Nitric oxide mediates inhibitory effect of interleukin-1beta on estrogen production in human granulosa-luteal cells.

OBJECTIVE: To investigate the effect of IL-1beta on NO production and steroidogenesis in human granulosa-luteal cells obtained from women undergoing in vitro fertilization procedures. SUBJECTS AND METHODS: To investigate the effect of IL-1beta, granulosa-luteal cells were cultured with various doses of IL-1beta (0, 0.05, 0.5, 5, 50, 100 ng/ml), IL-1beta (5 ng/ml) with NG-nitro-L-arginine-methyl ester (L-NAME), selective inhibitors of NOS, sodium nitroprusside (SNP), NO donors and Genistain, a tyrosine kinase inhibitor. RESULTS: IL-1beta induced a dose-dependent stimulation of NO production and inhibited the production of estradiol in a significant way in a dose-dependent manner. L-NAME significantly decreased NO production and increased the production of estradiol and progesterone. SNP significantly increased NO production and caused decreases in the production of both estradiol and progesterone. Genistain decreased NO production and significantly increased the production of estradiol and progesterone. Inducible NOS (iNOS) messenger RNA was present in granulosa-luteal cells before treatment with IL-1beta. CONCLUSIONS: IL-1beta stimulated NO production, and NO inhibited the production of estradiol.

Telomerase and proliferative activity in placenta from women with and without fetal growth restriction.

OBJECTIVE: To analyze telomerase and proliferative activity in placenta from women with and without fetal growth restriction (FGR). METHODS: Telomerase activity was analyzed in 30 first-trimester chorionic villi specimens (group A) and in 28 second- and third-trimester placenta specimens (group B) from women without FGR. Telomerase activity also was analyzed in 11 placenta specimens from women with asymmetric FGR (group C). The proliferative activity of these 69 specimens was assessed by immunohistochemical staining, using the MIB-1 monoclonal antibody. RESULTS: Telomerase activity was detected in 28 (93.3%) of 30 chorionic villi specimens and in 18 (64.3%) of 28 placenta specimens without FGR. In contrast, no telomerase activity was exhibited in the placenta specimens from any of the 11 women with asymmetric FGR by telomeric repeat amplification protocol assay. Telomerase activity also was detected by in situ telomeric repeat amplification protocol assay in trophoblastic cells from women without FGR but not in trophoblastic cells from women with asymmetric FGR. Thus, telomerase activity was detected significantly more often in groups A and B than in group C (P < .01). The rate of proliferative activity, evident as positive MIB-1 staining in trophoblastic cells, in groups A and B (28.1+/-1.7% and 7.0 +/-2.9%, respectively) was significantly higher than that in group C (1.9+/-0.6%; P < .01). CONCLUSION: Telomerase and proliferative activity were minimal in placenta from women with asymmetrical FGR, suggesting placental senescence with asymmetrical FGR.

[Early phase II trial of oral etoposide administered for 21 consecutive days in patients with cervical or ovarian cancer. ETP 21 Study Group--Cervical-Ovarian Cancer Group].

We conducted multi-site early phase II trial or oral etoposide administered for 21 consecutive days in patients with cervical or ovarian cancer in cooperation with 19 institutes. Fifty mg/body of oral etoposide was administered daily for 21 consecutive days. Cycles were repeated every 28 days. In cervical cancer, 24 patients were enrolled and 17 of them were evaluated. The overall response rate including CR and PR was 23.5% (4/17). In ovarian cancer, 18 patients out of 21 enrolled were evaluated. The overall response rate was 16.7% (3/18). The primary toxicity observed was myelosuppression such as leukopenia, neutropenia, hemoglobin decrease and thrombocytopenia. Other adverse effects were anorexia, nausea, vomitting, fatigue, alopecia and stomatitis. From these results we concluded that oral etoposide administered for 21 consecutive days was effective against cervical cancer.

Telomerase activity in human chorionic villi and placenta determined by TRAP and in situ TRAP assay.

Telomerase activity (TA) was analysed in human chorionic villi and placenta in normal and abnormal pregnancy using the telomeric repeat amplification protocol (TRAP) and in situ TRAP assay. Twenty chorionic villi specimens and 25 placenta specimens from normal pregnancies were examined as well as placenta specimens from 10 cases of intrauterine growth retardation (IUGR; nine asymmetric and one symmetric). TA was detected in 18 of the 20 (90 per cent) chorionic villi specimens and in 18 of the 25 (72 per cent) placenta specimens from normal pregnancy. However, no or only weak TA was exhibited in the placenta specimens of the nine asymmetric IUGR cases. In situ TRAP assay detected TA in trophoblastic cells from normal pregnancy, but not in trophoblastic cells from cases of asymmetric IUGR.

Unscheduled expression of cyclins by anti-cancer drug exposure.

The scheduled expressions of cyclins are observed in the normal cells or the tumor cells whose phenotype is characterized by scheduled expression of cyclins, while unscheduled expression of cyclins were reported in several leukemic and solid tumor cell lines by anti-cancer drugs. We studied the effects of cytotoxic concentrations of Taxol (TXL) on cyclin D1 and B1 expression on human ovarian cancer cell lines. In KFr13 cells, the control group showed low degree of cyclin D1 and moderate degree of cyclin B1 expression in all cell cycles, while 1 microM TXL exposure resulted remarkable cyclin D1 and B1 expression in G2+M phase cells. OVCAR-3 cells showed relatively high degree of cyclin D1 expression and mild to moderate degrees of cyclin B1 expression in control group. 1 microM TXL showed no significant changes in cyclin D1 expression, while decreased expression cyclin B1 in G0+1 and S and moderate degree of expression in G2+M.

[Cancer chemo-endocrine therapy and its cell biological basis].

The aim of our cell kinetic studies is to better understand the effects of chemo-endocrine therapy at the cell biological and molecular level. Cancer cell growth is characterized by uncontrolled proliferation, resulting in DNA distribution pattern in which, at any time, more cells are not G1 phase but in S, G2 and M phase of a shortened cycle. In a recent progress, flow cytometry (FCM) has become a powerful tool for the quantitative analysis of cell cycle parameters by measuring nuclear DNA content in large cell population with high speed. With the aid of FCM in earlier work about 60-80% of ovarian cancers were found to contain aneuploid cells. Now, multi-parameter FCM linked to a computer is available to measure fluorescent intensities not only no base total DNA (Propidium iodide) but also A-T (Hoechst 33342) and G-C (Mithramycin) base pairs in solid cancer nuclei. Since cisplatinum (CDDP) is the most important drug in the treatment of ovarian cancer, we have studied the relationship of CDDP cytotoxicity, pertubations cell cycle kinetis and DNA damage in ovarian adenocarcinoma cells in vitro & in vivo. We employed both CDDP sensitive cell line (KFt) and resistant cell line (KFr) derived from human serous cystoadenocarcinoma of the ovary by Kikuchi et al (JNCI 1986). Comparing cell kinetic pertubations of experimental cells demonstrates a decrease in G1 phase cells concomitant increase in S phase cells. The KFr cells had distinctly a shorter S-phase block up to 24 hrs not A-T but G-C preference in a quick response followed repairing of DNA damage to 48 hrs. However, some fractions of CDDP resistant cell population showed a later onset of G2, M phase accumulation. Comparison with the increase in early S phase cells of KFr in detailed analysis suggests only those damaged cells that are not killed immediately may proceed to G1 phase and start into DNA synthesis in S phase. Measurement of labeling index (L. I.) with Bromodeoxyuridine (BrdU) support our interpretation of differences between sensitivity and resistance to anti-cancer drug. Additionally, we discuss a targeting chemotherapy by coupling cytotoxic drugs with estrogen based on increasing DNA damage into apoptosis and interfares with DNA repair process.

Comparative cytogenetic studies of benign, borderline, and malignant epithelial ovarian tumors.

Comparative cytogenetic studies were performed in 40 cases of untreated epithelial ovarian tumors. Of these 40 tumors, 13 were classified as benign, 3 as borderline, and 24 as malignant, according to the WHO classification for ovarian tumors. Of 13 benign ovarian tumors, 4 (30.8%) showed chromosomal abnormalities. Of 4 ovarian tumors, 3 (75%) had single chromosomal abnormalities, and the remaining tumor (25%) retained multiple chromosomal abnormalities. Of 3 borderline-malignant ovarian tumors, 2 (66.7%) showed chromosomal abnormalities. Of 2 ovarian tumors, 1 (50%) indicated single chromosomal abnormalities, and the remaining tumor (50%) revealed multiple chromosomal abnormalities. Of 24 malignant ovarian tumors, 20 (83.3%) showed chromosomal abnormalities. Of these 20 ovarian tumors, 3 (15%) had single chromosomal abnormalities, and the other 17 (85%) exhibited multiple chromosomal abnormalities. These data indicate that the rate of chromosomal abnormalities, especially multiple abnormalities, increases following the progression of malignancy in epithelial ovarian tumors.

Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes.

We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials. Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate. Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections. The 3-D images were obtained by assembling these sections using a computerized image analysis device. The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere. The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase. The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase. The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.

[Phase II study of paclitaxel (BMS-181339) in patients with ovarian cancer by 3-hour intravenous infusion].

A phase II study of Paclitaxel in patients with ovarian cancer by 3-hour intravenous infusion was undertaken by a cooperative study group of 30 institutes. Of 66 cases enrolled, 57 cases were evaluable for efficacy, and 63 cases were evaluable for safety. In spite of the fact that all cases for efficacy evaluation were previously treated with chemotherapy including platinum-based drugs, 2 cases of complete response (CR) and 15 cases of partial response (PR) were observed, with a response rate of 29.8% (The 95% confidence interval of response rate was 18.4-43.4%). Paclitaxel also showed 28.2% (11/39) response rate in patients refractory to treatment by platinum-based drugs. Histologically, the response rates were 28.9% (11/38) in serous adenocarcinoma, 40.0% (2/5) in clear cell adenocarcinoma and 25.0% (1/4) in mucinous adenocarcinoma. As the major laboratory abnormalities, leukopenia, neutropenia and decrease in hemoglobin were observed with incidence rates of 98.4% (62/63), 95.2% (59/62) and 85.7% (54/63), respectively. However, these abnormalities were clinically manageable by either withdrawal of medication, administration of antibiotics, G-CSF or metachysis etc. In addition, thrombocytopenia, elevation in GOT and GPT were seen with moderate incidence. Peripheral neuropathy was a major adverse symptom with an incidence of 79.4% (50/63), followed by alopecia, myalgia, arthralgia and fever. However, the majority of these adverse reactions were less than grade 3. From these findings, we confirmed that 3-hour intravenous infusion of Paclitaxel was a clinically useful chemotherapeutic agent in patients with ovarian cancer.

[Recent problems on the endocrine and chemotherapy].

The endometrial carcinoma, as well as the breast cancers and some kinds of ovarian cancers, was considered to a hormone dependent carcinoma. Even if we tried to do the endocrine therapy using such as medroxy progesterone acetate (MPA), we could expect only 30% of anti-tumor effects on the endometrial carcinoma. Endocrine therapy was thought to have a different action mechanisms from the other anti-cancer drugs. Whereas cisplatin (CDDP) has strongly an effectiveness for ovarian cancers, but the drug resistance of cancer cells for CDDP was causing a serious problem. This paper will be discussed about the problems of the endocrine and chemotherapy from the point of view of cell cycle analysis. Additionally, we would like to describe about the new anticancer drug such as Taxol.

Numerical and structural chromosome abnormalities in an ovarian fibrothecoma.

Cytogenetic analysis of a fibrothecoma of ovary revealed numerical and structural chromosome abnormalities, i.e., 44,XX, dup(1)(p13p31),del(3)(p14) add (10p), -16, -22. This is the first report of numerical and structural abnormalities in a fibrothecoma of the ovary.

[A phase II study of BMS-181339 in patients with ovarian cancer. BMS-181339 Ovarian Cancer Study Group].

We conducted a multicenter Phase II study of BMS-181339 in patients with ovarian cancer. The facilities participating were 23 in number. The total number of cases registered for the study were 62; 57 of them entered for evaluation in drug efficacy, and 58 cases were evaluable in drug safety. All the cases were previously treated with chemotherapy including platinum-based drugs. The clinical responses of BMS-181339 were as follows: CR, 1 case; PR, 13 cases; MR, 3 cases; NC, 13 cases and PD, 27 cases. The response rate was 24.6% (95% CI: 14.1-37.8%). Histologically, the drug showed its efficacy on serous adenocarcinoma 28.2% (11/39), mucinous adenocarcinoma 20.0% (1/5) and clear cell adenocarcinoma 20.0% (1/5). In regional evaluation, the drug demonstrated its efficacy not only on endopelvic lesions 19.0% (4/21) and abdominal lesions 14.3% (2/14), but also on remote metastatic lesions such as hepatic metastasis 30.8% (4/13) and lung/pleura 33.3% (2/6). The drug also showed its efficacy on the cases 22.9% (8/35) refractory to the platinum-based drugs. Major adverse reactions were fever 63.8% (37/58), alopecia 59.3% (32/54), peripheral nerve disorders 28.1% (16/57) such as numbness of the extremities, nausea/vomiting 24.1% (14/58), arthralgia 20.7% (12/58) and diarrhea 20.7% (12/58) etc.. Abnormal alterations in laboratory test values were an incidence rates of 100% for both leukopenia and neutropenia. However, these symptoms were clinically manageable by transient withdrawal of medication, dose reduction and administration of antibiotics and G-CSF. In addition, decrease in hemoglobin 93.1% (54/58), decrease in platelet counts 31.0% (18/58), elevation in GOT 27.6% (16/58), in GPT 31.0% (18/58) and in LDH 20.7% (12/58) were seen, but no serious organopathy was observed. Thus, we confirmed that BMS-181339 was a clinically useful chemotherapeutic agent in patients with ovarian cancer.

[Comparative genomic hybridization(CGH)].

A new molecular cytogenetic method, comparative genomic hybridization(CGH), was reviewed. CGH produces a map of DNA sequence copy number on a normal metaphase spread after hybridization with the mixture of tumor DNA and normal reference DNA, which are detected with different fluorochromes, respectively. Then the ratio of two fluorochromes are analysed with digital image analyzer and reveals a real copy number of the DNA sequences. CGH surveys entire chromosomes at a time and would clarify oncogenes and tumor suppressor genes, which had not known.

[Analysis of DNA synthetic cells in endometrial carcinoma cases treated with progestogen].

It is well known that the proliferation of endometrial adenocarcinoma is inhibited by progestogens. We often use medroxyprogesterone acetate (MPA) as endocrine therapy for advanced endometrial carcinoma. In the present study, we administered 400-600mg of MPA/day on 14 days as a progestogen challenge test (PCT) to 37 cases of endometrial carcinoma. We analysed the variation in the percentage of S phase cells by flow cytometry and also conducted an immunohistochemistry analysis with anti-BrdU monoclonal antibody before and after the administration of MPA. The percentage of S phase cells in endometrial carcinoma tended to decrease with much greater variation after PCT than before. The percentage of BrdU positive cells tended to decrease after PCT. The cases with a good histological effect such as 1) basal vacuolization 2) clear staining cytoplasm 3) homogenus finding of cells and nucleus by PCT had a better prognosis than the cases with no histological effect due to PCT.

[Phase III study of DWA2114R for ovarian cancer].

A phase III study of DWA2114R for ovarian cancer was carried out in CAP regimen by a cooperative study group consisting of 42 institutions. The response rate of 31 cases for DWA2114R regimen was 38.7% and out of 30 cases for CDDP regimen 46.7%. No significant differences were observed between the two regimens in efficacy, adverse reactions, and abnormalities in laboratory findings except for red blood cell and creatinine clearance, but the incidence of thrombocytopenia was likely to be lower in DWA2114R than in CDDP regimen. Since the DWA2114R regimen did not need hydration, or require diuretics, DWA2114R is more useful in the treatment of ovarian cancer than cisplatin.