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Decreased Expression of 14-3-3 Is Associated with Advanced Disease in Human Epithelial Ovarian Cancer

Its Correlation with Aberrant DNA Methylation

¹ Departments of Obstetrics and Gynecology, and ² Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan; ³ Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, Kumamoto, Japan; and ⁴ Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical School, Saitama, Japan

	ABSTRACT

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Purpose: In this study, we examined the promoter methylation status and expression of 14-3-3 and evaluated its clinical significance in epithelial ovarian cancer.

Experimental Design: Twelve ovarian cancer cell lines; 2 ovarian surface epithelial cell lines; and 8 normal, 8 benign, 12 borderline, and 102 ovarian cancer tissues were examined. Methylation-specific PCR, quantitative reverse transcription-PCR, and immunohistochemistry were used to evaluate methylation status and expression of 14-3-3 gene and protein.

Results: Among the 12 ovarian cancer cell lines, the presence of a methylated band was detected in seven cell lines. Median values of relative 14-3-3 gene expression in cancers with methylation (3.27) were significantly lower than those without methylation (16.4; P < 0.001). Treatment of 5-aza-2'-deoxycitidine resulted in the demethylation of the promoter CpG islands and reexpression. All of the normal, benign, and borderline tissues were positive for 14-3-3 protein, and in ovarian cancer tissues, 73.5% (75 of 102) were positive for 14-3-3 protein and was almost consistent with methylation status. Negative immunoreactivity of 14-3-3 was significantly correlated with high age and serous histology, high-grade, advanced-stage residual tumor of >2 cm, high serum CA125, high Ki-67 labeling index, and positive p53 immunoreactivity. 14-3-3 immunoreactivity was significantly associated with overall survival (P = 0.0058).

Conclusions: Our findings suggest that 14-3-3 is inactivated mainly by aberrant DNA methylation and that it may play an important role in the pathogenesis of epithelial ovarian cancer.

	INTRODUCTION

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Epithelial ovarian cancer is the leading cause of death from gynecological malignancies in the great majority of developed countries (1) . This high mortality is considered to be, in large part, due to the advanced stage of the disease commonly present at the time of diagnosis, but many clinical studies have reported that there are some prognostic factors in ovarian cancer other than clinical stages, such as histology, the degree of primary surgical cytoreduction, and response to chemotherapy (1, 2, 3) . Other prognostic parameters have been also proposed in addition to those relatively established parameters. These include Ki67 index, progesterone receptor, and the preoperative serum maker CA125, and others (4, 5, 6) . The identification of new prognostic factors may further contribute to improve treatment and clinical outcome of ovarian cancer patients.

The cause of epithelial ovarian cancer is still unknown. Although BRCA1 and BRCA2 mutation have been identified as associated with susceptibility to ovarian cancer (7 , 8) , mutations in these genes account for only 2–3% of all ovarian cancers. The remaining cases are considered to be sporadic in nature and arise as a result of acquired alterations in oncogenes and tumor suppressor genes such as TP53 and PTEN (9, 10, 11) .

DNA methylation has an essential regulatory function in mammalian development, suppressing gene activity by changing chromatin structure (12 , 13) . It has become apparent that aberrant DNA methylation of promoter region CpG islands may serve as an alternate mechanism to genetic defects in the inactivation of tumor suppressor genes in human cancers (14 , 15) . Accordingly, the identification of gene targets of methylation-associated silencing could lead to novel genes involved in the initiation and progression of human neoplasia.

14-3-3 was originally identified as a p53-inducible gene that is responsive to DNA damaging agents (16) . Recent study demonstrated that 14-3-3 protein plays a crucial role in the G₂ checkpoint by sequestering the mitotic initiation complex, cdc2-cyclin B1, in the cytoplasm after DNA damage (17) . This prevents cdc2-cyclin B1 from entering the nucleus in which the protein complex would normally initiate mitosis. In this manner, 14-3-3 induces G₂ arrest and allows the repair of damaged DNA (16 , 17) . The expression of 14-3-3 is reported to be frequently lost in human breast, gastric, and lung cancers, and the inactivation is due to aberrant DNA methylation (18, 19, 20) . However, the expression of 14-3-3 and its mechanism have not been examined in epithelial ovarian cancer. Therefore, in this study, we examined the promoter methylation status and expression of 14-3-3 in epithelial ovarian cancer cells. We also evaluated the correlation between 14-3-3 expression and clinicopathological parameters in patients with epithelial ovarian cancer.

	MATERIALS AND METHODS

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Tissues and Cells.
Eight normal ovaries, 8 ovarian serous cystadenomas, 12 serous borderline tumors, and 102 ovarian cancer cases were obtained from patients after surgical therapy from 1988 to 2000 at Tohoku University Hospital, Sendai, Japan. In ovarian cancer patients, information regarding age, performance status on admission, histology, stage, grade, residual tumor after primary surgery, and overall survival were retrieved from the review of patient charts. Median follow-up time of the patients in this study was 59 months (4–120 months). Eighty-four (82.3%) of 102 patients received platinum-containing chemotherapy after operation. Patients who have early-stage (stage Ia) and low grade-disease (G1, G2) and patients who have poor performance status did not receive platinum-based chemotherapy. Performance status was defined according to WHO criteria (21) . Histology and stage were determined according to FIGO (International Federation of Gynecology and Obstetrics) criteria. Grade was evaluated by one of the authors (T. M.) using universal grading system in epithelial ovarian cancer (22) . Residual disease was determined by the amount of unresectable tumor that remained after primary cytoreductive surgery. Optimal cytoreduction was defined as no gross residual tumor greater than 2 cm in diameter, and suboptimal cytoreduction was defined as any gross residual disease remaining greater than 2 cm in diameter. Overall survival was calculated from the time of initial surgery to death, or the date of last contact. Survival times of patients still alive or lost to follow-up were censored in December 2002. All of these archival specimens were retrieved from the surgical pathology files at Tohoku University Hospital, Sendai, Japan. These specimens were all fixed in 10% formalin and embedded in paraffin. The research protocol was approved by the ethics committee of Tohoku University Graduate School of Medicine.

OVCAR3, Caov3, SKOV3, TOV112D, TOV21G, OV90, and ES2 (adenocarcinoma, OVCAR3, SKOV3; serous adenocarcinoma, Caov3, OV90; clear cell adenocarcinoma, TOV21G, ES2; endometrioid adenocarcinoma, TOV112D) cell lines were purchased from American Type Culture Collection. JHOS2, JHOS3, HTOA, OMC3, and JHOC5 (serous adenocarcinoma, JHOS2, JHOS3, HTOA; mucinous adenocarcinoma, OMC3; clear cell adenocarcinoma, JHOC5) cell lines were purchased from Riken cell bank (Tsukuba, Japan). Normal ovarian surface epithelial cell lines (OSE2 and OSE4) were established by one of the authors (M. N.; Ref. 23 ). Cell lines were maintained in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen), and were incubated in 5% CO₂ at 37°C. For 5-aza-2' deoxycytidine (5azaC) treatment, 1 x 10⁶ cells were seeded into T75 flasks and were treated with 0.5 µM or 1.0 µM 5azaC (Sigma) for 72 h.

Methylation-Specific PCR.
Methylation status of the samples was investigated by methylation-specific PCR as described in the literature (24) . Genomic DNA of ovarian cancer tissue was extracted using a laser capture microdissection and treated with proteinase K (0.5 mg/ml) for 48 h at 37°C. Genomic DNA from ovarian cancer cell lines was extracted using AquaPure Genomic DNA kit (Bio-Rad). The quality and integrity of the DNA was determined by the A260:280 ratio. One µg of genomic DNA was treated with sodium bisulfite using CpGenome DNA modification kit (Intergen) according to the instructions. Amplification was achieved in a 20-µl reaction volume containing 2 µl of 10x Ex Taq Buffer, 1.5 µl of 25 mM MgCl₂, 1 µM each primer, 1.5 µl of 2.5 mM dNTPs, and 1 unit of Takara Ex Taq polymerase (Takara, Japan). Hot-start PCR was performed in a thermal cycler (Takara) for 35 cycles, each of which consisted of denaturation at 96°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 90 s, followed by a final 10 min extension at 72°C. Primers used were 5'-TGGTAGTTTTTATGAAAGGCGTC-3' and 5'-CCTCTAACCGCCCACCACG-3' (104-bp) for the methylated reaction (M primers), and 5'-ATGGTAGTTTTTATGAAAGGTGTT-3' and 5'-CCCTCTAACCACCCACCACA-3' (106-bp) for the unmethylated reaction (U primers; Ref. 18 ). Universal methylated human male genomic DNA (Intergen) was used as a positive control for methylated reaction. Genomic DNA purified from MCF-7 breast cancer cell line was used as a positive control for unmethylated reaction (18) . A blank control containing all of the PCR components except template DNA was also included in all of the PCRs. Reaction products were separated by electrophoresis on a 3% agarose gel, stained with ethidium bromide, and visualized under UV illumination. Specimens with purely unmethylated promoters have positive PCR products by U primers but not with the M primers. Specimens that contain purely methylated promoters will have PCR products by using M primers but not with the U primers. Specimens that contain heterogeneous status of both methylated and unmethylated promoters have PCR products from both U primers and M primers.

Reverse Transcription and Real-Time Quantitative PCR.
Total RNA was isolated from cell lines by phenol-chloroform extraction using Isogen (Nippon Gene, Tokyo, Japan). RNA was treated with RNase-free DNase (Roche Diagnostics; 1 µg/µl) for 2 h at 37°C, followed by heat inactivation at 65°C for 10 min. A reverse transcription-PCR kit (SUPERSCRIPT II First-strand synthesis system, Invitrogen) was used and cDNA synthesis was carried out according to the instructions. cDNAs were synthesized from 2 µg of total RNA using random hexamer, and reverse transcription was carried out for 50 min at 42°C with SUPERSCRIPT II reverse transcriptase. Real-time quantitative PCR was performed using the iCycler system (Bio-Rad). For the determination of 14-3-3 cDNA content, a 25-µl reaction mixture consisted of 23 µl iQ SYBR Green MasterMix, 1 µM each primer, and 1 µl of template cDNA were prepared. The PCR conditions were as follows: initial duration at 96°C for 60 s, and followed by 35 cycles with denaturation at 96°C for 30 s, annealing at 64°C (both for 14-3-3 and ß-actin) for 30 s, and extension at 72°C for 30 s. The fluorescence intensity of the double-strand specific SYBR Green I, reflecting the amount of formed PCR-product, was read at 88°C after the end of each elongation step. Primers used were as follows: 14-3-3, 5'-CCTGCTGGACAGCCACCTCA-3' and 5'-TGTCGGCCGTCCACAGTGTC-3' (397-bp; Ref. 20 ); ß-actin, 5'-CCAACCGCGAGAAGATGA-3' and 5'-GGAAGGAAGGCTGGAAGAGT-3' (459-bp; Ref. 25 ). ß-actin cDNA fragments were amplified as internal positive controls. Normal human ovarian cDNA library (Stratagene, La Jolla, CA) was used as a normal control, cDNA from MCF-7 was used as a positive control, and water blank was used as a negative PCR control (data not shown). Control reactions in which reverse transcriptase was omitted were amplified under the same conditions to exclude DNA contamination (data not shown). Two independent quantitative PCR reactions were performed for each sample.

Immunohistochemistry.
Immunohistochemical analysis was performed using the streptavidin-biotin amplification method using a Histofine kit (Nichirei, Tokyo, Japan), and have been previously described in detail (26) . Polyclonal antibody for 14-3-3 (N-14), monoclonal antibody for p53 (B20.1), and monoclonal antibody for Ki-67 (MIB-1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Biomeda (Foster City, CA), and DAKO (Tokyo, Japan), respectively.

For antigen retrieval, the slides were heated in an autoclave at 120°C for 5 min in citric acid buffer [2 mM citric acid and 9 mM trisodium citrate dyhydrate (pH 6.0)]. The dilutions of primary antibodies for 14-3-3, p53 and Ki-67 were 1:100, 1:40, and 1:300, respectively. The antigen-antibody complex was visualized with 3,3'-diaminobenzidine (DAB) solution [1 mM DAB, 50 mM Tris-HCl buffer (pH 7.6), and 0.006% H₂O₂], and counterstained with hematoxylin. Nonneoplastic breast epithelial tissue was used as a positive control (18) . As negative controls, 0.01 M PBS and normal mouse IgG were used in place of primary antibodies.

The immunohistochemical expression of 14-3-3, p53, and Ki-67 was independently reviewed by two of the authors (J. A. and T. M.) who had no knowledge of the clinicopathological data. As immunoreactivities of 14-3-3 and p53 were relatively homogeneous and clearly distinguished for positive and negative, they were classified into two groups: +, positive carcinoma cells; and –, no immunoreactivity. Scoring of Ki-67 in carcinoma cells was counted independently by these same two authors, and the percentage of immunoreactivity in at least 500 carcinoma cells, i.e., labeling index, was determined. Whenever a difference of greater than 5% was observed between the two readings, slides were reviewed jointly, and a consensus was reached.

Statistical Analysis.
Statistical analysis was performed using Stat View 5.0 (SAS Institute Inc.) software. The statistical significance between 14-3-3 and characteristics of the patients was evaluated using Mann-Whitney U test, Kruskal-Wallis test, and Scheffe analysis. Correlation between 14-3-3 and Ki-67, p53 immunoreactivity was also assessed using Mann-Whitney U test. Univariate analysis of prognostic significance for prognostic factors was performed using a log-rank test, after each survival curve was obtained by the Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to evaluate the predictive power of each variable independently of the others. All of the patients who could be assessed were included in the intention-to-treat analysis. A result was considered significant when the P was less than 0.05.

	RESULTS

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Methylation Status and Expression of 14-3-3 in Ovarian Cancer Cells.
Among the 12 ovarian cancer cell lines in which 14-3-3 promoter methylation was investigated, the presence of a methylated band was detected in 7 cell lines, 2 of which were together with unmethylated band as shown in Fig. 1 . The methylated band was detected in all of the cell lines derived from clear cell adenocarcinoma (TOV21G, ES2, JHOC5), two of five of serous adenocarcinoma (Caov3, OV90, JHOS2, JHOS3, HTOA), one of one of endometrioid adenocarcinoma (TOV112D), and none of one of mucinous adenocarcinoma (OMC-3). Both of the OSE cells were negative for methylated band.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Methylation-specific PCR (MSP) for 14-3-3 in ovarian cancer and surface epithelial cell lines. Specimens with methylated promoters have positive PCR products in the Methylated Lane, and specimens with unmethylated promoters have positive PCR products in the Unmethylated Lane. Specimens that contain heterogeneous status of both methylated and unmethylated promoters have PCR products in both the Methylated and the Unmethylated Lanes. Methylation statuses of each cell line are defined as; +, purely methylated: –, purely unmethylated: ±, both methylated and unmethylated, and are shown in the Methylation status Lane. M-DNA (universal methylated human male genomic DNA) was used as a positive control for methylated reaction. MCF-7 (MCF7) breast cancer cell line was used as a positive control for unmethylated reaction.

View larger version (26K): [in this window] [in a new window]   Fig. 2. A, expression of the 14-3-3 gene in ovarian cancer cells. Mean of the two independent results of real-time quantitative reverse transcription (RT)-PCR of ovarian cancer and surface epithelial cell lines are shown. The number on top of each bar, relative 14-3-3 gene expression standardized by the amount of internal positive control (ß-actin). Normal ov (normal human ovarian cDNA library) was used as a normal control. MCF-7 was used as a positive control. B, reexpression of the 14-3-3 gene by 5-aza-2'-deoxycitidine (5azaC) treatment. OVCAR3 and OV90 cell lines that have only methylated bands were treated with 5azaC for 3 days. Methylation status and mRNA expression of 14-3-3 was assessed by methylation-specific PCR (MSP) and quantitative RT-PCR on day 0 (D0) and day 3 (D3) with or without 5azaC (0.5 µM and 1.0 µM) treatment (AzaC). Complete and partial demethylation was observed in OVCAR3 and OV90, respectively. The number on the top of each bar, relative 14-3-3 gene expression standardized by the amount of internal positive control (ß-actin).

Immunohistochemistry and Methylation Status of 14-3-3 in Ovarian Cancer Tissues.
Positive immunoreactivity for 14-3-3 was detected in the cytoplasm of epithelial cells, although p53 and Ki-67 were confined exclusively to the nuclei of epithelial cells (Fig. 3) . In normal, benign, and borderline tissues, all of the cases were positive for 14-3-3 immunoreactivity. In ovarian cancer tissues, 73.5% (75 of 102) and 36.3% (37 of 102) were positive for 14-3-3 and p53, respectively. Median Ki-67 labeling index was 17.2%.

View larger version (155K): [in this window] [in a new window]   Fig. 3. Immunohistochemistry for 14-3-3 in normal ovary, and in benign, borderline, and malignant ovarian tumors. Representative cases of immunohistochemistry for 14-3-3 in the ovarian surface epithelium (A), benign adenoma (B), borderline tumor (C), two cases of epithelial ovarian cancer (D, G), and serial sections of each case for Ki-67 (E, H) and p53 (F, I) are shown. Note that positive 14-3-3 case (D) is negative for Ki-67 (E) and p53 (F), whereas negative 14-3-3 case (G) is positive for Ki-67 (H) and p53 (I), x200 for all figures.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Methylation-specific PCR (MSP) for 14-3-3 gene in ovarian cancer tissues. The methylation status of 14-3-3 gene in microdissected ovarian cancer tissues was evaluated by MSP in the 10 cases of positive (Lanes 1–10), and 10 cases of negative (Lanes11–20) immunoreactivity. Definition of Methylated and Unmethylated Lanes are the same as in Fig. 1 .

View this table: [in this window] [in a new window]   Table 1 Correlation between 14-3-3 immunoreactivity and clinicopathological parameters in epithelial ovarian cancer

View larger version (15K): [in this window] [in a new window]   Fig. 5. Correlation between 14-3-3 expression and overall survival in patients with epithelial ovarian cancer.

	DISCUSSION

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The 14-3-3 gene is demonstrated to be induced after DNA damage in a p53-dependent manner (16) , and to play a role in the G₂ checkpoint by sequestering the Cdc2/cyclin B1 complex (17) . Similar to our results, several authors demonstrated epigenetic inactivation of the 14-3-3 gene in human cancers (18 , 20 , 27) . Experimental inactivation of the 14-3-3 gene causes a G₂ checkpoint defect, and results in the accumulation of chromosomal aberrations that increase the sensitivity to the DNA-damaging events (28 , 29) . In this context, it is of interest that serous histology in which significantly decreased expression of 14-3-3 protein was observed is highly sensitive to chemotherapeutic agents. The histological type-specific expression of the 14-3-3 gene suggests that serous adenocarcinomas develop unique differentiation, different from other histological subtypes. Although a high proportion of human cancers are likely to have a chromosomal instability phenotype, mutations of the genes involving the G₂-M-phase checkpoint have rarely been found, and the mechanism of chromosomal instability in most of them is still unknown. Further study on the correlation between genetic instability and 14-3-3 is needed.

In a number of previous reports, it is strongly postulated that the inactivation of 14-3-3 might play an important role in tumor progression. Ostergaard et al. (30) showed that less-differentiated bladder squamous cell carcinoma is characterized by decreased expression of some proteins, including 14-3-3. Suzuki et al. (19) reported that aberrant methylation of the 14-3-3 gene was frequently observed in poorly differentiated gastric adenocarcinoma. Umbricht et al. (31) reported that decreased expression of 14-3-3 was observed in 24 (96%) of 25 carcinomas, 15 (83%) of 18 of ductal carcinoma in situ, and 3 (38%) of 8 of atypical hyperplasias, and concluded that inactivation of 14-3-3-sigma occurs at an early stage in the progression of invasive breast cancer. Osada et al. (20) recently demonstrated frequent and type-specific inactivation of the 14-3-3 gene in small cell lung cancer. In our study, loss of 14-3-3 expression was significantly correlated with high-grade advanced-stage bulky residual tumor and high serum CA125 and high Ki-67 levels, all of these factors represent fundamental difference in pathogenesis in ovarian cancer. Our results, together with previous reports, suggest that the loss of 14-3-3 expression in ovarian cancer may have invasive and progressive characteristics.

Recent study suggests that promoter methylation increases with age in several genes in normal tissues, although the mechanism of age-related methylation is unknown (32 , 33) . Several factors may modulate age-related methylation, such as exogenous carcinogens, endogenously generated reactive oxygen species, and genetic differences in individual susceptibility (33) . In our study, 14-3-3 protein expression was decreased in elderly patients by immunohistochemistry. Although age-related methylation of 14-3-3 has not been reported, it is possible that expression of this gene is suppressed by methylation with age, and it may contribute to carcinogenesis in ovarian cancer.

In the present study, a clear correlation between aberrant methylation and silencing of 14-3-3 was observed except for one cell line (HTOA) and 2 of 10 primary ovarian cancer tissues, which showed unmethylated patterns despite loss of 14-3-3 expression. These results suggest that DNA methylation-independent mechanism may also be involved in the loss of 14-3-3 expression. Several other ways of gene inactivation such as loss of transcription factor are assumed, and Osada et al. (20) considered that 14-3-3 gene silencing might occur without methylation in only primary tissues. From our results, positive p53 immunoreactivity, which suggests loss of functional p53 protein (34 , 35) , seems to contribute to the loss of 14-3-3 expression in primary ovarian tissues. Possible methylation-independent mechanism remains to be elucidated to better understand the regulation of 14-3-3 expression.

In conclusion, aberrant CpG island methylation is an epigenetic change that is largely responsible for silencing of the 14-3-3 gene. Inactivation of 14-3-3 occurs in substantial proportion and may play a role as a potential tumor suppressor gene in epithelial ovarian cancer.

	FOOTNOTES

 
Grant support: Supported in part by a grant-in-aid for Scientific Research from the Ministry of Health and Welfare, a grant-in-aid from the Ministry of Education, Science and Culture, a grant-in-aid from Kurokawa Cancer Research Foundation, and a grant-in-aid from Japan Society of Gynecologic Oncology (JSGO).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Jun-ichi Akahira, Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan. Phone: 81-22-717-7254; Fax: 81-22-717-7258; E-mail: jakahira-tohoku{at}umin.ac.jp

Received 10/31/03; revised 12/ 3/03; accepted 12/19/03.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Akahira J, Yoshikawa H, Shimizu Y, et al Prognostic factors of stage IV epithelial ovarian cancer: a multicenter retrospective study. Gynecol Oncol, 81: 398-403, 2001.[CrossRef][Medline]
2. Del Campo JM, Felip E, Rubio D, et al Long-term survival in advanced ovarian cancer after cytoreduction and chemotherapy treatment. Gynecol Oncol, 53: 27-32, 1994.[CrossRef][Medline]
3. Omura GA, Brady MF, Homesley HD, et al Long-term follow-up and prognostic factor analysis in advanced ovarian carcinoma: the gynecologic oncology group experience. J Clin Oncol, 9: 1138-50, 1991.[Abstract]
4. Kaufmann M, Von Minckwitz G, Kuhn W, et al Combination of new biologic parameters as prognostic index in epithelial ovarian carcinoma. Int J Gynecol Cancer, 5: 49-55, 1995.[CrossRef][Medline]
5. Akahira J, Inoue T, Suzuki T, et al Progesterone receptor isoforms A and B in human epithelial ovarian carcinoma: immunohistochemical and RT-PCR studies. Br J Cancer, 83: 1488-94, 2000.[CrossRef][Medline]
6. Cooper BC, Sood AK, Davis CS, et al Pre-operative CA125 levels: an independent prognostic factor for epithelial ovarian cancer. Obstet Gynecol, 100: 59-64, 2002.[Abstract/Free Full Text]
7. Miki Y, Swensen J, Shattuck-Eidens D, et al A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science (Wash DC), 266: 66-71, 1994.[Abstract/Free Full Text]
8. Wooster R, Bignell G, Lancaster J, et al Identification of the breast cancer susceptibility gene BRCA2. Nature (Lond), 378: 789-92, 1995.[CrossRef][Medline]
9. Guttmacher AE, Collins FS. Breast and ovarian cancer. N Engl J Med, 348: 2339-47, 2003.[Free Full Text]
10. Newman B, Millikan RC, King MC. Genetic epidemiology of breast and ovarian cancers. Epidemiol Rev, 19: 69-79, 1997.[Free Full Text]
11. Ford D, Easton DF, Peto J. Estimates of the gene frequency of BRCA1 and its contribution to breast and ovarian cancer incidence. Am J Hum Genet, 57: 1457-62, 1995.[Medline]
12. Kass SU, Pruss D, Wolffe AP. How does DNA methylation repress transcription?. Trends Genet, 13: 444-9, 1997.[CrossRef][Medline]
13. Razin A, Ceder H. DNA methylation and gene expression. Microbiol Rev, 55: 451-8, 1991.[Abstract/Free Full Text]
14. Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet, 21: 163-7, 1999.[CrossRef][Medline]
15. Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res, 61: 3225-9, 2001.[Abstract/Free Full Text]
16. Hermeking H, Lengauer C, Polyak K, et al 14-3-3 is a p53-regulated inhibitor of G₂/M progression. Mol Cell, 1: 3-11, 1997.[CrossRef][Medline]
17. Chan TA, Hermeking H, Langauer C, Kinzler KW, Vogelstein B. 14-3-3 is required to prevent mitotic catastrophe after DNA damage. Nature (Lond), 401: 616-20, 1999.[CrossRef][Medline]
18. Ferguson AT, Evron E, Umbricht CB, et al High frequency of hypermethylation at the 14-3-3 locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA, 97: 6049-54, 2000.[Abstract/Free Full Text]
19. Suzuki H, Itoh F, Toyota M, et al Inactivation of the 14-3-3 gene is associated with 5' CpG island hypermethylation in human cancers. Cancer Res, 60: 4353-7, 2000.[Abstract/Free Full Text]
20. Osada H, Tatematsu Y, Yatabe Y, et al Frequent and histological type-specific inactivation of 14-3-3 in human lung cancers. Oncogene, 21: 2418-24, 2002.[CrossRef][Medline]
21. WHO. . Handbook for reporting results of cancer treatment, WHO Geneva WHO Publication No. 48. 1979.
22. Shimizu Y, Kamoi S, Amada S, et al Toward the development of a universal grading system for ovarian epithelial carcinoma. Prognostic significance of histopathologic features-problems involved in the architectural grading system. Gynecol Oncol, 70: 2-12, 1998.[CrossRef][Medline]
23. Nitta M, Katabuchi H, Ohtake H, et al Characterization and tumorigenicity of human ovarian surface epithelial cells immortalized by SV40 large T antigen. Gynecol Oncol, 81: 10-7, 2001.[CrossRef][Medline]
24. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA, 93: 9821-6, 1996.[Abstract/Free Full Text]
25. Akahira J, Suzuki T, Ito K, et al Differential expression of progesterone receptor isoforms A and B in the normal ovary, and in benign, borderline, and malignant ovarian tumors. Jpn J Cancer Res, 93: 807-15, 2002.[Medline]
26. Akahira J, Suzuki T, Ito K, et al Expression of 5-reductases in human epithelial ovarian cancer: its correlation with androgen receptor status. Jpn J Cancer Res, 92: 926-32, 2001.[Medline]
27. Lodygin D, Yazdi AS, Sander CA, Herzinger T, Hermeking H. Analysis of 14-3-3 expression in hyperproliferative skin diseases reveals selective loss associated with CpG-methylation in basal cell carcinoma. Oncogene, 22: 5519-24, 2003.[CrossRef][Medline]
28. Dhar S, Squire JA, Hande MP, Wellinger RJ, Pandita TK. Inactivation of 14-3-3 influences telomere behavior and ionizing radiation-induced chromosomal instability. Mol Cell Biol, 20: 7764-72, 2000.[Abstract/Free Full Text]
29. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in human cancers. Nature (Lond), 396: 643-9, 1999.
30. Ostergaard M, Rasmussen HH, Nielsen HV, et al Proteome profiling of bladder squamous cell carcinomas: identification of markers that define their degree of differentiation. Cancer Res, 57: 4111-7, 1997.[Abstract/Free Full Text]
31. Umbricht CB, Evron E, Gabrielson E, et al Hypermethylation of 14-3-3 (stratifin) is an early event in breast cancer. Oncogene, 20: 3348-53, 2001.[CrossRef][Medline]
32. Ahuja N, Li Q, Mohan AL, Baylin SB, Issa JP. Aging and DNA methylation in colorectal mucosa and cancer. Cancer Res, 58: 5489-94, 1998.[Abstract/Free Full Text]
33. Issa JP. CpG-island methylation in aging and cancer. Curr Top Microbiol Immunol, 249: 101-18, 2000.[Medline]
34. Iggo R, Gatter K, Bartek J, Lane D, Harris AL. Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet, 335: 675-9, 1990.[CrossRef][Medline]
35. Skilling JS, Sood A, Niemann T, Lager DJ, Buller RE. An abundance of p53 null mutations in ovarian carcinoma. Oncogene, 13: 117-23, 1996.[Medline]

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