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Mitotic-arrest deficiency 2 (MAD2) is a key component of spindle assembly checkpoint (SAC) function; SAC mediates spindle microtubule attachment to kinetochores on chromosomes and chromosomal segregation during mitosis. To determine whether MAD2 expression is associated with chemotherapy resistance in ovarian serous adenocarcinoma, we reviewed tumor samples from 41 cases of ovarian serous adenocarcinoma at Osaka City University Medical School Hospital (Osaka, Japan), 2000-2007. Of the 41 cases, 24 were recurrent and 17 were not recurrent. Expression of MAD2 was investigated in paraffin-embedded sections using a MAD2 antibody. Quantitative analysis of MAD2 expression gave mean weighted scores of 4.3 for the relapsed group and 7.2 for the relapse-free group; the expression was, thus, significantly greater in the relapse-free group compared to the relapsed group (P=0.023). When the 41 cases were classified into low- and high-expression, these classifications showed no significant difference in terms of progression-free survival (P=0.0685), however, overall survival for the low-expression group was significantly shorter than that of the high-expression group (P=0.0188). The present study implies that MAD2 expression levels can indicate sensitivity to anticancer agents, and risk for recurrence.

Uterine leiomyosarcomas (LMS) are difficult to distinguish from benign leiomyomas without surgery. In this study we performed transcervical needle biopsy on 475 patients, 8 LMS patients and 467 patients with non-sarcomas (non-LMS) in a high-risk group for LMS, and evaluated whether examinations performed with Ki-67 and CD34 immunohistochemical analyses in addition to the standard hematoxylin-eosin (H&E)-stained sections would improve preoperative diagnostic precision of the uterine smooth muscle tumors. Histopathologic analysis included three factors: degree of cytologic atypia, mitotic index and coagulative tumor cell necrosis (CTCN). We also evaluated cell proliferation with Ki-67 expression. In cases of suspected CTCN, we examined CD34 expression and counted positive blood vessels in the necrotic area. Three of the 8 LMS cases satisfied the diagnostic criteria of LMS by histopathologic evaluation with H&E-stained sections. We made a score list based on these analyses; scores for LMS specimens ranged from 6-14 points; non-LMS specimens scored 0-2 points. At the cut-off score of 6 points, the positive predictive value to distinguish LMS from non-LMS was 100%, showing that this scoring system, is a useful method for preoperative differentiation between LMS and non-LMS tumors.

In cancer-bearing animals, we previously demonstrated that skeletal muscle apoptosis may be involved in muscle wasting and that Bax may play a role in skeletal muscle cell apoptosis at an early stage. In this study, we investigated the occurrence of apoptosis in the liver, kidney, spleen, lung and heart during cancer cachexia as well as the associations between apoptosis and the expression levels of Bcl-2, Bcl-xL and Bax proteins. We also examined the relationship between normal organ apoptosis in cancer cachexia and multiple organ failure. We further studied the changes in body weight, lean body mass (LBM), apoptotic index (AI), DNA laddering pattern and expression levels of Bax, Bcl-2 and Bcl-xL in the liver, kidney, spleen, lung and heart in VX2 carcinoma-bearing rabbits. In the early stage of tumor bearing (20 days after implantation), the LBM was significantly reduced by 19.06+/-1.02% in the tumor-bearing group compared to an increase of 1.66+/-0.83% in the control group. The apoptotic indices of the liver, kidney, lung and spleen in the tumor-bearing group increased by 14.2+/-1.8%, 34.4+/-2.0%, 66.7+/-6.0% and 24.8+/-3.8%, respectively and were significantly higher than the corresponding indices in the control group. DNA laddering patterns characteristic of DNA fragmentation were visible on day 50 in the liver, kidney, spleen and lung in the tumor-bearing group. The expression of Bax increased in the tumor-bearing group and coincided with the occurrence of apoptosis. However, no significant changes were noted in the expression levels of Bcl-2 and Bcl-xL. These findings suggest the possibility that normal organ cell apoptosis related to Bax not only causes body weight loss but also multiple organ failure in cancer cachexia.

To better understand the molecular mechanisms of carcinogenesis induced in uterine endometrium by therapeutic anti-estrogenic Tamoxifen (TAM) exposure, 27 uterine tumors (4 benign endometrial polyps and 23 carcinomas) associated with TAM exposure were analyzed for the presence and spectrum of p53 and K-ras mutations. Although there was no significant difference between TAM-associated endometrial carcinomas and sporadic endometrial tumors in the frequency of these mutations, the spectrum of p53 mutations was characteristically unique to the TAM-associated tumors. The median duration of TAM exposure was significantly longer in patients with p53 mutations than those without p53 mutations (62 vs. 30 months, p=0.028). Our observation suggests that prolonged TAM exposure may directly inactivate the p53 gene by acting as a mutagen in a significant fraction of TAM-associated endometrial carcinomas.

HHUA, a rare endometrial cancer cell line expressing the estrogen receptor (ER), was adopted to investigate the expression of vascular endothelial growth factor (VEGF), erythropoietin (Epo), Bcl-2 and p53 under the administration of estradiol-17beta (E2). Based on quantitative real-time reverse transcription polymerase chain reaction assays, both VEGF and Bcl-2 mRNA levels decreased in a dose-dependent manner, although VEGF levels were increased in a time-dependent manner; no significant change was found for Epo and p53. An immunocytochemical study also showed the suppressed expression of VEGF and Bcl-2 under E2 induction. Both ERalpha and ERbeta mRNAs were detected in HHUA cells with ERbeta expression being predominantly higher than that of ERalpha, which is the converse of the pattern seen in normal endometria. The present study shows the E2-downregulated expression of VEGF and Bcl-2, and reveals a disrupted balance of ERalpha and ERbeta expression, which should be taken into consideration to understand the particularity of E2-regulated gene expression in HHUA cells.

Reproductive hormones exert their actions via receptors in diverse tissues. In this study, we examined the expression of estrogen receptors (ERalpha and ERbeta), progesterone receptor (PR), and follicle-stimulating hormone receptor (FSHR) in the adipose tissue and reproductive organs of mice following oral treatment with Dandelion T-1 extract (DT-1E) for 6 weeks. Quantitative assays by real-time reverse transcription polymerase chain reaction showed enhanced expression of ERalpha and ERbeta mRNA in the adipose tissue of mice fed a diet containing DT-1E compared with the control group fed a plain diet. Furthermore, following gonadotropin injection, higher mRNA expression of ERalpha, ERbeta and PR in the uterus and FSHR in the ovary was found in the DT-1E group compared with the control group. An immunohistochemical study also showed increased levels of the above-mentioned receptors in the DT-1E group. The present study shows that oral intake of DT-1E up-regulates ERalpha, ERbeta, PR and FSHR expression in mice, suggesting the potential application of DT-1E for the clinical treatment of reproductive hormone-related disturbances.

Recent changes in the lifestyle of young women have led to an increase in the rate of uterine cervical cancer. We investigated the clinicopathological characteristics of uterine cervical cancer in young women, and examined the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Tumor samples from 439 patients with uterine cervical cancer, who were initially treated at Osaka City University Medical School Hospital, Japan between 1995 and 2004, were stained immunohistochemically. The patients were classified into two groups according to age at onset: group Y included women aged < or =35 years, and group O included women aged > or =36 years. Group Y had more cases of squamous cell carcinoma, while group O had more advanced cases (P<0.05). Advanced cases (beyond stage Ib2) had a significantly worse prognosis in group Y than in group O (P<0.05). There were no differences between the two groups in the expressions of VEGF, MMP-2 and COX-2. However, in advanced cases (beyond stage Ib2), the expression of VEGF, MMP-2 and COX-2 was significantly greater in group Y than in group O (P<0.05). The above findings suggest that the expression of VEGF, MMPs and COX-2 is related to a worse prognosis for advanced uterine cervical cancer in young women.

We immunohistochemically examined the expression of the glucose transporters (GLUT)1, GLUT3 and GLUT4, in 154 tumor samples of epithelial ovarian carcinoma. In addition, we investigated the correlations between the expression of GLUTs and the vascular endothelial growth factor (VEGF), and microvessel count and clinical parameters. The rates of expression of GLUT1, GLUT3 and GLUT4 were 98.7%, 92.8% and 84.4%, respectively. GLUT1 and GLUT4 were both strongly expressed in serous adenocarcinoma, but weakly expressed in clear cell adenocarcinoma. The expressions of GLUT1 and GLUT4 correlated with the clinical disease stage. The expressions of GLUT1, GLUT3 and GLUT4 correlated positively with VEGF expression. The expression status for GLUT1, GLUT3, GLUT4 and VEGF did not represent a prognostic factor. These findings suggest that characteristic differences in the patterns of glucose uptake can exist according to the histological type and that GLUT1, GLUT3 and GLUT4 could be related to tumor angiogenesis in epithelial ovarian carcinoma.

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several human malignant tumors, in particular MMP-7, which is mainly produced by the cancer cell itself. We examined the expression of MMP-2, 7 and 9, and tissue inhibitors of metalloproteinase (TIMP)-1 and 2 in uterine endometrial carcinoma, and compared the expression with clinicopathological characteristics in uterine endometrial carcinoma (UEC). A group of 256 patients with UEC received surgery at the Osaka City University Medical School Hospital, and 196 tumor samples were immunohistochemically stained to examine the expression of MMP-2, 7 and 9, and TIMP-1 and 2. Additionally, the invasion ability of cell stain established from UEC was examined using an in vitro invasion assay. The expression of MMP-2, 7 and 9, and TIMP-1 and 2 was observed in the cytoplasm, and the expression of MMP-2 and 7, and TIMP-1 and 2 was observed in stromal cells around the tumor cells. The expression of MMP-7 was significantly stronger in higher-grade than lower-grade tumors (P<0.05). The invasion assay showed that the invasion of cells derived from UECs was significantly inhibited by TIMP-1 and 2. The disease-free interval was significantly shorter when MMP-7 expression was intense. This increased expression of MMP-7 in high grade UECs may be associated with tumor invasion and metastasis, and MMP-7 could serve as a prognostic maker in UEC.

We previously reported satisfactory therapeutic results of cisplatin-based cyclic balloon-occluded arterial infusion chemotherapy (BOAI) as neoadjuvant chemotherapy, which enabled treatment by hysterectomy in patients with advanced cervical cancer. We also reported expression of apoptosis among these patients and determined that the bax gene is related to this apoptosis. In the present study, we investigated the relationship between the effectiveness of BOAI therapy and expression of apoptosis regulatory genes and proteins in these cases. The subjects were 27 women with advanced cervical cancer classified as FIGO (International Federation of Gynecology and Obstetrics) stage III or higher who were admitted to the Department of Gynecology, Osaka City University Medical School Hospital between 2000 and 2003. All patients were treated by BOAI, and expression of cancer cell apoptosis was examined by the TUNEL method, expression of bax, bcl-2 and bcl-xL proteins were examined by immunohistochemistry, and expression of bax, bcl-2 and bcl-xL mRNA was examined by quantitative RT-PCR before and 3 days after BOAI. The effectiveness of BOAI therapy was thus determined. The 20 patients in whom BOAI was effective showed significantly higher expression of the bax protein and gene after BOAI, and cancer cell apoptosis was accelerated. On the other hand, the 7 patients in whom BOAI was ineffective showed significantly higher expression of the bcl-xL protein and gene after BOAI. These results suggest that bax/bcl-xL expression can be used as an indicator of the effectiveness of BOAI therapy.

Whether the human papillomavirus (HPV) status of the tumor affects the sensitivity to neoadjuvant chemotherapy, and the prognosis in advanced uterine cervical cancer (FIGO stage III or higher) remains unknown. We examined the HPV status of 43 patients who had received CDDP therapy by balloon-occluded arterial infusion (BOAI), as neoadjuvant chemotherapy for advanced uterine cervical cancer (squamous cell carcinoma) stage III or higher. DNA was extracted from formalin-fixed, paraffin-embedded tumor samples obtained by punch biopsy before the neoadjuvant chemotherapy. The detection of HPV and its typing were analyzed by a polymerase chain reaction (PCR)-based assay using consensus primers for the L1 consensus regions. HPV DNA was detected in all 43 patients (100%): 29 cases with HPV 16 (67.4%), 5 cases with HPV 33 (11.6%), 4 cases with HPV 31 (9.3%), 3 cases with HPV 35 (7.0%), 1 case with HPV 18 (2.3%) and 1 case with HPV 58 (2.3%). The HPV types were divided into 3 groups, HPV 16, HPV 33 and other HPV types (HPV 18, 31, 35, 58), and comparisons and examinations were performed among the 3 groups. Although the rates of tumor reduction and operation accomplishment after 3 courses of BOAI showed no significant differences among the 3 groups, there were significant differences in the survival rates. The survival rate of advanced uterine cervical cancer patients with HPV 33 infection was the highest, followed by that of patients with HPV 16 infection. The survival rates of patients with the other types of HPV infection were the worst among the 3 groups and significantly lower than those of patients with HPV 16 or HPV 33 infection. The differences in the curative effect after BOAI may depend on the different characters of the HPV types.

The mechanism of rebound body weight-gain after a restricted-diet state is unclear. We investigated the expression of angiogenic factors in human adipocytes with a changing nutritional state in culture medium, and attempted to ascertain the mechanisms involved in rebound weight-gain. Adipocytes were divided into three groups; the first group (control group) was cultured in medium with 10% fetal calf serum (FCS), the second (DR3% group) was cultured in medium with 3% FCS, and the third (DR6% group) was cultured in medium with 6% FCS. After being cultured for 48 h, each was next cultured with 12% FCS for a further 48 h. When made to change from a low nutrition state to a higher one, adipocytes changed from hypotrophic to hypertrophic. Simultaneously, vascular endothelial growth factor (VEGF) in the culture medium increased significantly. When investigated immunohistochemically, the expression of VEGF was similarly shown in the cytoplasm of adipocytes. The same tendency with the same quantity of mRNA was shown by RT-PCR. These results show that VEGF produced and secreted from adipocytes increases, when the cultivation state of adipocytes is changed from a low nutritional state to a higher one. VEGF produced and secreted from adipocytes may be related to rebound weight-gain.

Tetrahydrobiopterin (BH4) is an essential co-factor for nitric oxide (NO) synthase (NOS) and regulates the production of NO, or endothelium-derived relaxation factor. Although NOS is highly expressed in the placenta and NO plays a critical role in the regulation of feto-placental circulation, the mechanism maintaining the level of BH4 is not known. To investigate the de novo synthesis of BH4 in the human placenta, the activity of guanosine triphosphate cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) in the chorionic tissue during the first, second and third trimester of pregnancy was analyzed. GTPCH activity was the lowest of the three enzymes and became negligible after the second trimester. There was no significant change in PTPS activity throughout pregnancy. Although SR activity decreased significantly after the second trimester, the levels remained abundant throughout pregnancy. These results showed that GTPCH is a rate-limiting enzyme and the total activity of the de novo synthesis of BH4 is negligible in the mature placenta after the second trimester when fetal growth is accelerated. The present study suggests that the level of BH4 in the placenta depends principally on the system other than de novo synthesis. The salvage pathway is considered the most potent system, which is formed by the transfer of the substrates from the fetus and their enzymatic conversion to BH4 in the placenta.

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several malignant tumors in human. Especially so MMP-7, as it is mainly produced by the cancer cell itself. However, the expression of MMP-7 in renal cell carcinoma (RCC) has not been investigated. We examined expressions of MMP-7 and MMP-2 in RCC, and compared them with the clinicopathological characteristics in RCC. Tumor samples from 20 patients with RCC, who had surgery performed at Osaka City University Medical School Hospital, were immunohistochemically stained. Expression of MMP-2 was significantly stronger in advanced stage and in high grade tumors than in low stage and low grade tumors. MMP-7 was also expressed in RCCs, with its expression especially and significantly stronger in high grade than in low grade tumors (p=0.004). However, there was no significant difference of MMP-7 expression between advanced and low stages (p=0.1859). This increased expression of MMP-7 in high grade RCC might be associated with tumor invasion and metastasis.

Leukemia inhibitory factor (LIF) is produced locally in decidual tissues, but its direct effects on endometrial stromal cells have not been well characterized. In this study, we examined the direct effects of LIF on normal human endometrial stromal cells using an in vitro decidualization assay system with 8-Br-cAMP, a decidualization inducer. We found no effects of LIF on cell viability and prolactin secretion of unstimulated endometrial stromal cells. LIF dose-dependently enhanced cell viability, but not prolactin secretion of 8-Br-cAMP-stimulated cells. LIF dose-dependently enhanced the viability of stromal cells co-stimulated with 8-Br-cAMP and LIF without any significant effect on PRL secretion from the cells. Further, the extracellular matrix did not affect these stimulatory effects of LIF on cell viability of 8-Br-cAMP-stimulated endometrial stromal cells. These results indicate that LIF enhances the cell viability of PRL-non-secreting 8-Br-cAMP-stimulated stromal cells, and that the cell survival signals generated by LIF are independent of those generated by the extracellular matrix. LIF produced locally in decidual tissues may enhance cell viability in certain activated endometrial stromal cells in an autocrine or paracrine manner, and possibly protect against cell damage during embryo implantation and trophoblastic invasion.

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several human malignant tumors. Especially so MMP-7, as it is mainly produced by the actual cancer cells. However, the expression of MMP-7 in transitional cell carcinoma (TCC) of the urinary bladder has not been previously investigated. We examined expressions of MMP-7 and MMP-2 in TCC, and compared them with clinicopathological characteristics in TCCs. Tumor samples from 20 patients with TCC of the urinary bladder who had surgery performed at the Osaka City University Medical School Hospital were immunohistochemically stained. Expression of MMP-2 was significantly stronger in advanced stage and high grade tumors than in low stage and low grade tumors. MMP-7 was also expressed in TCC, having a tendency to be more strongly expressed in high than in low grade tumors. However, there was no significant difference of MMP-7 expression between advanced and low stages. This increased expression of MMP-7 in high grade TCC of the urinary bladder may be associated with tumor invasion and metastasis.

Cervical cancer can be classified into two histological types: squamous cell carcinoma (SCA) and adenocarcinoma (ACA). Reportedly ACA has poorer prognoses, metastasizes more easily to lymph nodes, and is more resistant to radiotherapy than SCA. To clarify the cause of characteristic differences between these histological types, we examined the expressions of apoptosis inhibiting and tumor-invasion related factors in both histological types. We reviewed the 34 cases of cervical cancer (17 ACA, 17 SCA) that had surgery as their initial treatment at Osaka City University Medical School Hospital between 1996 and 2001. The differences of survivin, and matrix metalloproteinase (MMP-2, and MMP-7) expressions between both histological types were immunohistochemically assayed, and the correlation between the expression of each protein and clinicopathological characteristics was analyzed. Survivin was expressed significantly stronger in ACA cases (p=0.035). The number of patients who expressed MMP-2 and MMP-7 simultaneously was significantly higher in SCA cases (p=0.039). MMP-2 and MMP-7 had tendencies to be expressed stronger in SCA (p=0.057 and p=0.084, respectively). These results suggest that the differences of the expression of survivin (an apoptosis inhibiting factor), MMP-2, and MMP-7 (tumor-invasion related factors) between ACA and SCA were causes of the characteristic differences between the two histological types.

Preoperative differential diagnoses between uterine sarcomas and leiomyomas are difficult. As telomerase activation is thought to be essential for the immortality of malignant cells, it is considered a potentially useful diagnostic marker. The aim of the present study was to evaluate the potential diagnostic use of measuring telomerase activity in needle biopsy samples to distinguish uterine sarcoma from leiomyoma. Sixty-two patients with suspected uterine sarcomas based on clinical findings or magnetic resonance imaging findings, and who were scheduled for surgery, underwent transcervical ultrasound-guided needle biopsy. Three samples were obtained per patient for histopathological examination and telomerase activity measurement. Telomerase activity was measured using the telomeric repeat amplification protocol and correlated with final histopathological findings of surgical specimens. Of the 62 patients, 6 leiomyosarcomas and 1 endometrial stromal sarcoma (high grade) were diagnosed by histopathology. In 6 of the 7 samples from uterine sarcomas, relatively high telomerase activity (22-102 units) was detected, whereas only low telomerase activity (11-18 units) existed in 3 of the remaining 55 samples from benign or borderline uterine smooth muscle tumors. At a cut-off value of 20 units, sensitivity, specificity, positive predictive, and negative predictive values for detecting uterine sarcoma were 86% (95% confidence interval, 59-100%), 100% (94-100%), 100% (54-100%) and 98% (95-100%), respectively. The results indicated that telomerase activity in needle biopsy samples is a useful diagnostic marker to distinguish uterine sarcoma from leiomyoma.

Specific chromosomal abnormalities, e.g. del(7q), t(12;14), 12 trisomy, and the rearrangement of 6p, are seen in approximately 30% of uterine leiomyomas. We investigated the association between the shrinkage effect of GnRH agonist on uterine leiomyomas and t(12;14), the second most frequent chromosomal abnormality in myomas. This study involved 42 women with uterine leiomyomas treated with a gonadotropin releasing hormone (GnRH) agonist before surgery. The volume of the largest myoma nodule was measured by MRI before and at 12 weeks after the beginning of GnRH agonist treatment, and the percentage change in volume was calculated. A specific chromosomal abnormality, t(12;14), was examined on thin sections of frozen leiomyomas by fluorescence in situ hybridization with chromosome-specific probes. Of the 42 tumors, 8 (19%) showed translocation. The mean (+/- SD) percentages change in volume of the largest myomas without and with translocation were -32+/-24 and 23+/-60%, respectively (p=0.006). The myomas showing translocation had significantly less reduction in size with GnRH treatment than did those without translocation. No myoma with trisomy 12 was found. On the basis of our results, we assumed that uterine leiomyomas showing t(12;14) are not so dependent on ovarian hormones for growth.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase as well as a cofactor of aromatic amino acid hydroxylases. However, its role in pregnancy is not yet understood. We evaluated the concentrations of BH4 throughout normal pregnancy and puerperium, and compared them with those of non-pregnant women by measuring its oxidation product biopterin. In addition, we also measured 6-pyruvoyl tetrahydropterin synthase (PTPS) activities, the rate-limiting enzyme in synthesizing BH4, in pregnant women at the 30th gestational week and non-pregnant women. Although the urinary biopterin levels did not remarkably change, plasma biopterin levels significantly decreased from the 10th gestational week to the 1st day of postpartum compared with those of non-pregnant women. There was no significant difference in PTPS activities between pregnant and non-pregnant women. However, the proportion of reticulocytes, which have been shown to possess high PTPS activity, is significantly higher in pregnant women than in non-pregnant women. Our results suggest that decreased plasma BH4 levels in pregnancy is caused by impaired PTPS activity.