Follicle Depletion Provides a Permissive Environment for Ovarian Carcinogenesis.

We modeled the etiology of postmenopausal biology on ovarian cancer risk using germ cell-deficient white-spotting variant (Wv) mice, incorporating oncogenic mutations. Ovarian cancer incidence is highest in peri- and postmenopausal women, and epidemiological studies have established the impact of reproductive factors on ovarian cancer risk. Menopause as a result of ovarian follicle depletion is thought to contribute to higher cancer risk. As a consequence of follicle depletion, female Wv mice develop ovarian tubular adenomas, a benign epithelial tumor corresponding to surface epithelial invaginations and papillomatosis frequently found in postmenopausal human ovaries. Lineage tracing using MISR2-Cre indicated that the tubular adenomas that developed in Wv mice were largely derived from the MISR2 lineage, which marked only a fraction of ovarian surface and oviduct epithelial cells in wild-type tissues. Deletion of p27, either heterozygous or homozygous, was able to convert the benign tubular adenomas into more proliferative tumors. Restricted deletion of p53 in Wv/Wv mice by either intrabursal injection of adenoviral Cre or inclusion of the MISR2-Cre transgene also resulted in augmented tumor growth. This finding suggests that follicle depletion provides a permissive ovarian environment for oncogenic transformation of epithelial cells, presenting a mechanism for the increased ovarian cancer risk in postmenopausal women.

Strategies for high-resolution imaging of epithelial ovarian cancer by laparoscopic nonlinear microscopy.

Ovarian cancer remains the most frequently lethal of the gynecologic cancers owing to the late detection of this disease. Here, by using human specimens and three mouse models of ovarian cancer, we tested the feasibility of nonlinear imaging approaches, the multiphoton microscopy (MPM) and second harmonic generation (SHG) to serve as complementary tools for ovarian cancer diagnosis. We demonstrate that MPM/SHG of intrinsic tissue emissions allows visualization of unfixed, unsectioned, and unstained tissues at a resolution comparable to that of routinely processed histologic sections. In addition to permitting discrimination between normal and neoplastic tissues according to pathological criteria, the method facilitates morphometric assessment of specimens and detection of very early cellular changes in the ovarian surface epithelium. A red shift in cellular intrinsic fluorescence and collagen structural alterations have been identified as additional cancer-associated changes that are indiscernible by conventional pathologic techniques. Importantly, the feasibility of in vivo laparoscopic MPM/SHG is demonstrated by using a "stick" objective lens. Intravital detection of neoplastic lesions has been further facilitated by low-magnification identification of an indicator for cathepsin activity followed by MPM laparoscopic imaging. Taken together, these results demonstrate that MPM may be translatable to clinical settings as an endoscopic approach suitable for high-resolution optical biopsies as well as a pathology tool for rapid initial assessment of ovarian cancer samples.

Expression of activated PIK3CA in ovarian surface epithelium results in hyperplasia but not tumor formation.

BACKGROUND: The Phosphatidylinositol 3'-kinase is a key regulator in various cancer-associated signal transduction pathways. Genetic alterations of its catalytic subunit alpha, PIK3CA, have been identified in ovarian cancer. Our in vivo data suggests that PIK3CA activation is one of the early genetic events in ovarian cancer. However, its role in malignant transformation of ovarian surface epithelium (OSE) is largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the Müllerian inhibiting substance type II receptor (MISIIR) promoter, we generated transgenic mice that expressed activated PIK3CA in the Müllerian epithelium. Overexpression of PIK3CA in OSE induced remarkable hyperplasia, but was not able to malignantly transform OSE in vivo. The consistent result was also observed in primary cultured OSEs. Although enforced expression of PIK3CA could not induce OSE anchorage-independent growth, it significantly increased anchorage-independent growth of OSE transformed by mutant K-ras. CONCLUSIONS/SIGNIFICANCE: While PIK3CA activation may not be able to initiate OSE transformation, we conclude that activation of PIK3CA may be an important molecular event contributing to the maintenance of OSE transformation initiated by oncogenes such as K-ras.

Vitamin A metabolism is impaired in human ovarian cancer.

OBJECTIVES: We have previously reported that loss in expression of a protein considered critical for vitamin A homeostasis, cellular retinol-binding protein 1 (CRBP1), is an early event in ovarian carcinogenesis. The aim of the present study was to determine if loss of vitamin A metabolism also occurs early in ovarian oncogenesis. METHODS: We assessed CRBP1 expression by immunohistochemistry in ovaries prophylactically removed from women with a genetic risk for ovarian cancer. Furthermore, we investigated the ability of normal, immortalized but nontumorigenic, and tumorigenic human ovarian epithelial cells to synthesize retinoic acid and retinaldehyde when challenged with a physiological dose of retinol, and determined expression levels of the retinoid-related genes, RARalpha, RXRalpha, CRABP1, CRABP2, RALDH1 and RALDH2 in these cells. RESULTS: Immunohistochemistry revealed loss of CRBP1 expression in potentially preneoplastic lesions in prophylactic oophorectomies. HPLC analysis of vitamin A metabolism showed production of retinoic acid in four independent, normal human ovarian surface epithelial (HOSE) cell cultures upon exposure to retinol. However, only one of two SV40-immortalized HOSE cell lines made RA, while none of the ovarian carcinoma cell lines produced detectable RA due to complete loss of RALDH2. CONCLUSIONS: The impaired conversion of retinol to RA in ovarian cancer cells and decreased CRBP1 protein expression in prophylactic oophorectomies support our hypothesis that concomitant losses of vitamin A metabolism and CRBP1 expression contribute to ovarian oncogenesis.

Platinum resistance: the role of DNA repair pathways.

Although platinum chemotherapeutic agents such as carboplatin, cisplatin, and oxaliplatin are used to treat a broad range of malignant diseases, their efficacy in most cancers is limited by the development of resistance. There are multiple factors that contribute to platinum resistance but alterations of DNA repair processes have been known for some time to be important in mediating resistance. Recently acquired knowledge has provided insight into the molecular mechanisms of DNA repair pathways and their effect on response to chemotherapy. This review will discuss the most important DNA repair pathways known to be involved in the platinum response, i.e., nucleotide excision repair (NER) and mismatch repair (MMR), and will briefly touch on the role of BRCA in DNA repair. The therapeutic implications of alterations in DNA repair which affect response to platinum in the treatment of patients with malignant disease, such as excision repair cross-complementation group 1 (ERCC1) deficiency and mismatch repair deficiency, will be reviewed.

Magnetic resonance imaging for detection and determination of tumor volume in a genetically engineered mouse model of ovarian cancer.

Our laboratory developed a transgenic mouse model of spontaneous epithelial ovarian cancer (EOC) in which tumors are initiated by expression of the early region of the Simian Virus 40 (SV40) under transcriptional control of the 5' upstream regulatory region of the Müllerian inhibiting substance type II receptor (MISIIR) gene. Female TgMISIIR-Tag-DR26 transgenic mice develop bilateral ovarian tumors with variable latency and survive an average of 152 days. In the absence of reliable methods for disease detection and evaluation of therapeutic response, preclinical studies of this transgenic mouse model of EOC would be limited to longitudinal experiments involving large numbers of animals with euthanasia as the endpoint. Therefore, a non-invasive method for detecting tumors, measuring tumor volume and calculating parameters relevant to the evaluation of therapeutic or preventive interventions (i.e., tumor growth rates, tumor initiation, tumor regression and the time for tumors to reach a given size) is required. We developed and optimized a non-invasive Magnetic Resonance Imaging (MRI) scanning protocol to obtain high resolution abdominal images that is well tolerated by mice. Superior contrast and contrast to noise ratio (CNR) was found with Gd-DTPA contrast enhanced T(1)-weighted sequences. Image sets in both the axial and coronal orientations for redundant measurements of normal ovary and ovarian tumor volume can be acquired in approximately 20 minutes. Accuracy of MRI-based ovary and tumor volume determinations was verified by standard volume measurements at necropsy. Serial imaging studies were performed on 41 ovarian cancer bearing TgMISIIR-Tag-DR26 transgenic mice to quantitate tumor progression over time in this model. A chemotherapy study was conducted on TgMISIIR-Tag-DR26 transgenic mice using a standard combination therapy consisting of cisplatin and paclitaxel. Our results demonstrate that MRI is well tolerated and can be repeated in serial imaging studies, permitting quantitative analysis of tumor growth and progression and response to therapeutic interventions.

RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer.

The mammalian target of rapamycin (mTOR) is thought to play a critical role in regulating cell growth, cell cycle progression, and tumorigenesis. Because the AKT-mTOR pathway is frequently hyperactivated in ovarian cancer, we hypothesized that the mTOR inhibitor RAD001 (Everolimus) would inhibit ovarian tumorigenesis in transgenic mice that spontaneously develop ovarian carcinomas. We used TgMISIIR-TAg transgenic mice, which develop bilateral ovarian serous adenocarcinomas accompanied by ascites and peritoneal dissemination. Fifty-eight female TgMISIIR-TAg mice were treated with 5 mg/kg RAD001 or placebo twice weekly from 5 to 20 weeks of age. To monitor tumor development, mice were examined biweekly using magnetic resonance microimaging. In vivo effects of RAD001 on Akt-mTOR signaling, tumor cell proliferation, and blood vessel area were analyzed by immunohistochemistry and Western blot analysis. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by approximately 84%. In addition, ascites formation, together with peritoneal dissemination, was detected in only 21% of RAD001-treated mice compared with 74% in placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. Treatment with RAD001 diminished the expression of vascular endothelial growth factor in tumor-derived cell lines and inhibited angiogenesis in vivo. RAD001 also attenuated the expression of matrix metalloproteinase-2 and inhibited the invasiveness of tumor-derived cells. Taken together, these preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer.

A reduction of cyclooxygenase 2 gene dosage counters the ovarian morphological aging and tumor phenotype in Wv mice.

Menopausal ovaries undergo morphological changes, known as ovarian aging, which are implicated in the high incidence of ovarian cancer occurring during the perimenopausal and immediate postmenopausal periods. The germ cell-deficient Wv mice recapitulate these postmenopausal alterations in ovarian morphology and develop tubular adenomas. We demonstrate that a reduction of cyclooxygenase 2 gene dosage rescued the ovarian aging phenotype of the Wv mice, whereas homozygous deletion was accompanied by a compensatory increase in ovarian cyclooxygenase 1 expression and prostaglandin E(2) synthesis. Cyclooxygenase inhibitors also reduced the tumor phenotype in a preliminary study. These findings suggest that increased cyclooxygenase activity contributes to the preneoplastic morphological changes of the ovarian surface epithelium, which can be reversed by a reduction of gene dosage achieved by either genetic or pharmacological approaches.

10th Biennial Helene Harris Memorial Trust meeting.

Improving the prognosis of ovarian cancer patients is a major challenge to scientists and clinicians. At a recent multidisciplinary meeting in Washington DC, advances in identification of precursor lesions, progress in disease biomarkers and animal models, the promise of nanotechnology, and strategies for manipulation of the innate and adaptive immune response offered prospects for real progress in this difficult-to-treat disease.

Altered expression and loss of heterozygosity of the LOT1 gene in ovarian cancer.

OBJECTIVES: Previously, we demonstrated that the LOT1 (PLAGL1/ZAC1) gene encodes a zinc-finger transcription factor and has growth suppressive effects in carcinoma cell lines. The gene is localized on chromosome 6q24-25, a common site for loss of heterozygosity (LOH) in many solid tumors including ovarian cancer. In this study, we evaluated the LOT1 gene expression and allelic deletion in the tumor tissues in order to provide additional evidence to support the gene's potential role in cancer. METHODS: The LOT1 gene expression was analyzed in malignant ovarian epithelium obtained from frozen human ovarian tumor tissues using laser capture microdissection (LCM) and real-time PCR techniques. Highly frequent single nucleotide polymorphic (SNP) sites within the LOT1 gene were identified and used for PCR and direct sequencing to determine the occurrence of allelic imbalance in a series of surgically resected ovarian and breast carcinomas. RESULTS: The analysis revealed that LOT1 mRNA expression was not detectable in 12 of 31 (39%) cases of ovarian cancer and was variable between the remaining 19 tumor specimens. These findings are consistent with the previous data that showed altered expression of LOT1 in different human ovarian carcinoma cell lines. In addition, we analyzed the occurrence of LOT1 allelic deletion in different ovarian tumor genomic DNA samples that included papillary serous (majority), mucinous, and primary peritoneal adenocarcinomas of low- to high-grade and the corresponding normal lymphocytes. The informative samples showed 12 out of 33 or about 36.4% LOH of this gene based on allelic loss of one or more polymorphic sites within the LOT1 genomic sequences. Similarly, primary breast carcinomas, which included invasive ductal (majority), spindle cell, mucinous, giant cell, and atypical medullary carcinomas, were examined on genomic DNA from patients for the allelic loss of LOT1. The informative cases showed 4 out of 10 samples or 40% LOH of this candidate tumor suppressor gene locus in breast cancer. CONCLUSIONS: The altered expression and LOH of the LOT1 locus support the gene's potential role, at least in part, in the pathogenesis of ovarian cancer and possibly in other types of cancer.

Characterization of a carcinogenesis rat model of ovarian preneoplasia and neoplasia.

Animal models of ovarian cancer are crucial for understanding the pathogenesis of the disease and for testing new treatment strategies. A model of ovarian carcinogenesis in the rat was modified and improved to yield ovarian preneoplastic and neoplastic lesions that pathogenetically resemble human ovarian cancer. A significantly lower dose (2 to 5 mug per ovary) of 7,12-dimethylbenz(a)anthracene (DMBA) was applied to the one ovary to maximally preserve its structural integrity. DMBA-induced mutagenesis was additionally combined with repetitive gonadotropin hormone stimulation to induce multiple cycles of active proliferation of the ovarian surface epithelium. Animals were treated in three arms of different doses of DMBA alone or followed by hormone administration. Comparison of the DMBA-treated ovaries with the contralateral control organs revealed the presence of epithelial cell origin lesions at morphologically distinct stages of preneoplasia and neoplasia. Their histopathology and path of dissemination to other organs are very similar to human ovarian cancer. Hormone cotreatment led to an increased lesion severity, indicating that gonadotropins may promote ovarian cancer progression. Point mutations in the Tp53 and Ki-Ras genes were detected that are also characteristic of human ovarian carcinomas. Additionally, an overexpression of estrogen and progesterone receptors was observed in preneoplastic and early neoplastic lesions, suggesting a role of these receptors in ovarian cancer development. These data indicate that this DMBA animal model gives rise to ovarian lesions that closely resemble human ovarian cancer and it is adequate for additional studies on the mechanisms of the disease and its clinical management.

Loss of surface and cyst epithelial basement membranes and preneoplastic morphologic changes in prophylactic oophorectomies.

BACKGROUND: The authors suggested that the loss of collagen IV and laminin-containing basement membrane and the loss of Disabled-2 (Dab2) expression were two critical events associated with morphologic dysplastic changes of the ovarian surface epithelium as a step in tumorigenicity. Both the basement membrane and Dab2, a candidate tumor suppressor of ovarian carcinoma, were involved in epithelial cell surface positioning and organization. The authors speculated that the purging of the basement membrane may be similar to the proteolysis during gonadotropin-stimulated ovulation, a cyclooxygenase 2 (Cox-2)-mediated process. METHODS: Prophylactic oophorectomy is used to prevent breast and ovarian carcinoma in high-risk populations. These ovarian tissue specimens often contain an increased presence of morphologically abnormal lesions that are believed to be preneoplastic. The authors evaluated archived prophylactic oophorectomy specimens to verify whether the loss of Dab2 expression and the removal of the basement membrane that occur at the ovarian surface and inclusion cyst epithelia are molecular markers of preneoplastic lesions. Of the 36 samples containing identifiable ovarian surface epithelial components on slides, immunostaining was employed to evaluate the intactness of the basement membrane (periodic acid-Schiff [PAS], collagen IV, and laminin) and the expression of Dab2 and Cox-2. Expression of Cox-1 and Cox-2 also were evaluated in cultured ovarian surface epithelial cells prepared from ovarian tissue specimens removed from patients who underwent prophylactic surgery. RESULTS: The morphologically normal ovarian surface epithelium typically contained a collagen IV- and laminin-positive basement membrane, which also was detected by PAS staining. Many morphologically altered areas, such as papillomatosis, invaginations, inclusion cysts, stratification, adenomas, and microscopic adenocarcinomas, were found in these specimens. Both the morphologically altered and adjacent morphologically normal epithelia consistently exhibited loss of basement membrane and/or Dab2 expression and an increase in Cox-2 staining. Frequently, an increase in Cox-2 staining was correlated with the loss of epithelial basement membrane in morphologically normal areas. CONCLUSIONS: The loss of Dab2 and basement membrane and the overexpression of Cox-2 were observed in presumptive neoplastic precursor areas of oophorectomy specimens obtained from a population at high risk for ovarian carcinoma. Transient loss of collagen IV and laminin in the basement membrane of the preneoplastic epithelium and the loss of Dab2 expression are common early events associated with morphologic alteration and tumorigenicity of the ovarian surface epithelium. The authors concluded that Cox-2 overexpression may play a role in the purging of basement membrane of the ovarian surface epithelium, mimicking the process of ovulation. Further experiments may be able to test the hypothetical model derived from these histologic observations.

Anomalous expression of epithelial differentiation-determining GATA factors in ovarian tumorigenesis.

Tumor cells often appear in a deviant differentiated stage, and dedifferentiation is a hallmark of malignancy; however, the causative mechanism of the global changes in dedifferentiation is not understood. The GATA transcription factors function in cell lineage specification during embryonic development and organ formation. The transcriptional targets of the GATA factors in early embryonic development include Disabled-2 and collagen IV, markers for epithelial lineages. GATA-4 and GATA-6 are expressed strongly and are localized in the nucleus in ovarian surface epithelial cells in tissues or primary cell cultures. By immunohistochemistry, we found that 82% of the 50 tumors analyzed had lost GATA-6 function, either by a complete absence of expression or by cytoplasmic mislocalization. The frequent loss of GATA-6 was also confirmed in a panel of ovarian surface epithelial and tumor cell lines. Although GATA-4 is absent only in a small percentage (14%) of ovarian tumors, it is lost in the majority of established cell lines in culture. The loss of GATA-6 correlates with the loss of Disabled-2, collagen IV, and laminin, markers for epithelial cell types. Loss of GATA factors was also found in an in vitro model for spontaneous transformation of rat ovarian epithelial cells. Repression of GATA-6 by small interfering (si)RNA approach in cultured cells leads to dedifferentiation as indicated by the loss of Disabled-2 and laminin expression. Restoration of GATA factors expression by ectopic transfection suppresses cell growth and is incompatible with the maintenance of the cells in culture. However, restoration of GATA-4 and GATA-6 expression is not able to induce expression of endogenous Disabled-2 in tumor cells, suggesting that the loss of GATA factors and dedifferentiation are irreversible processes. In conclusion, we observed the inappropriate expression and cellular localization of the GATA transcription factors in ovarian tumor tissues and cancer cell lines, and we have demonstrated that down-regulation of GATA factor expression leads to dedifferentiation. We propose that alterations of GATA transcription factor expression and aberrant nucleocytoplasmic localization may contribute to the anomalous epithelial dedifferentiation of the ovarian tumor cells.

Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells.

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.

Generation of tumors in transgenic mice expressing the SV40 T antigen under the control of ovarian-specific promoter 1.

OBJECTIVE: The ovarian-specific promoter, OSP-1, which was cloned from the transcript of a rat retrovirus-like element specifically expressed in ovarian tissue, was tested for its ability to drive ovary-specific transcription in transgenic mice. METHODS: Transgenic mice were generated with the lacZ reporter gene (OSP-lacZ) or the early region of SV40 virus (OSP-TAg) placed under the control of the OSP-1 promoter. OSP-lacZ and OSP-TAg transgenic animals were examined, respectively, for the expression of lacZ (OSP-lacZ) or the development of tumors (OSP-TAg). RESULTS: The expression of lacZ in the resulting OSP-lacZ mice was restricted to the ovary as determined by X-gal staining of multiple organs. Immunohistochemical detection of beta-galactosidase showed lacZ expression mainly in the granulosa cells and ovarian surface epithelial cells. OSP-TAg mice developed tumors in a variety of tissues, including unilateral granulosa cell tumors in two of three female founder mice. In the contralateral ovary of one mouse with a granulosa cell tumor, there were alterations in the ovarian surface epithelial cells suggestive of preneoplasia. CONCLUSIONS: Although the OSP-1 promoter was able to restrict reporter gene expression to the ovary in transgenic mice, the expression of TAg in the OSP-TAg mice resulted in ovarian tumors as well as tumors in numerous other organs. This indicated that although transcription from the OSP-1 promoter occurs predominantly in the ovary, this promoter is sufficiently leaky in cells in other tissues to permit their tumorigenic conversion by SV40 TAg.

Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer.

In women, >80% of malignant ovarian tumors are of epithelial origin. Early detection of these tumors is very challenging,and extensive i.p. dissemination is common by the time of diagnosis. The lack of adequate geneticmouse models of ovarian carcinomas significantly delays advances in early detection and treatment. We report that female transgenic mice expressing the transforming region of SV40 under control of the Mullerian inhibitory substance type II receptor gene promoter develop bilateral ovarian tumors in approximately 50% of cases. Histologically, these tumors are poorly differentiated carcinomas with occasional cysts and papillary structures present at the surface of the ovary. These tumors disseminate i.p., invade omentum, and form ascites as do human ovarian carcinomas. The epithelial origin of these tumors is supported by detection of cytokeratins 8 and 19, and the absence of alpha-inhibin, a protein characteristically expressed in normal granulosa cells and most granulosa cell tumors. Cell lines derived from the ascites exhibit the properties of epithelial ovarian cancer, such as anchorage-independent growth, tumorigenicity in immunocompromised mice, expression of epithelial cell markers, and organotropic implantation. The availability of a transgenic mouse model of disseminated ovarian carcinoma and respective cell lines should advance our understanding of this neoplasm, and serve as a useful tool for the evaluation of emerging detection and treatment strategies.

Loss of cellular retinol-binding protein 1 gene expression in microdissected human ovarian cancer.

PURPOSE: We have previously found that cellular retinol-binding protein 1 (CRBP1),involved in retinol transport and metabolism, is down-regulated in an in vitro rat model of ovarian cancer and in several human ovarian cancer cell lines. The aim of this study was to determine the clinical relevance of this change to human ovarian cancer. EXPERIMENTAL DESIGN: A cohort of 48 frozen human serous ovarian carcinomas was evaluated for CRBP1 gene expression. Malignant ovarian epithelial cells were selectively procured by laser capture microdissection, and their CRBP1 expression was determined by real-time PCR. Immunohistochemistry for CRBP1 was performed on paraffin sections of ovarian tumors using polyclonal affinity-purified rabbit anti-CRBP1 antibody. RESULTS: In 35% of ovarian cancer patient samples, there was no detectable CRBP1 expression by real-time PCR. The expression of CRBP1 in microdissected serous ovarian carcinomas was not related to either tumor stage (P = 0.6839) or grade (P = 0.9599). Quantitative PCR results were confirmed by immunohistochemistry using an antibody against CRBP1. CONCLUSIONS: The loss of CRBP1 expression in clinical ovarian tumor specimens is consistent with our previous findings in the rat model and human ovarian cancer cell lines. It appears to be an early event in ovarian carcinogenesis because there was no statistically significant difference in its frequency between tumor stages and grades. Our findings suggest that the loss of CRBP1 expression contributes to the ovarian cancer oncogenesis via altered vitamin A metabolism.

Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines.

Ovarian cancer is the leading cause of death among women from gynecological malignancies inthe United States. Resistance to the chemotherapeutic agent cisplatin isa major limitation for the successful treatment of ovarian cancer. In an effort to overcome the cisplatin resistance problem in ovarian cancer treatment, we have sought to enhance cisplatin cytotoxicity by perturbing the nucleotide excision repair (NER) pathway. The NER pathway is responsible for repairing cisplatin bound to DNA. Expression of one of the NER components, ERCC1, is correlated with cisplatin drug resistance. Hence, we targeted ERCC1 by antisense RNA methodologies, and we show that we could sensitize a relatively sensitive A2780 cell line and also the highly resistant OVCAR10 cell line to cisplatin by expressing antisense ERCC1 RNA in them as measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The A2780 cell lines expressing antisense ERCC1 had 1.9-8.1-fold enhancements in cisplatin sensitivity. The OVCAR10 antisense ERCC1 cell lines had IC(50) values ranging from 2.28 microM to 2.7 microM cisplatin as compared with 9.52 micro M for control OVCAR10 cells. The OVCAR10 antisense ERCC1 cells also show reduced DNA-damage repair capacity as assessed by host cell reactivation. Furthermore, immunocompromised mice transplanted with the antisense cell lines survived longer than the mice bearing control cells after response to cisplatin treatment. These data suggest that it is possible to substantially enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines and to enhance the survival capacity of mice in an ovarian cancer xenograft model.