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Annexin V is a Ca2+-dependent phospholipid-binding protein belonging to the annexin family whose regulation is currently not well understood. In this study, we utilized anisomycin, a protein synthesis inhibitor that activates MAP kinases (MAPKs), to examine the role of MAPKs in annexin V expression in the MCAS ovarian carcinoma cell line. A one-step real-time TaqMan-based reverse transcriptase-PCR method was developed to quantify annexin V mRNA expression. We found that annexin V was induced 13.3-fold by anisomycin and that this superinduction was attenuated by pretreatment with the MEK inhibitors, U0126 and PD98059, but not with the p38 MAPK inhibitor, SB203580. In addition, immunoblotting showed that anisomycin stimulated the phosphorylation of ERK1/2 as well as p38 MAPK and that the phosphorylations were blocked by the three kinase inhibitors. Taken together, these results suggest that anisomycin superinduces annexin V mRNA expression through the ERK1/2 MAPK pathway, but not through the p38 MAPK pathway. [Copyright &y& Elsevier]

Abstract: Introduction: The purpose of this study was to determine histopathological factors for para-aortic lymph node (PALN) metastasis in patients with endometrioid uterine cancer. Methods: A total of 355 patients (Stage I, n =269; II, n =24; and III, n =62) (FIGO 2009) underwent primary radical surgery including complete systematic pelvic lymph node (PLN) and PALN dissection in Tohoku Gynecologic Cancer Unit (TGCU) between 1993 and 2004. Logistic regression analysis was used to determine the independent prognostic factors for PALN metastasis. Results: Multivariate analysis revealed that PLN metastasis (p <0.0001) and ovarian metastasis (p =0.0080) related with PALN metastasis. Moreover, among the sites of PLN metastases, obturator lymph node (LN) [risk ratio (RR): 16.9, 95% confidence interval (CI): 4.3–66.4, p <0.0001] and common iliac LN (RR: 7.1, 95% CI: 1.1–44.5, p =0.0375) related with PALN metastases. In detection of PALN metastasis, combination of obturator LN and/or common iliac LN and/or ovarian metastasis (A) revealed 75.9% sensitivity (22/29) and 97.8% negative predictive value (NPV) (304/311). However, by combination of obturator LN metastasis and/or common iliac LN metastasis and/or grade 3 and/or deep myometrial invasion (B), the detection of PALN metastasis was 100.0% sensitivity (29/29) and 100.0% NPV (198/198). Also, 55.8% (198/355) of patients could have avoided PALN dissection by combination B. Conclusions: These results suggest that PALN dissection is necessary when combination B is positive by pre- and intra-operative assessments. Further prospective randomized controlled studies need to be conducted in a larger patient population to establish the strategy for detecting PALN metastasis utilizing pre-/intra-operative assessments. [Copyright &y& Elsevier]

Abstract: Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus–oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge. [Copyright &y& Elsevier]

Abstract: Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus–oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos. [Copyright &y& Elsevier]

Abstract: Objective: The purpose of this study was to determine whether para-aortic lymphadenectomy improves disease-related survival (DRS) in stage IIIc endometrial cancer. Methods: A total of 63 patients with stage IIIc endometrial carcinoma underwent primary radical surgery in the Tohoku Gynecologic Cancer Unit from 1993 to 2004. All patients had modified radical hysterectomy, bilateral salpingo-oophorectomy, systemic pelvic lymph node (PLN) adenectomy, and with or without para-aortic lymph node (PAN) adenectomy, followed by adjuvant chemotherapy. DRS was analyzed using Kaplan–Meier curves and the log-rank test. Independent prognostic factors were determined by multivariate Cox regression analysis using a forward stepwise selection. Results: There were no statistical differences in age distribution and histopathological prognostic factors between PLN adenectomy group (n =25) and PLN+PAN adenectomy group (n =38). On univariate analysis, architectural grade (p =0.026), peritoneal cytology (p =0.033), and the number of PLN positive sites (≤1 or ≥2) (p =0.010) were related to poor DRS. On multivariate Cox regression analysis, the number of positive PLN sites was related to DRS (p =0.040). In positive PLN≥2 sites group (n =33), PAN adenectomy significantly improved DRS compared to PLN adenectomy alone (p =0.011). The incidence of initial PAN recurrence was higher in the PLN adenectomy group (6/25) than in the PLN+PAN adenectomy group (1/38) (p =0.013, Odds Ratio=11.68). Conclusions: The number of positive PLN site is an independent prognostic factor in stage IIIc endometrial cancer. PAN adenectomy decreased the incidence of PAN recurrence and may improve DRS in patients with ≥2 positive PLN sites. A large prospective clinical trial needs to be conducted to establish the strategy of PAN adenectomy before or intra-operative treatment. [Copyright &y& Elsevier]

Abstract: Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos. [Copyright &y& Elsevier]

Abstract: Objective. : Some endometrial cancer patients without clinical evidence of extrauterine spread die as a result of recurrence even after curative operation. These recurrences may arise from occult tumor cells that are not detected by conventional methods. The goal of this study was to develop a quantitative method for the detection of disseminated tumor cells (DTCs) in the peripheral blood (PB) and lymph nodes (LNs) of patients with endometrial cancer. Methods. : Ninety-eight PB samples from 30 patients and 218 LNs from 14 patients were studied. Real-time quantitative analysis was performed using a LightCycler instrument and a TaqMan probe for cytokeratin 19 (CK19) as a marker gene. Results. : This method resulted in the reproducible quantitation of 10 to 106 MCF-7 cells (CK19-expressing breast cancer cell line) per 106 peripheral blood nucleated cells. CK19 mRNA expression was detected in 28 PB samples and in 62 LNs. Only three preoperative PB samples and one postoperative PB sample (from four patients) and 33 LNs (from six patients) were PCR-positive. The PCR-positive rate of LNs was higher in patients with pathologically metastatic (path-positive) LNs than in patients with path-negative but PCR-positive LNs. Furthermore, the CK19 mRNA background expression rate was higher in the LNs of path-negative but PCR-positive patients than in LNs of path-negative and PCR-negative patients. Conclusions. : Real-time qRT-PCR with TaqMan probes is a sensitive, specific and rapid method for the detection of DTCs in PB and LNs. Additional studies with larger numbers of patients and adequate follow-up would be of benefit. [Copyright &y& Elsevier]

Human papilloma viruses (HPV), particularly type 16, have been associated with cervical cancer. It has been noted that the average onset of cervical cancer is occurring in younger women coupled with a higher prevalence of cervical HPV infection. However, the correlation between HPV 16 infection and the early onset of cervical cancer is still unclear. We hypothesize that HPV infection is an indicator of early onset of cervical cancer. To test this hypothesis, cervical smears from 197 women were evaluated by the polymerase chain reaction for HPV 16. These data revealed that the HPV 16-positive women were significantly younger than the HPV 16-negative women. Moreover, the average age of HPV 16-positive women with CIN 3 or invasive cancer was significantly younger compared with the other groups. These data clearly suggest that HPV 16 infection is a significant risk factor for the progression for cervical cancer in a young population of women. [Copyright &y& Elsevier]

Apoptosis is an essential physiologic process used in almost all tissues to remove damaged or superfluous cells. However, the early embryos are unique because no cell death is found up to the blastocyst stage during normal development. Survivin, a member of the IAP family, is capable of binding to caspases to modulate their functions. Here, we investigated the expression of survivin, and its role in preventing apoptosis in mouse preimplantation embryos. Transcripts for survivin and a splice variant lacking exon 2 were detected from unfertilized oocytes up to hatched blastocyst stage. At the protein level, survivin was also detected at all stages of early embryos. The antisense approach was used to demonstrate the role of survivin on embryo development. Development of early embryos treated with antisense survivin oligonucleotides was arrested at the morula or early blastocyst stage with disruption of tubulin formation and abnormal nuclei, associated with apoptosis. The effect of the antisense was enhanced by cotreatment with an apoptosis-inducing reagent, staurosporine. In contrast, apoptosis induced by the antisense treatment was inhibited by caspase-3 and -9 inhibitors. These results indicate that survivin is an essential antiapoptotic gene expressed in preimplantation embryos and could protect the embryos from apoptosis by inhibiting an apoptotic pathway involving caspases. [Copyright &y& Elsevier]