Measurement of endometrial thickness in premenopausal women in office gynecology.

Purpose: To define the median endometrial thickness (ET) in office gynecology is thought to be important for clinical practice. However, there are few reports about ET that have included the general female population on a large scale. The median ET was determined prospectively in premenopausal women who attended office gynecology for cervical cancer screening. Methods: In total, 849 women were enrolled. The median ET was determined by using transvaginal ultrasound and the relationships between the ET and various clinical factors were analyzed. Results: The participants' median age was 38.5 years. The median ET was 8.6 mm (90% and 95% quantiles: 13.8 and 15.8 mm). The ET was not related to their age, symptoms, obstetric history, geographical location, or risk factors for endometrial cancer. In the women with a menstrual cycle length of 28-30 days, the ET was 7 mm on days 1-6, but it increased from 5.4 mm immediately after menstruation (day 7 or 8) to 9.2 mm on days 13-14. Subsequently, the ET increased further to 11.1 mm on day 18. Conclusion: In all the women, the upper limit of the ET was 13.8 mm and 15.8 mm in the 90% and 95% quantile, respectively, in office gynecology.

Clinical features of ovarian large-cell neuroendocrine carcinoma: Four case reports and review of the literature.

The objective of the present study was to present 4 recently encountered ovarian large-cell neuroendocrine carcinoma (LCNEC) cases, and to evaluate their clinicopathological features in the context of the previously reported 29 LCNEC cases. First, we described the clinical features of 4 recently encountered cases. Routine H&E staining and immunohistochemistry for CD56, synaptophysin and chromogranin A were performed on sections of both the LCNEC and epithelial carcinoma components. Clinical data for the total of 33 LCNEC cases were summarized, and the Kaplan-Meier survival curve was estimated. Our cases were observed in women aged 42-81 years. One case is clinically classified as FIGO stage IV with multiple metastases, and the others are classified as FIGO stages Ic, IIc and IIIb by post-surgical findings. Pathological features, assessed by H&E staining, were similar to lung LCNEC, and at least one neuroendocrine marker was positive staining in both LCNEC and the epithelial component. One case was pure type LCNEC and the others were mixed carcinoma. Paclitaxel/carboplatin chemotherapy was performed for all cases and 3 of the 4 treatments were effective. The prognoses of our cases were as follows: 1 in stage Ic died from the disease after only 2 months, but the others survived, with or without recurrence, for 32-64 months, whereas the total 5-year survival of the 33 LCNEC cases was 34.9%. In summary, our 3 LCNEC cases revealed ordinary chemo-sensitivity, resulting in a better prognosis than those previously described, apart from 1 case which exhibited aggressive behavior. For the future, a retrospective survey to elucidate the prognostic factors and prospective clinical studies to evaluate the efficacy of treatment modalities of ovarian LCNEC are necessary, particularly for aggressive LCNEC cases.

Intra-arterial infusion chemotherapy using cisplatin with radiotherapy for Stage III squamous cell carcinoma of the cervix.

PURPOSE: To examine the effectiveness of concomitant intra-arterial infusion chemotherapy (IAIC) using cisplatin (CDDP) with radiotherapy for Stage III squamous cell carcinoma of the cervix. MATERIALS AND METHODS: We analyzed 29 cases of Stage III squamous cell carcinoma of the uterine cervix treated with radiotherapy and IAIC of CDDP from 1991 to 2006. External-beam therapy was given to the whole pelvis using four opposing parallel fields with an 18-MV linear accelerator unit. A central shield was used after 30-40 Gy with external whole-pelvic irradiation, and the total dose was 50 Gy. High-dose-rate brachytherapy was given with (192)Ir microSelectron. The dose at Point A was 6 Gy per fraction, 2 fractions per week, and the total number of fractions was either 3 or 4. Two or three courses of IAIC were given concomitantly with CDDP 120 mg or carboplatin 300 mg. RESULTS: We confirmed excellent medicine distribution directly by using computed tomographic angiography. The 5-year overall survival rate for Stage III patients was 62%, the cause-specific survival rate was 70%, and the local relapse-free survival rate was 89%. Local recurrence, distant metastasis, and occurrences of both were 7%, 38%, and 3%, respectively. The incidence of severe acute hematologic adverse reactions (Grade > or =3) was 27% for all patients; however, all recovered without interruption of radiotherapy. Severe nonhematologic effects (Grade > or =3) were 3%, including nausea and ileus. Only 1 patient's radiotherapy was interrupted for a period of 1 week because of ileus. Severe late complication rates (Grade > or =3) for the bladder, rectum, and intestine were 3%, 3%, and 10%, respectively. CONCLUSION: A combination of IAIC and systemic chemotherapy should be considered to improve the prognosis of patients with Stage III squamous cell carcinoma of the cervix.

Distribution of platinum in the female genital tract and efficacy of radiotherapy combined with transcatheter arterial infusion of cisplatin for locally advanced stage IIIb carcinoma of the uterine cervix.

The current main treatment for locally advanced stage III/IV cervical cancer involves chemoradiotherapy. In this study, we investigated the distribution of platinum in the female genital tract by intra-arterial infusion of platinum (carboplatin 150 mg) during surgery and examined the therapeutic effects of radiotherapy with transcatheter arterial infusion (TAI) of cisplatin for locally advanced carcinoma of the uterine cervix. From January 1991, we randomly selected 26 patients with locally advanced stage IIIb cervical cancer to receive radiotherapy combined with TAI of 120 mg/body cisplatin twice a month at an interval of 4 weeks. Radiotherapy routinely involved 50 Gy of external beam irradiation to the whole pelvis and 12-24 Gy (point A dose) of intracavitary irradiation using a remote afterloading system. The mean platinum concentration in the cervical cancer was 1.77 microg/g wet tissue (wt) and high value, but the genital tract also contained the same platinum concentration. The platinum concentration in each regional lymph node was 1.10-1.48 microg/g wt, and its level of platinum was equal to that in the female genital tract. The effective histologic response rate was 88.5% (23/26). The median follow-up period was 38 months. The cumulative survival rate was 74.0%. Serious acute adverse reactions interfering with treatment were not observed. Based on these results, intra-arterial infusion of platinum produced a therapeutic effect on the primary cervical cancer site and the other parts of the female genital tract. We concluded that radiotherapy with TAI of cisplatin achieved superior therapeutic efficacy in locally advanced stage IIIb cervical cancer.

Preclinical analysis of the antitumor efficacy of TS-1 using human uterine cervical cancer tumor xenografts.

We investigated the antitumor activity of TS-1 in comparison with that of UFT and cisplatin (CDDP) against cervical cancer using xenografts of a human uterine cervical squamous cell cancer cell line, CaSki, transplanted into female Balb/cA JcL-nu mice. CaSki cell xenografts were prepared by subcutaneous (s.c.) implantation of 3x10(6) cells/animal into the right dorsal region of the mice. The tumor volume was measured twice a week and the relative tumor volume (RTV) was calculated. We divided the animals into four groups according to the treatment administered; TS-1 (10 mg/kg orally, once daily for 14 consecutive days), UFT (24 mg/kg orally, once daily for 14 consecutive days), CDDP (7.6 mg/kg injected intravenously once on the 1st day) and control (no treatment) groups. The antitumor effects of the drugs were measured. On the 35th day after the completion of treatment, the mean tumor volume in the mice treated with TS-1 or CDDP changed from 132.873+/-11.783 mm(3) to 706.401+/-613.122 mm(3) and 133.809+/-19.366 mm(3) to 722.630+/-855.509 mm(3), respectively. The mean tumor volume in the groups treated with TS-1 or CDDP was significantly lower compared to that in the control group (p<0.001; one-tailed Student's t-test). The relative inhibition of the tumor growth was 65.31 in the TS-1 group, 48.31 in the UFT group and 64.51 in the CDDP group. We conclude that TS-1 administered orally for 14 consecutive days showed the highest antitumor activity.

UFT and its metabolite gamma-butyrolactone (GBL) inhibit angiogenesis induced by vascular endothelial growth factor in advanced cervical carcinoma.

OBJECTIVE: The aim of this study was to evaluate the potential role of UFT and its metabolite gamma-butyrolactone (GBL) for inhibition of angiogenesis induced by vascular endothelial growth factor (VEGF) in advanced cervical carcinoma by the determination of serum GBL and VEGF, and by immunohistochemical staining to assess VEGF protein expression, before and after UFT therapy. METHODS: The subjects were 35 patients with an advanced cervical carcinoma and five healthy volunteers between 2002 and 2003 at Hiroshima University Hospital, under informed consent. The patients received two courses of oral fluoropyrimidine (UFT) therapy at a dose of 600 mg/day for 5 and 2 days off treatment. Serum GBL and VEGF was measured before and after UFT therapy by the gas chromatography mass spectrometry and ELISA-kit in 22 patients and five healthy volunteers, respectively. Immunohistochemical detection of VEGF protein was done in 35 cervical cancers. Results The mean serum GBL level before and after UFT therapy was 21.9 +/- 2.3 and 79.3 +/- 6.2 ng/ml, respectively, and it was significantly increased after UFT administration (P < 0.0001). The mean serum VEGF level before and after UFT therapy was 95.3 +/- 28.1 and 67.5 +/- 11.2 pg/ml, respectively, and it was decreased by UFT administration. In 20 out of 33 (66.6%) patients who were detected with VEGF protein, VEGF protein expression was decreased by UFT therapy. The Delta GBL value (GBL after UFT--GBL before UFT therapy) showed a significant inverse correlation with Delta VEGF value (VEGF after therapy--VEGF before therapy) (r2 = 0.940). CONCLUSIONS: Our findings suggest that UFT and its metabolite GBL inhibit angiogenesis induced by VEGF to have an antitumor effect on cervical cancer.

Differential regulation of DEC2 among hypoxia-inducible genes in endometrial carcinomas.

In this study, we demonstrate an important role of activation of the hypoxia-inducible factor-1 (HIF-1) pathway in endometrial carcinogenesis and tumor phenotype development of endometrial carcinoma, and suggest a unique role of the HIF-1-target gene, differentiated embryo chondrocyte 2 (DEC2), in carcinogenesis. Hypoxia caused an increase in HIF-1alpha protein expression in 4 endometrial carcinoma cell lines. The expressions of its 5 target genes - DEC1, DEC2, carbonic anhydrase-9 (CA9), vascular endothelial growth factor (VEGF), and solute carrier family 2, member 1 (SLC2A1) - also reactively increased in most of the cell lines, except for DEC2 in the SNG-M cells. The expression levels of DEC2, CA9, and SLC2A1 were significantly higher in the 4 atypical hyperplasia tissues and 82 endometrial carcinomas compared with those in the 21 normal endometria. Clinicopathological analyses of carcinoma patients revealed a significant correlation of the VEGF and SLC2A1 expression with the status of lymph-vascular involvement and lymph node metastasis. The expression levels of CA9 and VEGF were significantly higher in the tumors of post- as opposed to pre-menopausal patients. The SLC2A1 expression was also related to the FIGO stage, but the DEC2 expression was inversely related to the FIGO grade. The activation of the HIF-1 pathway could be related to endometrial carcinogenesis, and the component, DEC2, could have different expression-regulatory mechanisms and unique roles in carcinogenesis.

Expression of tumor-associated differentially expressed Gene-14 (TADG-14/KLK8) and its protein hK8 in uterine endometria and endometrial carcinomas.

To clarify the biological behavior of TADG-14/KLK8, we investigated TADG-14/KLK8 mRNA by semiquantitative RT-PCR and hK8 expression by immunohistochemistry using 37 normal endometria and 44 endometrial carcinoma tissues. TADG-14/KLK8 mRNA expression levels were significantly higher in proliferative compared to secretory phase endometria (p = 0.0143). Levels of TADG-14/KLK8 mRNA expression correlated with hK8 protein levels. hK8 was detected in 73.3% (11/15) of endometria with a significantly higher detection rate in the proliferative compared to secretory and atrophic phase endometria (p = 0.0002). High expression of hK8 was found in 61.4% of endometrial carcinomas compared to 35.1% of endometrial tissue samples (p = 0.0187). hK8 expression was significantly higher in stage I (p = 0.0433, 0.0038) and grade 1/2 (G1/2) of the tumors (p = 0.0195, 0.0044). We suggest that expression of TADG-14/KLK8may be regulated by sex steroid hormones in endometria. Our results indicate that elevated TADG-14/KLK8 expression is an early event in endometrial carcinogenesis, and may potentially serve as a useful early biomarker for the detection of endometrial carcinomas in menopausal women.

Prediction of individual response to platinum/paclitaxel combination using novel marker genes in ovarian cancers.

We attempted to identify potent marker genes using a new statistical analysis and developed a prediction system for individual response to platinum/paclitaxel combination chemotherapy in ovarian cancer patients based on the hypothesis that expression analysis of a set of the key drug sensitivity genes for platinum and paclitaxel could allow us to predict therapeutic response to the combination. From 10 human ovarian cancer cell lines, genes correlative in the expression levels with cytotoxicities of cisplatin (CDDP) and paclitaxel were chosen. We first selected five reliable prediction markers for the two drugs from 22 genes already known as sensitivity determinants and then identified another 8 novel genes through a two-dimensional mixed normal model using oligomicroarray expression data. Using expression data of genes quantified by real-time reverse transcription-PCR, we fixed the best linear model, which converted the quantified expression data into an IC(50) of each drug. Multiple regression analysis of the selected genes yielded three prediction formulae for in vitro activity of CDDP and paclitaxel. In the same way, using the same genes selected in vitro, we then attempted to develop prediction formulae for progression-free survival to the platinum/paclitaxel combination. We therefore constructed possible formulae using different sets of 13 selected marker genes (5 known and 8 novel genes): Utility confirmation analyses using another nine test samples seemed to show that the formulae using a set of 8 novel marker genes alone could accurately predict progression-free survival (r = 0.683; P = 0.042).

Relative expression levels of Th1 and Th2 cytokine mRNA are independent prognostic factors in patients with ovarian cancer.

The aim of the present study was to examine mRNA expression levels of Th1 (TNF-alpha , IFN-gamma, and IL-12p40) and Th2 (IL-6 and IL-10) cytokines for any association with clinicopathological characteristics of epithelial ovarian cancer. mRNA was isolated, and cDNA prepared from 40 samples of epithelial ovarian cancers. Expression level of each cytokine mRNA was examined by the real-time PCR technique (GAPDH gene, internal control). Expression ratio (target gene/GAPDH) was used to evaluate gene expression. Results were analyzed against clinical stage, histological grade, and histological type. Prognostic significance of expression levels of each combination of Th1/Th2 values was assessed. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) expression levels were significantly higher in serous adenocarcinoma than in non-serous adenocarcinoma (p<0.05), but with no difference between individual cytokine mRNA expression levels and clinical stage or histological grade. Log-rank testing showed that high TNF-alpha mRNA expression (p=0.033) and the diameter of largest residual lesion at initial surgery (p=0.012) significantly correlate with longer survival in advanced stage (II/III/IV) ovarian carcinomas. In examining all combinations of Th1/Th2 expression values, the most significant association was between high IFN-gamma.IL-12p40/IL-6 expression levels and better prognosis in advanced stage (II/III/IV) ovarian carcinomas (p=0.004). In multivariate analysis, high IFN-gamma.IL-12p40/IL-6 expression (p=0.009) and the diameter of residual lesion (p=0.011) remained significantly associated with survival, whereas high TNF-alpha expression lost significance. In conclusion, Th1 and Th2 cytokines might play an important role in regulating the immune reaction in epithelial ovarian cancer cells. IFN-gamma.IL-12p40/IL-6 expression may be a useful prognostic molecular marker for patients with advanced ovarian cancer.

Predicting the chemosensitivity of ovarian and uterine cancers with the collagen gel droplet culture drug-sensitivity test.

We investigated the utility of the collagen gel droplet culture drug-sensitivity test (CD-DST) for predicting the response of gynecological cancers to chemotherapy. Eighty-three cancer patients were enrolled in this study: 26 ovarian, 29 cervical and 31 endometrial cancers. The CD-DST was performed at various concentrations of drugs. We calculated the T/C ratio, where T is the total volume of the treated culture and C is the total volume of the control culture, and a T/C ratio of 50% or less was defined as sensitive in vitro. The efficacy rate (%) was defined as the number of cultures with a T/C ratio of 50% or less, divided by the total number of evaluable cultures. True-positive cases were defined as clinical responders (complete+partial responses) and true-negative cases were defined as clinical non-responders. The overall tumor evaluation rate was found to be 79.1%. The appropriate drug concentrations were selected as 1.0 microg/ml for cisplatin, 20.0 microg/ml for carboplatin, 1.0 microg/ml for paclitaxel and 0.1 microg/ml for docetaxel by the linear regression equations. The in vitro sensitivity for each drug showed a significant correlation with clinical response rates (r=0.592, p=0021). We therefore conclude that the CD-DST can be used to predict the response to anti-cancer drugs, and may also provide important information by contributing to the development of new chemotherapy regimens.

Expression of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in uterine endocervical glands and in cervical adenocarcinomas.

To investigate the potential role of human kallikrein 7 (hK7/SCCE) and its inhibitor antileukoprotease (ALP/SLPI) in the development and progression of uterine cervical adenocarcinoma, we examined hK7 and ALP protein expression by immunohistochemistry in 70 cervical adenocarcinomas and 13 normal cervical tissues. Positive hK7 expression rates in normal endocervical glands and in cervical adenocarcinomas were 46.2 and 80%, respectively. A significantly higher hK7 expression rate was observed in cervical adenocarcinomas compared to normal endocervical glands (p=0.0099). In contrast, positive ALP detection rates in normal endocervical glands and in cervical adenocarcinomas were 100 and 15.7%, respectively. A significantly lower ALP detection rate was observed in cervical adenocarcinomas compared to normal endocervical glands (p<0.0001). There was a significant inverse correlation between hK7 and ALP expression status (p=0.0010). However, no statistically significant differences in hK7 or ALP expression status were found with respect to age, clinical stage, histological grade and lymph node metastasis status in cervical adenocarcinoma cases. Log-rank testing showed that advanced clinical stage and positive lymph node metastasis significantly correlated with poor patient survival (p=0.0005 and p<0.0001, respectively), whereas no correlation was found between hK7 or ALP expression and survival. These results suggest that increased expression of hK7 and decreased expression of ALP might play an important role in cervical adenocarcinoma development.

Clinicopathologic study of 56 patients with endometrial cancer during or after adjuvant tamoxifen use for their breast cancers.

OBJECTIVES: The aim of this study was to describe the clinicopathologic features and prognosis of endometrial cancer patients diagnosed during or after tamoxifen treatment for breast cancer. METHODS: Fifty-six tamoxifen-related endometrial cancers were identified from 10 hospitals in Japan. Past users were defined as endometrial cancer patients diagnosed more than 12 months after the cessation of tamoxifen treatment for breast cancer. All other users were classified as recent users. RESULTS: Age at diagnosis of the endometrial cancer ranged from 29 to 81 years. Sixteen (29%) and 19 (34%) patients were nulliparous and overweight, respectively. When the patients were divided into two groups: 30 recent and 26 past users, the distribution of various clinical characteristics, except for age at the time of diagnosis for endometrial cancer and the interval between the diagnoses of two cancers, was similar for two groups. The daily dose, duration and cumulative dose also showed no significant difference between the two groups. Past users had histopathologically more invasive tumors showing prognostically more unfavorable subtypes than recent users. The background lesions including endometrial polyps and diffuse cystic changes were similar for the two groups. The cumulative 3-year survival was significantly worse for past users than for recent users (74.8% and 96.4%, respectively, P < 0.04). In multivariate analysis including recentness of tamoxifen use and age at diagnosis of endometrial cancer, the significance of past user disappeared. CONCLUSIONS: Past users had a worse prognosis of endometrial cancer with more invasive histologic features than recent users, probably because they included more elderly patients.

Human papillomavirus DNA status after loop excision for cervical intraepithelial neoplasia grade III - A prospective study.

The aims of the study were to investigate the relationship between human papillomavirus (HPV) DNA status and recurrence of cervical intraepithelial neoplasia (CIN) after loop excision (LEEP/LLETZ). Women (n=161) who underwent loop excision for CIN III and who were followed up prospectively for at least 4 years were the study cohort. Cervical smear cytology and testing for HPV DNA was performed at 3, 6 and 12 months prospectively and thereafter at intervals of 6-12 months, using the PCR method with a consensus primer targeting the L1 region. There has been no recurrence in 141 (81.6%) out of 161 subjects, while squamous intra-epithelial lesions (SIL) of low or high grade on cytology and CIN grade I-III on histology have been detected in 20 subjects. Prior to loop excision, HPV DNA was detected in 17 subjects who developed recurrence (9 had type 16, 2 each had types 18 and 52, and 1 each had types 31, 51, 58, and unknown). Within 3 months postoperatively, 12 (70.7%) subjects became negative for HPV, but 2 remained positive for the same type (1 each had types 16, 18), along with high-grade SIL on cytology, and CIN III on histology within 6 months, so repeat loop excision was performed. On the other hand, cytological findings were normalized in all transiently infected subjects within 18-36 months. Our results suggest that loop excision has improved HPV infection in many cases of CIN III and the persistent infection with a high-risk type of HPV is a predictor of the recurrence of CIN grade III.

Telomerase activation in endometrial epithelial cells by paracrine effectors from stromal cells in primary cultured human endometrium.

Uterine endometrium displays telomerase activity in a menstrual cycle-dependent manner, despite its somatic origin. This study was performed to elucidate the regulation of telomerase in human endometrium. Telomerase activity and human telomerase reverse transcriptase mRNA expression in proliferative endometrium were significantly stronger than those in secretory endometrium. Their expression was only detected in epithelial cells, although stromal cells also showed proliferation. The growth of epithelial cells decreased day by day in accordance with the decline of telomerase activity. Telomerase activity was significantly stronger in co-cultures of epithelial and stromal cells than in cultures of epithelial cells alone. Moreover, the telomerase activity of co-cultured cells was increased by estradiol or basic fibroblast growth factor, whereas that of epithelial cells cultured alone showed no change. Thus, human endometrium shows reversible telomerase activation during the menstrual cycle, unlike cancer tissues. Also, the telomerase activity of uterine endometrial epithelial cells might be modulated by paracrine effectors released from stromal cells, and not only by the direct action of sex steroids such as estradiol and progesterone.

Overexpression of cyclooxygenase-2 protein and its relationship to apoptosis in cervical carcinoma treated with neoadjuvant chemotherapy.

The aims of this study were to examine the overexpression of COX-2 protein and its relationship to apoptosis in cervical carcinoma patients treated with neoadjuvant chemo-therapy (NAC), and to assess the potential role of COX-2 as a predictor of the response to NAC in a series of patients with cervical carcinoma. For immunohistochemical analysis, cervical cancer tissue samples were collected before NAC and 3 weeks after NAC using transcatheter arterial infusion of cisplatin from 40 patients who underwent surgery for advanced cervical carcinoma in stages IB, IIA and IIB and from 5 normal cervical tissues between 1991 and 2000 at the Department of Obstetrics and Gynecology, under informed consent. Patients were randomly assigned to receive one or two arterial infusions of cisplatin. COX-2 protein expression was detected by immunohistochemical staining and was classified as no expression for tumors with negative or <10%, while > or =10% positive staining was defined as overexpression. Detection of apoptosis was done by the TUNEL method. The percentage of cells with DNA fragmentation (apoptotic index, AI) was calculated before NAC and 3 weeks after NAC. The AI ratio (AI after NAC/AI before NAC) was also calculated. COX-2 expression was not detected in the normal cervix. Overexpression of COX-2 protein was detected in 18 out of 40 (45.0%) cervical cancers. A higher incidence of COX-2 protein overexpression was observed in patients with adenocarcinoma than in those with squamous cell carcinoma (p=0.1797, Fisher's exact text). The average AI value before and after NAC was 8.85 versus 11.82 respectively. In COX-2 protein-negative patients with squamous cell carcinoma, the AI ratio was 0.96+/-0.46 following one arterial infusion of cisplatin and 3.19+/-2.72 following two infusions of cisplatin. There was a significant positive correlation between apoptosis and the number of infusions of cisplatin (p=0.0098, Mann-Whitney, U-test). Our findings suggest that COX-2 protein expression could be used as a predictor of chemoresistance and that assessment of the COX-2 status could be useful to identify cervical cancer patients who may benefit from NAC.

Comparison of forearm endothelial function between premenopausal and postmenopausal women with or without hypercholesterolemia.

We sought to determine whether menopausal status or postmenopausal hypercholesterolemia affects forearm resistance artery endothelial function. We studied the forearm resistance artery endothelial function in 75 Japanese women: 25 premenopausal volunteers, 25 postmenopausal women with normal serum low-density lipoprotein (LDL) cholesterol concentrations, and 25 hypercholesterolemic postmenopausal women. Excluded from the study were patients with hypertriglyceridemia, hypertension, or diabetes, cigarette smokers. The forearm blood flow (FBF) during reactive hyperemia and after sublingual nitroglycerin (NTG) administration was measured by strain-gauge plethysmography. The serum concentrations of lipoprotein (a) [Lp(a)] were significantly higher in the hypercholesterolemic postmenopausal group than in the other two groups (P<0.01). These lipid parameters were similar between the premenopausal and postmenopausal women with normal cholesterol. The FBF responses to reactive hyperemia were significantly lower in the postmenopausal hypercholesterolemic women than in the other two groups (P<0.01). The reactive hyperemia also was impaired in the postmenopausal group with normal cholesterol as compared with the premenopausal group (P<0.01). Increases in FBF after NTG were similar between the three groups. By stepwise multivariate analysis, menopausal status and serum LDL cholesterol was the significant predictor of forearm endothelial function. These findings suggest that reactive hyperemia is impaired in forearm resistance arteries after menopause, especially in postmenopausal women with hypercholesterolemia.

Validation of a simple gas chromatographic-mass spectrometric method for the determination of gamma-butyrolactone in human plasma.

A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.

Expression of cyclooxygenase-2 and its relationship to p53 accumulation in ovarian adenocarcinomas.

To investigate cyclooxygenase-2 (COX-2) expression and its relationship to p53 accumulation in ovarian adenocarcinomas, COX-2 and p53 protein expressions were examined by immunohistochemistry in 86 ovarian adenocarcinomas and six normal ovaries. In addition, COX-2 mRNA expression level was examined by semi-quantitative PCR in 36 ovarian adenocarcinomas. Neither COX-2 expression nor p53 accumulation were detected in normal ovarian surface epithelium or germinal inclusion cyst epithelial cells. In contrast, COX-2 protein expression was detected in 31.4% of adenocarcinomas, and p53 protein accumulation was found in 30.2% of adenocarcinomas. A significantly higher COX-2 expression rate was observed in endometrioid adenocarcinomas than in either mucinous (p=0.019) or clear cell (p=0.021) adenocarcinomas, and a significantly higher p53 accumulation rate was observed in serous adenocarcinomas compared to clear cell adenocarcinomas (p=0.015). p53 accumulation correlated with advanced clinical stage (stage I vs. stage II/III/IV: p=0.007), whereas no correlation was found between COX-2 expression and clinical stage. There was a significant positive correlation between COX-2 expression and p53 accumulation status (p=0.003). Log-rank testing showed that p53 accumulation was significantly correlated with poor patient survival (p=0.004), whereas no correlation was found between COX-2 expression and survival. COX-2 mRNA expression was detected in 72.2% of ovarian adenocarcinomas, and a significant correlation between COX-2 mRNA expression status and immunoreactivity (p=0.023) was observed. These results suggest that COX-2 expression might play an important role in ovarian cancer development and that COX-2 expression in ovarian adenocarcinomas is frequently associated with p53 protein accumulation. COX-2 overexpression in ovarian cancer cells might partly be caused by dysfunctional p53.

GnRH agonist inhibits human telomerase reverse transcriptase mRNA expression in endometrial cancer cells.

We investigated the relationship between the antiproliferative effect of GnRH agonist and telomerase activity using the endometrial cancer cell line HEC-1A. The subjects were 38 endometrial cancer, and 2 atypical endometrial hyperplasia patients. GnRH-R expression was detected using RT-PCR. HEC-1A cells were incubated with 10(-7)-10(-4) M GnRH agonist (leuprolide acetate), and cell proliferation was determined using MTT assay. The telomerase activity was detected by the TRAP assay and expression of human telomerase reverse transcriptase (hTERT) was assessed by RT-PCR. GnRH-R mRNA was detected at 94.7% (36/38) in endometrial cancer and in both of the atypical endometrial hyperplasia and in HEC-1A cells. Cell proliferation of HEC-1A showed significant inhibition at leuprolide acetate concentrations of 10(-6) M or higher compared with untreated control culture (p<0.05). The telomerase activity showed no marked difference compared with untreated culture. However, hTERT mRNA expression showed a decrease in the leuprolide-treated cells. It is suggested that the mechanism of the antitumor effect of GnRH agonist involved the inhibition of hTERT mRNA expression in the endometrial cancer cells.