: HOSE was harvested from 42 women undergoing laparoscopy or laparotomy for benign gynecologic disorders, infertility problems, or pregnancy. Of the 42 women, 12 were postovulatory and 20 were receiving hormonal regimens. Cells were harvested with a sterile brush inserted through a laparoscopic port or with a sterile cell scraper at laparotomy., Results: Two HOSE populations were identified, ranging in size from 8 to 10 microm and from 15 to 20 microm, respectively. The cells measuring 15-20 microm exhibited slight anisonucleosis, more prominent nucleoli, fine cytoplasmic metachromasia, and an overall reparative or squamoid morphology. Cells were single or arranged in small clusters, sheets, or papillae. They coexpressed cytokeratin and vimentin but did not overexpress p53. Cellularity and proliferation (up to 3.2% +/- 0.8) were higher and papillae more frequent in postovulatory and cyst-bearing ovaries, including polycystic ovaries, suggesting underlying ovarian or hormonal influences. Representative HOSE brushings yielded a mean of 23,133 cells per patient (range, 4250-64,500 cells), equivalent to an estimated 0.58, 0.46, and 0.14 microg of nuclear protein, cell RNA, and nuclear DNA, respectively. Within 7-10 days of explantation, HOSE cells formed confluent monolayers with immunohistochemical and ultrastructural epithelial features., Conclusions: The current study defined baseline features of HOSE cells important to pathologists and clinicians evaluating women at risk for ovarian epithelial cancer and to researchers investigating the pathobiology of this aggressive gynecologic malignancy., (Copyright 2003 American Cancer Society.)

Aurora-A kinase is frequently overexpressed/activated in human ovarian and breast cancers. A rat mammary tumor model study indicates that alterations of Aurora-A are early events during mammary tumor development (T. M. Goepfert et al., Cancer Res., 62: 4115-4122, 2002), suggesting that Aurora-A plays a pivotal role in transformation. However, the molecular mechanism by which Aurora-A induces ovarian and breast cell transformation remains elusive. Here we show that ectopic expression of Aurora-A induces telomerase activity in human ovarian and breast epithelial cell lines HIOSE118 and MCF-10A. The mRNA and promoter activities of human telomerase reverse transcriptase (hTERT) are stimulated by Aurora-A. Furthermore, we have demonstrated that the c-Myc binding sites of hTERT promoter are required for Aurora-A-induced hTERT promoter activity. Ectopic expression of Aurora-A up-regulates c-Myc. Knockdown of c-Myc by RNA interference attenuates Aurora-A-stimulated hTERT expression and telomerase activity. To our knowledge, these findings demonstrate, for the first time, that Aurora-A induces telomerase activity and hTERT by up-regulation of c-Myc and provides an additional mechanism for the role of Aurora-A in malignant transformation in addition to its cell cycle control.

Calcium signaling has been linked to activation of Pyk2, a calcium-dependent, focal adhesion kinase-related, non-receptor tyrosine kinase. Signaling via Pyk2 can activate c-jun NH(2)-terminal kinase (JNK). Calcium has also been shown to activate phosphatidylinositol triphosphate kinase and/or JNK. Here, we show that calcium signaling in ovarian surface epithelial cells not only induces telomerase activity via JNK but also activates Pyk2. Moreover, telomerase activation by Pyk2 requires JNK activation. In contrast, a kinase-deficient Pyk2 construct failed to activate either JNK or telomerase. Finally, we demonstrate that Pyk2 is capable of driving the human telomerase reverse transcriptase promoter, resulting in telomerase activation. These data suggest a novel role of Pyk2 for telomerase regulation.

Characterization of intracellular signaling pathways should lead to a better understanding of ovarian epithelial carcinogenesis and provide an opportunity to interfere with signal transduction targets involved in ovarian tumor cell growth, survival, and progression. Challenges toward such an effort are significant because many of these signals are part of cascades within an intricate and likely redundant intracellular signaling network (Fig.1). For instance, a given signal may activate a dual intracellular pathway (ie, MEK1-MAPK and PI3K/Akt required for fibronectin-dependent activation of matrix metalloproteinase 9). A single pathway also may transduce more than one biologic or oncogenic signal (ie, PI3K signaling in epithelial and endothelial cell growth and sprouting of neovessels). Despite these challenges, evidence for therapeutic targeting of signal transduction pathways is accumulating in human cancer. For instance, the EGF-specific tyrosine kinase inhibitor ZD 1839 (Iressa) may have a beneficial therapeutic effect on ovarian epithelial cancer. Therapy of this cancer may include inhibitors of PI kinase (quercetin), ezrin and PIP kinase (genistein). The G protein-coupled family of receptors, including LPA, also is an attractive target to drugs, although their frequent pleiotropic functions may be at times toxic and lack specificity. Because of the lack of notable toxicity, PI3K/Akt pathway inhibitors such as FTIs are a promising targeted therapy of ovarian epithelial cancer. Increasing insight into the oncogenic pathways involved in ovarian epithelial cancer also is helping clinicians to understand better the phenomenon of chemoresistance in this malignancy. Oncogenic activation of gamma-synuclein promotes cell survival and provides resistance to paclitaxel, but such a resistance is partially overcome by an MEK inhibitor that suppresses ERK activity. Ovarian epithelial cancer is a complex group of neoplasms with an overall poor prognosis. Comprehension of this cancer pathobiology suffers because of an incomplete understanding of precursor lesions and the absence of an orthotopic animal model until very recently. It can be predicted with confidence, however, that the discovery of potent inhibitors of signal transduction and the development of discovery tools, such as proteomics and metabolomics, may change the way by which clinicians may now address basic biomedical questions in this insidious and lethal disease.

Telomerase activity is present in >90% of all tumors and appears to be regulated by the phosphatidylinositol 3-kinase signaling pathway. Here we demonstrate that Akt is not involved in the signaling cascade for telomerase regulation in ovarian surface epithelial cells. However, we showed that c-Jun NH2-kinase induces telomerase activity, that inhibition of JNK by JIP abrogates telomerase activity, and that JNK expression activates transcription of a reporter gene fused to the hTERT promoter sequence. Consequently, our data show that JNK is a key regulator of telomerase activity and, hence, may provide new perspectives on tumorigenesis that could be exploited for novel therapeutic strategies.

BACKGROUND: The frequently occurring 185delAG mutation occurs in the amino-terminal zinc RING domain of the breast and ovarian cancer susceptibility gene, BRCA1. We sought to determine differential cell viability and apoptotic response of human ovarian surface epithelial cells with and without the 185delAG mutation. RESULTS: BRCA1wt and BRCA1+ cells were treated with staurosporine. Cell proliferation assays showed BRCA1wt cells grew to a greater extent compared to BRCA1+ cells. Trypan blue exclusion assays confirmed this observation. Western immunoblot analysis revealed that caspase 3 levels were higher after staurosporine treatment in BRCA1+ cells than in wild type cells, while full length DNA Fragmentation Factor 45 levels were lower in BRCA1+ cells. While there was no significant difference in levels of excision repair cross complementing protein1 (ERCC1) with BRCA1 status, BRCA1+ cells demonstrated cleavage of polyribose ADP polymerase (PARP) before wild type cells. CONCLUSIONS: Disruption of the BRCA1 RING domain caused altered cell viability and caspase-dependent apoptotic response after chemotoxic stress.

Though the potential of telomerase as an anti-cancer target is evident, information about regulation of telomerase remains fragmentary. In the present study, we examined the role of calcium, an essential cellular signaling molecule, in the regulation of telomerase. We found that calcium induced de novo telomerase activity in telomerase-negative ovarian surface epithelial (OSE) cell lines but not in primary cultures of OSE. In addition, we showed that calcium elevated endogenous telomerase levels in a telomerase-positive ovarian cancer cell line. The use of calcium channel blockers or calcium chelators inhibited this calcium-mediated induction of telomerase activity. Furthermore, cadmium and chromium appeared to cause a moderate induction of telomerase activity while several other metal salts did not. Our data provide the first example of calcium-induced telomerase activity in human cell lines, provide a novel avenue for possible intervention of telomerase, and permit development of therapeutic agents for adjunctive chemotherapy.

Information about telomerase regulation is incomplete, especially since various studies suggest complexity in telomerase regulation. Given the important association between telomerase and cancer, it is imperative to design and develop a model system in which telomerase activity can be regulated and studied. We employed ultraviolet (UV) radiation or dimethyl sulfoxide (DMSO) to transiently induce telomerase activity in a telomerase-positive cell line and, most importantly, in a telomerase-negative cell line. UV- or DMSO-induced telomerase activity was associated with increased hTRT, but not hTR, mRNA transcription in the telomerase-negative cells. However, no changes in hTRT or hTR mRNA transcription were noted with UV- or DMSO-induced telomerase activity in the telomerase-positive cells. Inhibition of protein synthesis or the phosphotidyl inositol 3-kinase (PI3K) pathway suppressed telomerase induction and/or activity in all cell lines examined, suggesting telomerase activity was dependent on protein synthesis and PI3K-mediated phosphorylation. Furthermore, enhanced telomerase activity was limited to UV and DMSO, since a variety of chemotherapeutic agents failed to induce telomerase activity. Therefore, our data provide a useful culture model system to study telomerase regulation in telomerase-negative and -positive cell lines and from which to obtain information about telomerase as a target for cancer intervention., (Copyright 2001 Academic Press.)

Objective: Increased telomeric instability in normal ovarian surface epithelium may contribute to ovarian carcinogenesis in women from families with a high frequency of breast/ovarian cancer. To test this hypothesis, we compared proliferative potential, mean telomeric length, and telomerase activity in SV-40 large T-antigen transfected cell lines derived from normal ovarian surface epithelium of women with and without a familial history of breast/ovarian cancer., Methods: Telomeric instability was examined in SV-40 large T-antigen transfected cell lines of normal ovarian surface epithelium from patients with (FHIOSE, N = 5) and without (NFHIOSE, N = 11) a history of familial breast/ovarian cancer. The duration and total attainable number of population doublings, mean telomeric length, rate of telomeric loss, and telomerase activity were determined by cell counts, Southern blot analysis, and PCR ELISA., Results: FHIOSE cells attained fewer population doublings than NFHIOSE cells and doubled at approximately half the rate of NFHIOSE cells, indicating a reduced proliferative capacity in FHIOSE cells. While telomerase activity was not detected in FHIOSE or NFHIOSE cell lines, mean telomeric lengths in FHIOSE were generally 1 kb shorter than in NFHIOSE cells and the rate of telomeric loss as a function of population doublings was up to threefold greater in FHIOSE cells., Conclusions: Increased telomeric instability and reduced growth potential suggest greater proximity to replicative senescence in ovarian surface epithelium from women with a familial history of breast/ovarian cancer. Consequently, an accumulation of genetic aberrations due to accelerated cellular aging may contribute to the enhanced susceptibility for malignant transformation and earlier onset in heritable ovarian cancer., (Copyright 1999 Academic Press.)

Telomeres play an important role in maintaining chromosomal stability and are often shortened in transformed cells. p53 is the most commonly mutated gene in cancers and its status is thought to reflect the level of genomic stability. We measured telomeric length by Southern blot analysis in cells from cancer-prone syndromes and in selected cancer cells with altered p53 status. Mean telomeric lengths in the cancer-prone syndromes Li-Fraumeni syndrome, Fanconi's anemia, and ataxia telangiectasia, were shorter in the affected individuals than in their unaffected parents. We also found that altered p53 expression in selected cancer cell model systems may be associated with shortened telomeric length, but did not appear to be associated with significant alterations in telomerase activity.

Telomerase is a ribonucleoprotein that elongates telomeric repeats de novo. We examined the possibility that telomerase activity is cell cycle regulated by examining telomerase activity in cell cycle synchronized Chinese hamster ovary (CHO) B11 cells. Overall telomerase activity was similar in growing and quiescent cells. Further, cells synchronized in G1, S, or G2/M showed similar levels of telomerase activity. However, a detailed analysis of cells within S phase showed that there was a higher level of telomerase activity in early S phase when compared with other points in the cell cycle. These results suggest a relationship between telomerase activity and cell cycle regulation.

Telomerase adds (TTAGGG)n hexanucleotide repeats to the ends of mammalian telomeres. This compensates for telomeric loss with successive rounds of cellular replication. Telomerase activity is detected in many neoplastic cells, but not in most normal somatic cells. To determine whether telomeric length and telomerase activity are associated with cellular differentiation, we measured telomeric lengths and telomerase activity in embryonic NT2 precursor cells prior to and following differentiation into post mitotic hNT neurons. This system allows for studies in a direct neuronal cell lineage and, thus, provides a unique model for studying the role of neuronal telomerase activity. Our results show that telomerase activity was present in precursor cells, but not in neuronal cells. Telomeres were consistently longer in NT2 cells than in hNT cells. These results suggest that changes in telomeric length and loss of telomerase activity play a role in neuronal cellular differentiation.

We have established a method for the detection of DNA damage and its repair in human telomeres, the natural ends of chromosomes which are necessary for replication and critical for chromosomal stability. We find that ultraviolet light-induced pyrimidine dimers in telomeric DNA are repaired less efficiently than endogenous genes but more efficiently than inactive, noncoding regions. We have also measured telomeric length, telomeric DNA damage, and its repair in relation to the progression of aging. Telomeres are shorter in fibroblasts from an old donor compared to fibroblasts from a young donor, shortest in cells from a patient with the progeroid disorder Werner syndrome, and relatively long in fibroblasts from a patient with Alzheimer disease. Telomeric DNA repair efficiency is lower in cells from an old donor than in cells from a young donor, normal in Alzheimer cells, and slightly lower in Werner cells. It is possible that this decline in telomeric repair with aging is of functional significance to an age-related decline in genomic stability.

The human ovarian surface epithelium (OSE), or ovarian mesothelium, is functionally complex as seen by its capacity to proliferate, migrate, and contribute to ovulation and ovulatory repair in response to cyclic hormonal and environmental changes. We wished to determine whether this phenotypic versatility is reflected in cell-extracellular matrix interactions in primary and low-passage culture. Comparisons of cultures maintained on different substrata revealed that these cells form cohesive monolayers on plastic, while fibrin clots enhance cell dispersion, and thus may provide a migratory cue. The cells invaded Matrigel as multicellular aggregates, while collagen gels mediated a morphologic epithelial-mesenchymal conversion. On plastic, the cells produced extracellular matrix components characteristic of epithelial basement membrane (laminin and collagen type IV), as well as stroma (collagen types I and III). In addition, ovarian surface epithelial cells secreted serine proteases and matrix metalloproteinases. The levels of chymotrypsin- and elastase-like proteases were dictated by the substratum: low levels were secreted by cells grown on plastic, intermediate levels on collagen gels and fibrin clots, and most protease was produced on Matrigel. The rate of cell proliferation varied with the substrata and was inversely related to protease secretion. Integrin expression was greatest on plastic and least on collagen gels where integrins were downregulated with time. alpha 6/beta 4 was absent from all cells while varying levels of alpha 2, alpha 3, alpha 5, beta 1, and vitronectin receptor were detected depending on the culture substratum employed. In low-passage cultures of human ovarian surface epithelial cells, then, cell shape, growth, protease production, and integrin expression are modulated by the extracellular matrix. The cells, in turn, alter extracellular matrix by synthesis, lysis, and physical remodeling, and express both stromal and epithelial characteristics. The broad repertoire of these functions may be related to their mesodermal origin, and may reflect the expression of a dual, epithelio-mesenchymal phenotype by relatively immature, uncommitted cells. The results demonstrate the great complexity and versatility of these interactions which render OSE cells capable of participating in numerous physiological and pathological processes.

Background: The ovarian surface epithelium (OSE) is a modified mesothelium that gives rise to most human ovarian carcinomas. In culture, OSE cells tend to assume atypical morphologies that make it difficult to accurately identify normal OSE cells and to recognize pathologic changes. The present study was undertaken to improve the accuracy of OSE identification and to distinguish phenotypic variations of normal OSE cells from early (pre)neoplastic changes., Experimental Design: The expression of epithelial and stromal markers was compared between OSE cultures in low passage, three simian virus 40-immortalized OSE lines (IOSE lines) and two ovarian carcinoma lines, using immunofluorescence microscopy, immunocytochemistry, and Western blots, with fibroblasts and vascular endothelial cells as controls., Results: Whereas keratin remained a convenient and specific epithelial marker for normal OSE, it was not expressed by all cells, and it diminished with passages in culture. E-cadherin and desmoplakins were absent in cultured OSE, mucin was detected in few cells, and microvilli diminished within one to two passages. Laminin and collagen IV were uniformly expressed and stable with time but were also found in endothelial cells. In contrast to endothelial cells, OSE lacked Factor VIII and did not bind Ulex Europaeus Lectin. The three IOSE lines were more stable than OSE morphologically, and keratin was expressed consistently in 100%, 90%, and 0% of the cells, respectively. All IOSE cells produced laminin and collagen IV but lacked E-cadherin. Microvilli persisted in 50% of the cells in one IOSE line and were lacking in the others. The antibody to breast/ovarian carcinoma, 2G3, reacted with few OSE cells but with significantly more IOSE cells. All fibroblast markers tested (vimentin, collagen types I and III, and prolyl-4-hydroxylase) were expressed in OSE and IOSE cultures, concurrently with the epithelial markers. There was no consistent relationship between any of the markers and cell morphology., Conclusions: Cultured OSE is more accurately identified if the demonstration of keratin is supplemented by 2G3, laminin, or the lack of endothelial markers. The modulation to a fibroblast-like morphology by OSE cells may reflect the expression of their dual epithelio-mesenchymal phenotype rather than epithelio-mesenchymal conversion. Possible indicators of early neoplastic change in immortalized OSE cells include reduced morphologic plasticity and increased 2G3 binding.

A spontaneously immortalized, yet non-tumorigenic rat ovarian surface epithelial (ROSE 199) cell line, deposits large amounts of extracellular matrix (ECM) in response to crowding. The characteristics and components of ROSE 199-derived cell-free ECM were compared after three different preparative techniques: treatment with 20 mM ammonium hydroxide, with 1% sodium deoxycholate, or by repeated freeze-thaws. The ECMs were analyzed by histochemistry, immunofluorescence, electron microscopy, and Western immunoblotting. Components of ROSE 199 ECM included laminin, fibronectin, and collagen types I and III. Even though ROSE 199 is an epithelial cell line, striated collagen fibers formed a major part of its matrix. Thus, ROSE 199 matrix consists of both basement membrane and stromal matrix components. This matrix supported the adhesion, spreading, and growth of several cell types without altering their morphology or growth pattern, and enhanced the attachment of some cell types that spread on plastic only with difficulty. Immunofluorescence, electron microscopy, and dry weight determinations indicated that a greater proportion of matrix was retained in preparations obtained by ammonium hydroxide or freeze thaw techniques than after sodium deoxycholate treatment. Ammonium hydroxide and freeze-thaw treated matrices were also superior to sodium deoxycholate preparations as evidenced by enhanced initial cellular adhesion and spreading compared to cells plated on plastic. Residual nuclear material did not seem to affect the biological activity of this matrix. ROSE 199 extracellular matrix provides a novel, complex substratum for cell culture and for studies of matrix functions and synthesis.

Objective: After ovulation the human ovarian surface epithelium proliferates at the wound edges, migrates over the ovulatory defect, and contributes to its repair primarily by the action of proteolytic enzymes and by the deposition of new matrix material. We examined the potential for human ovarian surface epithelial cells to physically remodel extracellular matrix in culture, similar to collagen gel lattice contraction by fibroblasts, a well-known culture model for wound repair, as an additional role of human ovarian surface epithelium in wound repair., Study Design: Human ovarian surface epithelium cells from ovarian biopsies of 11 patients were grown in culture and plated onto a combination of collagen gel and rat ovarian surface epithelial-derived extracellular matrix. The degree of matrix contraction was measured as the percentage of the original culture diameter., Results: Human ovarian surface epithelial cells surrounded and contracted the combination of matrices into a dense matrix organoid. The degree of organoid contraction was related to the number of human ovarian surface epithelial cells plated per organoid and to the inclusion of fibroblasts within the collagen gel but was not affected either by adding epidermal growth factor and hydrocortisone to the culture medium or by reducing the serum component of the medium., Conclusion: Human ovarian surface epithelial organoids may be useful for the study of normal and abnormal ovarian events such as ovulatory wound repair and cyst formation.

Objective: We aimed to improve the availability of experimental models for the study of human ovarian surface epithelium., Study Design: Low-passage cultures of human ovarian surface epithelium were transfected with SV40 large- T antigen and the resulting lines were characterized., Results: Three immortalized lines were obtained. They formed epithelial monolayers resembling ovarian surface epithelium in serum-free medium, expressed large-T antigen and overexpressed p53, produced laminin, and were CA 125 negative. Two lines expressed keratin. On plastic surfaces, the growth rate and growth potential of immortalized ovarian surface epithelium were increased over the growth of ovarian surface epithelium, but on extracellular matrices their growth and morphologic features resembled ovarian surface epithelium. The lines were not tumorigenic in Nu/Nu mice., Conclusion: The immortalized ovarian surface epithelium lines resemble cells early in neoplastic progression and should be useful to study ovarian carcinogenesis.

The ovarian surface epithelium (OSE) is known to contribute to postovulatory repair of the ovarian cortex by proliferation and migration over the site of follicular rupture, and by deposition of a basement membrane. We examined the production of other extracellular matrix components in culture by OSE cells of the rat (ROSE), using immunofluorescence microscopy, electron microscopy, and proline incorporation. We compared recently explanted cells in low passage, the immortal line ROSE 239, whose growth pattern resembles low passage cultures, and the immortal line ROSE 199, which forms ridges and papillae. The epithelial nature of all three cell types was confirmed by the presence of keratin and laminin. All three cell types secreted collagen types I and III and at least one (ROSE 199) produced highly polymerized banded fibrils, which are characteristic for stromal or interstitial extracellular matrix. Simultaneous production of collagen types I and III, keratin, and laminin by cloned subpopulations ruled out an origin of the lines in mixed epithelial/fibroblast populations. The results demonstrate that OSE has the capacity to synthesize major components of connective tissue stroma. They suggest that this epithelium, in addition to its postulated proteolytic role, may also express synthetic activity in the remodelling of the ovarian cortical stroma. A capacity of OSE cells to produce stromal components autonomously might be an important factor in the formation of ovarian surface papillae and in neoplastic progression of OSE-derived ovarian carcinomas.

The ovarian surface epithelium (OSE) is thought to give rise to over 85% of human ovarian carcinomas. In spite of its clinical importance, no animal models for the in vivo investigation of this tissue exist, and available culture methods have yielded limited success. In this study, OSE cells from 55 normal ovarian biopsy specimens were used to improve and simplify the methodology for OSE culture and to define the influence of clinical parameters on cultured OSE cells. An improved explanation method was developed which takes advantage of the tenuous attachment of OSE to underlying tissues: the surface epithelium was scraped off the ovarian surface with a rubber scraper, generating epithelial fragments which produced monolayers in culture, with little contamination by other cell types. The scrape method is superior to the explant method previously described (Siemens CH, Auersperg N: J Cell Physiol 134:347, 1988) in terms of speed, simplicity, higher purity of cultures, and increased cell yield. An improved nutrient medium (199/MCDB105/15%FBS) resulted in OSE lines that maintained the original epithelial phenotype for up to 12 population doublings. OSE, detached from the ovary, remained viable if frozen in liquid nitrogen either before culture or in primary culture on strips of plastic, providing OSE independently of the availability of surgical specimens. Growth was not influenced by diagnosis (nonmalignant gynecologic disorders), patient age (mean range: 40.5, 20 to 62 years), or the presence of inclusion cysts or large follicles in the biopsy specimen. This culture system provides conditions for in depth studies of OSE physiology and pathology.

A method of classification is presented, which divides histochemical visualization reactions into categories based on general reaction types. This scheme is dependent upon the reaction between two elements, the substrate and the probe. The substrate represents a tissue component(s) with one or more reactive groups that can combine directly with the probe. Alternatively, the substrate reactive groups are chemically modified (activation) with a suitable reagent before reaction with the probe. Probes are of three types: those that yield a coloured product, those that result in a colourless product, and those that produce a coloured product only after a further reaction. Methods used in carbohydrate histochemistry are divided into one, two and three probe reactions. Two probe reactions are further subdivided into sequences involving one or two coloured products (one and two dye sequences); three probe reactions into sequences involving one, two or three coloured products (one, two and three dye sequences). This classification permits the rationalization and organization of methods, and provides a framework for the examination of existing methods and the development of new ones.

Undifferentiated cervical carcinomas vary considerably in their intercellular organization and patterns of invasion. In spite of its clinical significance, the basis for such variation is poorly understood. We investigated the cellular properties that may be responsible for this diversity, using as a model two human cervical carcinoma cell lines that were derived from the same tumor specimen and the same clone. It was shown previously that, in spite of their common origin, each line forms a histologically distinct type of undifferentiated carcinoma when heterotransplanted in vivo: cells of line C-4I grow as compact expanding masses with central necrosis, while tumors of line C-4II infiltrate host tissues as small, well-vascularized, dispersed cell groups. The characteristic behavior of each line was retained in culture, where C-4I cells formed highly multilayered cohesive colonies, while C-4II cells formed diffuse, monolayered colonies and shed into the culture medium. These observations as well as ultrastructural data suggested that each line may be arrested at a different stage of stratified squamous differentiation. In the present study, this hypothesis was tested by examining specific differentiation markers. An analysis of the cultures by immunofluorescence microscopy and immunoblotting revealed that keratin was more abundant in the compact C-4I line than in the dispersed C-4II line. C-4I cells expressed keratins 5, 6, 8, 16, 18, and 19, while C-4II expressed only keratins 8, 16, 18, and 19. In the multilayered C-4I colonies, involucrin-positive cells occurred in the apical cell layers only. In C-4II, involucrin-positive cells occurred in monolayers and domes, and they were most consistently located apically in crowded cultures. Laminin was secreted by both lines, but only C-4II cells deposited a fibronectin matrix. The results suggest that C-4I cells resemble normal cervical cells at the spinous stage of stratified squamous differentiation, while C-4II cells resemble basal/suprabasal cells. The different growth patterns of the tumors, formed by the lines in vivo, therefore likely reflect functional and behavioral differences that normally exist between spinous and basal cervical epithelial cells. The results suggest that differentiation-related functional properties may lead to histological diversity among cervical carcinomas that are categorized as undifferentiated by histopathological criteria.