Sulfatase activity in normal and neoplastic endometrium.

BACKGROUND: Dehydroepiandrosterone sulfate (DHEAS) is metabolized to active androgens and estrogens, which may have a role in the development of endometrial cancer. METHODS: We studied DHEAS conversion to dehydroepiandrosterone (DHEA) in normal and neoplastic endometrium utilizing gas chromatography-mass spectral (GC-MS) analysis. Endometrial homogenate was incubated with known amounts of DHEAS for 4 h at 37 degrees C. Methanol extract was separated from debris by centrifugation, concentrated to 200 microl and 1 microl injected into the GC-MS instrument, equipped with a CP-Sil 8 column. DHEAS and DHEA areas were calculated by autoquantization and DHEA/DHEAS ratio was used for comparing sulfatase activity among normal endometrium (n = 6), Stage I endometrioid carcinoma (EC) (n = 15), Stage I mixed mesodermal Mullerian tumor (MMMT) (n = 6) and Stage I uterine papillary serous carcinoma (UPSC) (n = 7). RESULTS: DHEA/DHEAS ratios in normal endometrium, EC, MMMT and UPSC were 1.45 +/- 1.10, 5.63 +/- 3.27, 2.88 +/- 0.99, and 3.04 +/- 1.76, respectively. Sulfatase activity was significantly higher in EC when compared with normal endometrium (p < 0.001), MMMT (p < 0.05), and UPSC (p < 0.05). The enzyme activity did not differ significantly between low-grade and high-grade EC tumors (5.8 +/- 2.77 and 5.49 +/- 3.84, respectively, p > 0.05). CONCLUSION: Stage I EC have higher sulfatase activity than normal endometrium, and Stage I MMMT and UPSC tumors.

Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer.

OBJECTIVE: Matrix metalloproteinases (MMPs) and their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), play a key role in tumor cell invasion, angiogenesis, and growth. The aim of this study was to determine the expression and cellular distribution of MMP-26, TIMP-3, and TIMP-4 in endometrial cancers and benign endometrium throughout the menstrual cycle and the correlation with tumor histological subtype, stage, and grade. METHODS: Immunohistochemical analysis using polyclonal antibodies generated against pro- and active MMP-26, and mono- and polyclonal antibodies specific to TIMP-3 and TIMP-4, respectively, was performed. RESULTS: MMP-26, TIMP-3, and TIMP-4 are expressed in endometrial carcinomas (N = 86) and benign endometrium (N = 50) from various stages of the menstrual cycle. Semi-quantitative analysis of staining intensity indicated that endometrial carcinomas expressed more MMP-26, TIMP-3, and TIMP-4 compared to benign endometrium from the postmenopausal period, but not from the secretory phase of the menstrual cycle. The highest staining intensity was associated with endometrial epithelial cells, followed by vascular endothelial cells, myometrial smooth muscle cells, and endometrial stromal cells. Increased staining intensity of MMP-26 and TIMP-3 correlated with grade III tumors and MMP-26 and TIMP-4 with the depth of myometrial invasion in tumors histologically characterized as endometrioid adenocarcinoma, clear-cell, and papillary serous carcinoma staged/graded based on FIGO criteria. CONCLUSION: MMP-26 and TIMP-4 are expressed in endometrium and endometrial carcinoma and their elevated expression and correlation with myometrial invasion suggests that MMP-26 and TIMP-4 may play a key role in endometrial tumor progression.

u-PA expression in benign, borderline and malignant ovarian tumors.

The malignant potential of solid tumors is related to their ability to invade adjacent tissue and to metastasize. The plasminogen activation system is one of the critical factors in tumor progression since it is involved in tumor invasion and metastasis. This study was performed to examine the expression of u-PA in benign, borderline and malignant tumors of the ovary by immunohistochemical evaluation on formalin-fixed, paraffin-embedded specimens applying monoclonal antibody 3689 directed to the b-chain of u-PA. Normal epithelial cells of the ovary (n = 5) showed no staining of u-PA but some stromal cells were slightly stained. Invasive carcinomas (n = 16) and borderline tumors (n = 15) showed a moderate to strong diffuse cytoplasmic staining. Benign tumors (n = 20) showed a variety of staining. The observation of randomly positive u-PA stromal cells is noteworthy. The percentage of u-PA-positive tumors was higher in carcinomas than in other tumors. There was no correlation with other known risk factors of malignancy such as differentiation, stage or type of tumor. In conclusion there are noticeable differences in u-PA expression among ovarian tumors and u-PA increase in ovarian tumors can be attributed to an increased diffuse cytoplasmic content in the neoplastic epithelial cells.

DNA methylation in ovarian cancer. II. Expression of DNA methyltransferases in ovarian cancer cell lines and normal ovarian epithelial cells.

OBJECTIVE: The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers. METHODS: DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells. CONCLUSIONS: We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer.

Tumor angiogenesis and thymidine phosphorylase expression in ovarian carcinomas including serous surface papillary adenocarcinoma of the peritoneum.

To clarify biological and clinical significance of tumor angiogenesis in the development of ovarian carcinoma, we investigated the relationship between tumor vascularity, the expression of thymidine phosphorylase (dThdPase), which is an angiogenic factor identical to platelet-derived endothelial cell growth factor (PD-ECGF), and patient outcome in ovarian carcinoma, including serous surface papillary carcinoma (SSPC). Primary tumor specimens (stages I-IV) from 54 patients were examined. Intratumoral microvessel density (IMVD) and dThdPase expression were evaluated immunohistochemically using anti-CD34 and anti-dThdPase antibodies, and results were correlated with clinicopathologic parameters and prognosis. IMVD for the 54 tumors ranged from 22.5 to 120.7 (number/0.73686 mm2/field). Twenty-three tumors were positive, and 31 tumors were negative for dThdPase expression. IMVD positively correlated with the expression of dThdPase (p < 0.01), tumor size, and peritoneal metastases (p < 0.05). However, there was no statistical correlation between IMVD, dThdPase expression, and clinical outcome. Of the 54 patients examined, 30 were diagnosed with International Federation of Gynecology and Obstetrics (FIGO) stage III or IV primary ovarian carcinoma, and 9 were diagnosed with SSPC. There were no significant differences between the two groups with respect to clinicopathologic features, IMVD, dThdPase expression, or patient outcome. In conclusion, angiogenic activity may be necessary for the growth of metastatic implants in ovarian carcinoma and SSPC.

Expression of gamma-glutamyl transpeptidase in stage III and IV ovarian surface epithelial carcinomas does not alter response to primary cisplatin-based chemotherapy.

OBJECTIVE: Gamma-glutamyl transpeptidase activity has been shown to be essential for the nephrotoxicity of cisplatin. The purpose of this study was to determine whether expression of gamma-glutamyl transpeptidase in ovarian carcinomas is necessary for the antitumor effect of cisplatin. STUDY DESIGN: Tumor tissue from 18 patients with stage III or IV ovarian serous papillary carcinoma or poorly differentiated adenocarcinoma was analyzed for expression of gamma-glutamyl transpeptidase by histochemical or immunohistochemical staining. Response to cisplatin-based combination chemotherapy was evaluated on the basis of clinical response, progression-free interval, and survival. RESULTS: Gamma-glutamyl transpeptidase expression in the tumors ranged from 0% to 100% of the tumor cells gamma-glutamyl transpeptidase positive. Patient survival ranged from 15 months to 9 years. Twelve of the 18 patients had a complete response to the initial course of cisplatin-based combination chemotherapy. There was no statistically significant correlation between either response or time to relapse and gamma-glutamyl transpeptidase expression. However, there was a correlation between high levels of gamma-glutamyl transpeptidase in the tumor and acute ototoxicity in patients treated with cisplatin. Expression of high levels of gamma-glutamyl transpeptidase in the tumor was also found to be associated with shorter patient survival, suggesting that gamma-glutamyl transpeptidase might have a role in resistance to drugs used in second- and third-line therapy. CONCLUSIONS: Expression of gamma-glutamyl transpeptidase in ovarian serous papillary or poorly differentiated adenocarcinomas is not necessary for the antitumor activity of cisplatin. A correlation was found between high levels of gamma-glutamyl transpeptidase in the tumor and both increased ototoxicity from cisplatin and decreased patient survival. These data suggest that administering an inhibitor of gamma-glutamyl transpeptidase activity to block the nephrotoxicity of cisplatin would not interfere with its therapeutic effect.

Biochemical characterization of primary peritoneal carcinoma cell adhesion, migration, and proteinase activity.

Primary papillary serous carcinoma of the peritoneum (PPC) is clinically and histologically similar to advanced stage epithelial ovarian carcinoma. PPC classically presents with widespread intraperitoneal dissemination, superficial invasion, and minimal ovarian involvement. Surgical cytoreduction and combination chemotherapy utilized for patients with epithelial ovarian carcinoma have produced varying results for patients with PPC. These differences in response may be secondary to the stage of disease or due to biological differences in metastatic behavior between these carcinomas. In this study, short-term primary cultures of PPC and epithelial ovarian carcinoma (OVCA) were compared to enable biochemical comparison with respect to components of the metastatic cascade including adhesion, migration, and proteinase activity. These data demonstrated similar properties in adhesive profiles of PPC and OVCA, with preferential adhesion to type I collagen and vitronectin. Matrix-degrading proteinases including matrix metalloproteinases (MMP)-2, MMP-9, and urinary-type plasminogen activator were produced by both cell types. PPC migration was stimulated by multiple extracellular matrix proteins, whereas OVCA cells demonstrated maximal migration on type I collagen coated surfaces. Together our data suggest biochemical similarities between PPC and OVCA with respect to individual components of the metastatic cascade.

Deregulated expression of cyclin D1 and other cell cycle-related genes in carcinogen-induced rat mammary tumors.

Dysregulation of cyclin expression has been reported for several human malignancies, including breast cancer. To further investigate the role of cyclin genes in mammary tumorigenesis we analyzed the expression of cyclins D1, E and A and other cell cycle-related proteins in a series of nine N-methyl-N-nitrosourea-induced primary rat mammary tumors. Western blot analysis revealed a 10- to 15-fold increase in the level of cyclin D1 protein in most (7/9) of the tumors, when compared with normal rat mammary gland. The two tumors that did not show this increase also displayed negligible levels of the retinoblastoma protein. A moderate increase, 1.5- to 2-fold, in the level of cyclin E was observed in four tumors and three tumors displayed abnormal low molecular weight cyclin E-related proteins. None of the tumors showed amplification of the cyclin D1 or E genes when studied by Southern blot analysis. All nine tumors showed a 2- to 6-fold increase in the level of cyclin A protein. Most of the tumors also displayed a marked increase in levels of the CDK2 and CDK4 proteins. These changes did not appear to be simply a consequence of increased cell proliferation, as assessed by proliferating cell nuclear antigen analysis. Thus, aberrant expression of cyclins and other cyclin-related genes occurs frequently in mammary tumorigenesis in both rodents and humans.