Relationship between the expression of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase and survival in epithelial ovarian cancer.

The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14+/-0.62 and 0.59+/-0.06 in tumor tissue, and 0.71+/-0.14 and 0.16+/-0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11+/-0.02) than in normal controls (0.38+/-0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients' prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.

15-Lipoxygenase-2 is differentially expressed in normal and neoplastic ovary.

Ovarian cancer is a major cause of lethality from gynecological malignancies, and there is a lack of reliable and specific serum markers for this disease. Eicosanoid-related enzymes have previously been implicated in the pathogenesis of various types of cancer, but little is known about the relevance of lipoxygenase isoforms in ovarian cancer and the results on cyclooxygenases are conflicting. For this study, we quantified the expression of eicosanoid-related enzymes (cyclooxygenase-1 and cyclooxygenase-2, 15-lipoxygenase-1 and lipoxygenase-2, 5-lipoxygenase) in normal and malignant human ovarian tissue by real-time polymerase chain reaction and found a 22-fold elevated expression of 15-lipoxygenase-2 in malignant specimens when compared with normal ovarian tissue (P=0.001). In ovarian carcinoma metastases, expression of the enzyme was also augmented (20-fold upregulation, P=0.004). For 15-lipoxygenase-1 and cyclooxygenase-2, we did not observe differential expression, but there was a trend for increased steady-state concentrations of cyclooxygenase-1 (P=0.1 for ovarian carcinoma, P=0.011 for metastases) and 5-lipoxygenase (P=0.1 for ovarian carcinoma, P=0.018 for metastases, respectively). These data indicate that expression of 15-lipoxygenase-2 mRNA is strongly augmented during ovarian carcinogenesis and that the enzyme may constitute a suitable candidate as a tumor marker.

Multifocal biliary cystadenocarcinoma of the liver: CT and pathologic findings.

Biliary cystadenocarcinoma is a rare tumor that originates from the hepatobiliary epithelium. Although this tumor can affect any portion of the biliary tree, intrahepatic location is more common. It is usually a slow growing tumor and often asymptomatic until it reaches a considerable size. The lesion is most often solitary and large when discovered; multiple lesions or metastases within the liver are very rare. A 63-year-old man was referred to our institute for weight loss, abdominal discomfort, worsening bulky symptoms in the right upper abdominal quadrant, and an increase in serum aminotransferases that had been present for several months. Spiral CT of the abdomen demonstrated two lesions, a larger one and a distant intrahepatic lesion, with a multiloculated cystic aspect, a thin peripheral capsule, multiple solid peripheral portions, and irregular septa enhancing in the portal phase after intravenous administration of iodinated contrast medium. The diagnosis of multifocal cystadenocarcinoma of the liver was confirmed by surgical laparoscopy and biopsy of the lesion. The patient was treated with chemotherapy.

Comparison of four new cell lines from patients with adenocarcinoma of the ovary.

Permanent human tumor cell lines COLO 110, COLO 316, COLO 319, and COLO 330 were established from four patients with serous cystadenocarcinoma of the ovary. COLO 110 was derived from primary tumor tissue; COLO 316, COLO 319, and COLO 330 were derived from cells in malignant effusions. COLO 110 and COLO 316 grew as monolayers of epithelioid cells in culture; COLO 319 and COLO 330 grew as vermiform, floating colonies of epithelioid cells in culture. Epithelial-like morphology was confirmed by transmission electron microscopy. All four cell lines had marker chromosomes and double minute chromosomes. Giemsa banding revealed chromosomes 1, 3, 6, and 7 were involved in markers in all four lines, and chromosomes 2, 4, 5, 9, 11, and 15 were involved in markers in three of the cell lines. Marker chromosomes with possible homogeneous staining regions were observed in COLO 319. Estrone was elaborated by three of the lines, but neither chorionic gonadotropin, carcinoembryonic antigen, nor estrogen or progesterone receptor proteins were detected. Each cell line demonstrated a distinctive isozyme phenotype. These cell lines are maintained in active culture and in a cell bank for distribution to other investigators.

Fibronectin secretion from human peritoneal tissue induces Mr 92,000 type IV collagenase expression and invasion in ovarian cancer cell lines.

Our previous study showed that human peritoneal conditioned medium (CM) increased the matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of ovarian cancer cells (NOM1). In an effort to identify this MMP-9-stimulating factor, we examined the effects of extracellular matrix components, such as type IV collagen, laminin, and fibronectin, on ovarian cancer cells. We found that fibronectin increased the MMP-9 activity of NOM1 cell CM in a concentration-dependent manner and that the peritoneal CM contained high level of fibronectin. An increase of MMP-9 activity in NOM1 cell CM by the peritoneal CM was almost completely blocked by 20 microg/ml of anti-integrin alpha5/FnR antibody and RGD polypeptides. Furthermore, after immunoprecipitation by antifibronectin antibody supernatant of the peritoneal CM did not increase MMP-9 activity in NOM1 cells. Fibronectin and the peritoneal CM also increased MMP-9 activity and expression in NOM1 cell lysate, and these effects were blocked by anti-integrin alpha5/FnR antibody. Invasiveness of NOM1 cells was enhanced by fibronectin and the peritoneal CM in a concentration-dependent manner, and anti-integrin alpha5/FnR antibody blocked these effects. These results suggested that fibronectin secreted from peritoneum increased MMP-9 activity and expression, and, in turn, invasiveness of ovarian cancer cells.

Cytologic evidence for gene amplification in methotrexate-resistant cells obtained from a patient with ovarian adenocarcinoma.

To analyze if methotrexate (MTX) resistance arises from gene amplification in a patient treated clinically with MTX, the cytogenetic and drug sensitivity profile of the tumor colony forming units (TCFUs) from a 58-year-old woman with stage III well-differentiated ovarian serous adenocarcinoma was studied. This patient had not received treatment directed against her tumor for nine months before this study, but had received oral-dose MTX (2.5 mg, twice weekly) for three years for the treatment of psoriasis. Analysis of TCFUs grown in nucleoside-free media demonstrated MTX resistance at concentrations of up to 100 micrograms/mL (2.2 X 10(-4)M). Cytologic evidence for dihydrofolate reductase (DHFR) gene amplification in TCFUs was determined by in situ hybridization, using radiolabeled cDNA to DHFR mRNA. Results localized the DHFR sequences to an abnormally staining region present on chromosome 4q. This study supports the notion that alterations in gene dosage (that is, gene amplification) play a role in the development of drug resistance in spontaneous human cancers.

In situ steroid sulfatase activity in human epithelial carcinoma cells of vaginal, ovarian, and endometrial origin.

The enzymatic hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroisoandrosterone, respectively, was studied in cells that were derived from four different malignant tumors of the lower reproductive tract of women, viz. a squamous cell vaginal carcinoma, an ovarian carcinoma, and two endometrial adenocarcinomas. These cells had the capacity to hydrolyze both steroid sulfoconjugates. Estrone sulfate was more efficient as a substrate than dehydroepiandrosterone sulfate, since the amount of product formed from estrone sulfate was approximately 3-fold greater than that formed from dehydroepiandrosterone sulfate. Some kinetic parameters of steroid sulfatase were determined in the four cell types and were found to be very similar, as were the rates of hydrolysis. Sulfatase activity was linear with incubation time for at least 2 h and with cell number up to 3.2 X 10(6) cells/mL. The apparent pH optimum of steroid sulfatase, determined by the use of cell sonicates and estrone sulfate as the substrate, was between 6.0 and 7.5. The apparent Km values of steroid sulfatase for estrone sulfate in both squamous vaginal carcinoma cells and ovarian carcinoma cells were both 5 microM, and those for dehydroepiandrosterone sulfate in squamous vaginal carcinoma cells and endometrial adenocarcinoma cells were 6 and 4 microM, respectively. The optimal temperature of steroid sulfatase in squamous vaginal carcinoma cells was 50 C; at this temperature, enzymatic activity was more than twice that at 37 C. The steroid sulfatase pathway that is operative in carcinoma cells in vitro to produce free steroids from steroid sulfate precursors also may serve to produce free steroids in vaginal, endometrial, and ovarian carcinomas in vivo and, perhaps, maintain and stimulate tumor growth.

Expression of O6-alkylguanine-DNA-alkyltransferase in situ in ovarian and Hodgkin's tumours.

The cellular expression of O6-alkylguanine-DNA-alkyltransferase (ATase) may be an important factor in determining tumour sensitivity to certain alkylating agents. In a comparative study, we have examined the inter- and intracellular distribution of ATase in tumour biopsies of a series of patients with Hodgkin's disease and ovarian cancer using a rabbit antihuman ATase antiserum. The antibody recognises the ATase protein on western blots of cell-free extracts of a number of ovarian tumours with ATase activities varying from 20 to 420 fmol/mg protein as determined by in vitro assay and there was a linear correlation between ATase activity and the intensity of the band on western blots (r = 0.993). Immunohistochemical staining was seen in all of the ovarian tumours examined and was confined to the nucleus. This is in contrast to the Hodgkin's tissue, where staining was much reduced and present in both nuclei and cytoplasm. The results suggest that in ovarian tumours the general resistance to nitrosourea chemotherapy may be related to the high cellular expression of ATase protein: this is in contrast to the more chemosensitive Hodgkin's disease. This raises the possibility that it might be feasible to predict sensitivity or resistance to these alkylating agents by immunohistochemical staining of tumour or tissue specimens.

Inhibition of telomerase activity and cell proliferation by a reverse transcriptase inhibitor in gynaecological cancer cell lines.

Telomerase is a ribonucleoprotein which has a RNA template to bind and extend telomere ends, so prolonging the life of tumour cells. The aim of this study was to determine whether transcriptase function of telomerase could be inhibited by the reverse transcriptase inhibitors (RTI); azydothymidine (AZT), dideoxyinosine (ddI) and AZT-5' triphosphate (AZT-TP). We examined their effects on the proliferation of cancer cells and the antitumour effects of cisplatin in vitro. The three agents did not cause major changes in telomerase activity or telomere length in MCAS cells. However, in HEC-1 cells changes in telomerase activity and telomere length were observed that were dependent on the RTI concentration and duration of exposure. ddI and AZT-TP reduced telomerase activity and shortened the length of the telomere. In the presence of RTI, the antitumour effects of cisplatin were enhanced. This was particularly evident in HEC-1 cells where there was a marked reduction in cell proliferation, appearance of morphological changes and senescent-like cells in the presence of ddI or AZT-TP. In MCAS cells, TP53 expression was increased by ddI and AZT-TP, while p21 expression was unchanged. In HEC-1 cells the expression of both TP53 and P21 was increased by ddI. Continuous administration of RTI enhanced the cell growth inhibition of cisplatin. RTI also inhibited the proliferation of some cells.

Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro.

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.

Immunohistochemical localization of lactate dehydrogenase isoenzyme 1 in germ cell tumors of the testis.

In this study, antiserum to lactate dehydrogenase isoenzyme 1 (LD 1) was used to determine immunohistochemical patterns of localization in a variety of germ cell neoplasms of the testis. All 29 seminomas and teratocarcinomas and four of seven embryonal carcinomas were positive for LD 1. These results suggest that elevated serum LD 1 levels noted in patients with germ cell neoplasms may be derived directly from neoplastic tissue and explain the relationship between serum LD 1 levels and tumor burden. A variety of neoplasms from other organs also were evaluated in order to determine the specificity of LD 1 staining for testicular tumors.

Structural study of the sugar chains of alpha-amylases produced ectopically in tumors.

About thirty percent of two alpha-amylases produced from a serous papillary cystadenocarcinoma of the ovarium (case 1) and a bronchioloalveolar adenocarcinoma of the lung (case 2) was glycoproteins containing 1 mol of asparagine-linked sugar chain, respectively. The structures of the sugar moieties were found by sequential enzymatic degradation and methylation analysis to be as follows: [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3)][GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(6)]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6][NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc. Structures of asparagine-linked sugar chains were the same in the tumors of cases 1 and 2 and were incomplete in comparison with those of the parotid amylase.

Relationship between aromatase activity and steroid receptor levels in ovarian tumors from postmenopausal women.

Aromatase activity, as well as steroid receptors, exists in nonfunctional ovarian tumors. Steroid receptor status has been reported to be related to prognosis in ovarian cancer patients. We determined aromatase activity and progesterone receptor (PR) and estrogen receptor (ER) levels in 43 ovarian tumors obtained from postmenopausal women. Aromatase activity was detected in 35 tumors (81%), PR in 21 tumors (49%) and ER in 13 tumors (30%). Eighty-three percent (10/12) of mucinous cystadenoma tissues showed positive PR with high aromatase activity, while 93% (13/14) of malignant tumors showed negative PR and low aromatase activity. Aromatase activity was detected in 95% (20/21) of PR-positive tumors, being greater than in PR-negative tumors (P < 0.002). There was a positive correlation between aromatase activity and PR (rs = 0.49, P < 0.001). However, there was no correlation between aromatase activity and ER. In 17 patients (43%), the serum estradiol level was higher than 30 pg/ml and there was a positive correlation among estradiol, estrone, androstenedione and testosterone. However, serum steroid levels were not correlated with aromatase activity, PR or ER. Aminoglutethimide inhibited aromatase activity of benign and malignant ovarian tumors, uterine myoma, choriocarcinoma cells and purified human placental P-450arom in a similar manner. These results suggest that aromatase activity is correlated with PR in ovarian tumors of postmenopausal women. In addition to steroid receptor status, aromatase activity may be a useful prognostic factor in ovarian cancers.

Radioimmunoassay of placental alkaline phosphatase in ovarian cancer sera and tissues.

A specific radioimmunoassay for human placental alkaline phosphatase has been developed using the 125I-labeled enzyme, highly purified with a fast protein liquid chromatography system and an absorbed rabbit antiserum. The sensitivity of this assay was 0.2 U/L. Serum levels of over 0.2 U/L were found in 27% of ovarian cancer patients, and most of these elevated enzyme levels occurred with more advanced stages of the disease. On the other hand, almost all ovarian cancer tissue contained detectable levels of the enzyme. Serous adenocarcinoma, endometrioid adenocarcinoma, and dysgerminoma had particularly large amounts. Placental alkaline phosphatase was more frequently detected in tissue than in the serum of ovarian cancer, and therefore may be a useful target in immunodetection and immunotherapy and in studying the histopathology of ovarian cancer.

Immunochemical studies of human placental alkaline phosphatase in normal and neoplastic tissues.

Human placental alkaline phosphatase (hPLAP) is normally present in plasma membranes of syncytiotrophoblast microvilli. hPLAP is expressed by many malignant tumors and is considered to be useful as a tumor marker. The objective of the work was to develop immunochemical methods for detection of this marker. hPLAP was isolated from placenta using a procedure with preparative isoelectric focusing as a final purification step, and was used to obtain rabbit antisera. Anti-hPLAP antibodies were obtained by affinity chromatography on the immobilized hPLAP antigen and were used in immunoenzyme tests for detection of hPLAP in sera and tissues of cancer patients with different malignancies. Anti-hPLAP antibodies were also used for localization of hPLAP on the histological level. The described methods may be of use in cancer diagnostics and therapy.

Glutathione S-transferase pi immunostaining of cisplatin-resistant ovarian cancer cells in ascites.

OBJECTIVE: To clarify immunohistochemically the correlation between glutathione S-transferase pi (GST pi) expression of surgically resected specimens and clinical response in ovarian cancer and to evaluate ascites cytology using GST pi staining. STUDY DESIGN: Eighty-seven patients with ovarian cancer underwent initial debulking surgery and received cisplatin-based chemotherapy after surgery. Immunostaining for GST pi was performed on formalin-fixed sections of the patients' tumors. The cytologic slides of 24 cases were acquired for evaluation of GST pi staining. RESULTS: Of 87 surgically resected specimens, 55 (63.2%) were GST pi positive. Twenty-five of 28 patients (89.3%) who showed no response to chemotherapy had GST pi-positive tumor cells. The predictive value of positive GST pi staining for drug resistance was 75.8% (25/33). Of 18 cases that were GST pi positive in surgically resected specimens, 17 were positive in ascites cytology. Five cases were negative in both resected specimens and ascites cytology. There was a significant correlation in the GST pi labelling index between resected specimen and ascites cytology from the same case; the correlation coefficient was .701 and P value < .001. CONCLUSION: Overexpression of GST pi is related to resistance to cisplatin, and GST pi staining of ascites cytology can be used in pretherapeutic assessment of patients with ovarian cancer.

[Neoplasm of the ovary as a cause of hyperamylasemia]. / Neoplasia de ovario como causa de hiperamilasemia.

A case of hyperamylasemia produced outside the pancreas and of exceptional origin, is presented. The place of origin is an ovarian carcinoma whose first manifestation was acute abdominal pain. The blood level of amylase was slightly increased mainly in its salivary fraction detected by the chromogenic/inhibition technique. This level, followed the tumor evolution and decreased in parallel to the tumor's response treatment.

[Comparative study on activity of adenylate cyclase in normal human ovary with ovarian tumors].

It has been documented that the binding activity sites of human chorionic gonadotropin (HCG) receptor and kd in human ovarian tumors are different from those in the normal ovary. And some observations suggest that the HCG receptor depends on adenylate cyclase (AC) for its physiological functions. We studied the activity of AC in normal human ovary and ovarian tumors. Five human ovarian specimens and eighteen ovarian tumor specimens were obtained from women patients undergoing gynecological surgery. Ovaries were homogenized and sonicated. The homogenates were centrifuged at 1000 x g for 15 min. After sucrose density gradient ultracentrifugation (78000 x g, 4 h), the membrane fraction was collected from interface between 33% and 37%. The membrane protein approximately 10 micrograms, ATP 1 mmol/L, 3H-ATP 5 x 10(4) cpm, sulphydryl-ethyl alcohol 10 mmol/L, in a final volume of 200 microliters of Tris-HCl buffer (50 mmol/L), pH7.5, containing MgSO4 5 mmol/L, were incubated at 35 degrees C for 10 min. The reaction was stopped by boiling water bath for 3 min. The AC activity (U/mg): of human normal ovary, 67 +/- 8, of cystadenocarcinoma serous, 146 +/- 70; of cystadenocarcinoma mucinous, 289 +/- 83.