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Circulating tumor cells (CTC) are newly discovered biomarkers of cancers. Although many systems detect CTC, a gold standard has not yet been established. We analyzed CTC in uterine cervical cancer patients using an advanced version of conditionally replicative adenovirus targeting telomerase-positive cells, which was enabled to infect coxsackievirus-adenovirus receptor-negative cells and to reduce false-positive signals in myeloid cells. Blood samples from cervical cancer patients were hemolyzed and infected with the virus and then labeled with fluorescent anti-CD45 and anti-pan cytokeratin antibodies. GFP (+)/CD45 (-) cells were isolated and subjected to whole-genome amplification followed by polymerase chain reaction analysis of human papillomavirus (HPV) DNA. CTC were detected in 6 of 23 patients with cervical cancers (26.0%). Expression of CTC did not correlate with the stage of cancer or other clinicopathological factors. In 5 of the 6 CTC-positive cases, the same subtype of HPV DNA as that of the corresponding primary lesion was detected, indicating that the CTC originated from HPV-infected cancer cells. These CTC were all negative for cytokeratins. The CTC detected by our system were genetically confirmed. CTC derived from uterine cervical cancers had lost epithelial characteristics, indicating that epithelial marker-dependent systems do not have the capacity to detect these cells in cervical cancer patients.

Positron emission tomography (PET) with fluorodeoxyglucose F18 (18 F-FDG) is useful for detecting malignancies, but benign lesions occasionally have false-positive 18 F-FDG uptake. Here, we report the cases of five postmenopausal women with solid ovarian tumors suspected to be ovarian cancer on magnetic resonance imaging and 18 F-FDG uptake. Mean age of the five patients was 57 years (range, 53-65 years). Average early standardized uptake value (SUV) of 18 F-FDG was 5.76 (range, 2.2-12.0) and delayed SUV was 6.56 (range, 2.4-13.8). In all five patients, frozen section diagnosis at surgery was thecoma, and bilateral salpingo-oophorectomy was performed. On immunohistochemistry, immunoreactive glucose transporter 5 (GLUT5) expression was detected in thecoma tissues. This case shows that thecoma sometimes has positive 18 F-FDG uptake on positron emission tomography-computed tomography (PET-CT), indicating the need for caution regarding false-positive PET-CT in patients with benign solid ovarian tumor.

Dienogest (DNG) is a selective progesterone receptor (PR) agonist and oral administration of DNG is used for the treatment of endometriosis. DNG is considered to act on PR to down-regulate pathophysiological factors associated with endometriosis. PR exists as two major isoforms, PR-A and PR-B, and their physiological functions are mostly distinct. It was suggested that PR isoform expression patterns are altered in endometriosis, but it is unknown whether the pharmacological effects of DNG are exerted through PR-A, PR-B or both. In the present study, we investigated the pharmacological effects of DNG through these PR isoforms on the expression of CYP19A1 which encodes aromatase and inflammatory and neuroangiogenesis factors associated with the pain and progression of endometriosis. We used immortalized human endometriotic epithelial cell lines that specifically express PR-A or PR-B in a spheroid cell culture system, and treated them with DNG. We evaluated messenger RNA (mRNA) expression of CYP19A1, prostaglandin (PG)E2 synthase (cyclooxygenase (COX)-2 and microsomal PGE2 synthase (mPGES)-1), inflammatory cytokines (interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1) and neuroangiogenesis factors (vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)) using real-time polymerase chain reaction. In addition, PGE2 production was measured by enzyme immunoassay. We found that DNG down-regulated mRNA expression of CYP19A1, COX-2, mPGES-1, IL-6, IL-8, MCP-1, NGF and VEGF, and PGE2 production in human endometriotic epithelial cell lines that specifically express either PR-A or PR-B. These results demonstrate that DNG activates both PR-A and PR-B and down-regulates the expression of pathophysiological factors associated with pain and progression of endometriosis. Our results suggest that DNG exerts therapeutic efficacy against the pain and progression of endometriosis regardless of PR isoform expression patterns.

Background: Recently developed detection system for circulating tumour cells (CTCs) using a telomerase-specific replicative adenovirus generated nonspecific green fluorescent protein (GFP) signals because of the co-presence of white blood cells (WBCs) nonspecifically infected by viruses. Here, we established a unique detection system for CTCs that completely excludes nonspecific signals. Methods: Blood obtained from the patients was subjected to haemolytic processes to eliminate red blood cells. The cell pellets were then infected with OBP-401, fixed, incubated with fluorescence-labelled anti-CD45 antibody to mark white blood WBCs, and examined on slides under a microscope. Results: Preparatory experiments with cancer cells artificially added to healthy donor samples confirmed that CD45 labelling could distinguish GFP-positive cancer cells from WBCs. In 53 patients with gynaecological cancers, CTCs were detected in 21 patients (39.6%) when CD45-positive cells were excluded as WBCs among GFP-positive cells. No CTCs were detected in samples from healthy volunteers. There was no significant correlation between CTC counts and known clinicopathological factors. The CTCs rapidly vanished after surgery or chemotherapy in most patients whose treatments were effective. In contrast, the persistence of CTCs even after treatments was tightly associated with poor response to the treatments (P

Purpose: Although the KRAS mutation is one of critical genetic alterations in endometrial carcinogenesis, the downstream targets are not known. Experimental Design: In this study, we investigated the molecular targets of KRAS signals, using tumorigenic cells with oncogenic KRAS mutation established from telomerase reverse transcriptase (TERT)-immortalized endometrial epithelial cells. Results: We first confirmed that the RAF-ERK pathway, but not the PI3K-Akt pathway, was activated in KRAS tumorigenic cells. However, the introduction of constitutively active MAP/ERK kinase into immortalized cells to mimic RAF-ERK activation failed to obtain tumorigenic phenotypes, indicating the existence of other carcinogenic pathways triggered by KRAS. Recent evidence suggestive of linkage with KRAS signals prompted us to examine the involvement of NF-κB in endometrial carcinogenesis. We found that the DNA-binding activity of NF-κB was markedly elevated in KRAS tumorigenic cells compared with TERT-immortalized cells. Furthermore, the ability of NF-κB to activate the target gene promoters significantly increased in KRAS tumorigenic cells. Introduction of a mutant IκB that is resistant to degradation and thereby enhances the inhibitory effect on NF-κB largely abrogated the transformed phenotypes of KRAS tumorigenic cells. Thus, oncogenic KRAS signals contributed to the tumorigenic phenotypes of endometrial cells by activating the transcription function of NF-κB. Conclusions: These findings clearly show that NF-κB activation is a novel target of oncogenic KRAS in endometrial carcinogenesis, implying the potential utility of NF-κB inhibitors for endometrial cancer chemoprevention, especially with KRAS mutation. Clin Cancer Res; 17(6); 1341–50. ©2011 AACR.

Maintenance of an adequate laparoscopic view by an assistant is a fundamental aspect for performing effective laparoscopic surgery. We describe a self training model for obtaining optimal laparoscopic view using a web camera and a bench trainer. Trainees held a needle-holder with the right hand and introduced a needle into the trainer, sutured a sponge pad, and withdrew the needle; while the left hand held the web camera for monitoring the operating field. They were videotaped during the procedure and were scored before and after training by independent observers using an objective assessment of a technical skill scoring system. Results showed the scores after training were significantly higher than before training (P=0.0021) . Thus, we conclude the self-training method could improve the skill in manipulating the laparoscope by an assistant.

Purpose: Tamoxifen is an anti-estrogenic drug that is widely used for endocrine-dependent breast cancer as adjuvant hormonal therapy, and its use has been reported to be frequently associated with high levels of serum estradiol. Since the population of premenopausal women receiving tamoxifen therapy is growing in Japan, we retrospectively analyzed the incidence of ovarian hyperstimulation by tamoxifen therapy in Japanese women. Methods: Eleven patients who received surgical therapy for endocrine-dependent breast cancer and showed high values of serum estradiol during post-operative tamoxifen therapy were recruited in this study and evaluated by examining the serum concentration of follicular stimulating hormone (FSH) and follicular development. Results: The mean age, serum concentrations of estradiol and FSH, and follicular diameter were 41.3years old, 1015.8pg/mL, 11.8mIU/mL, and 3.47cm, respectively. In 6 cases, multiple follicular development was observed, while the other cases showed single follicular development with a mean serum estradiol level of 848.6pg/mL and follicular diameter of 4.46cm. There was no significant difference in age or FSH concentration between the two groups. The mean periods from the start of the single administration of tamoxifen to the initial detection of a high estradiol concentration was 716.5days. Conclusions: These findings indicate that tamoxifen could stimulate the ovarian function even after 2-year treatment. Since single and multiple follicular developments with large sizes were observed, dual mechanisms through the inhibition of both negative and positive feedback to the hypothalamic-pituitary-axis can be proposed to explain the adverse effects of tamoxifen on ovarian function. © 2015, Yamazaki et al.

We experienced a laparoscopic forceps' fracture and successfully retrieved the very small part. Laparoscopic forceps' fractures are known complications of laparoscopic surgery. To prevent this complication, forceps should be maintained in good condition. It is also important to know how to manage laparoscopic forceps' fractures quickly.

Objective To investigate the impact of estrogen contained in oral contraceptives (OCs) on the action of progestin on ovarian endometrioma epithelial cells. Design Experimental in vitro study and immunohistochemical analysis. Setting University hospital. Patient(s) Patients who underwent surgery due to ovarian endometrioma. Intervention(s) Not applicable. Main Outcome Measure(s) Telomerase-immortalized epithelial cells derived from ovarian endometrioma were treated with norethindorone (NET; 80 nmol/L) or levonorgestrel (LNG; 20 nmol/L) with or without 17β-ethynylestradiol (EE; 0.6 nmol/L) for 96 hours, and the cell growth was monitored. Estrogen receptor (ER) α, progesterone receptor (PR) A, and PRB expressions in clinical samples of ovarian endometrioma epithelial cells were analyzed with the use of immunohistochemistry. Result(s) NET or LNG effectively suppressed cell growth, and addition of EE significantly enhanced the growth suppression. This EE-mediated enhancement of cell growth suppression was observed only in cells that expressed ERα and therefore was ERα dependent. Western blot analysis revealed that expression of PRB was significantly induced by the addition of EE. Immunohistochemical analysis confirmed that ERα expression and PRB expression are significantly correlated, indicating that progestin-sensitive cells with PRB expression are predisposed to react with estrogen stimulation. Conclusion(s) These findings suggest that EE contained in OCs plays a supportive role in progestin-induced growth inhibition of ovarian endometrioma epithelial cells. In the absence of estrogen priming, concurrent estrogen action was essential for rapid induction of PR to achieve maximal progestin effect.

A small subset of cells with CD133 expression is thought to have increased chemoresistance and tumorigenicity, features of cancer stem cells (CSCs); the molecular mechanisms by which these properties arise remain unclear. We characterized CD133+ endometrial cancer cells based on microarray analyses of Ishikawa cells. Of the genes upregulated in CD133+ cells compared with CD133- cells, we noted several key factors involved in the aggressive behavior of cells, including ABCG2 and matrix metalloproteinase (MMP). Flow cytometric analyses identified a side-cell population (SP) with CSC features in Ishikawa cells, and they were found to be more enriched in CD133+ cells than CD133- cells. In particular, CD133+/SP cells exhibited higher proliferative and colony‑forming activity than CD133+/non-SP cells. Matrigel invasion assay revealed that CD133+ cells have enhanced invasive capacity with elevated MT1-MMP expression. siRNA‑based knockdown of MT1-MMP largely abolished the invasive capacity of CD133+ cells, but not CD133- cells due to low levels of constitutive MT1-MMP1 expression. These findings demonstrate that increased chemoresistance and tumorigenic potential of CD133+ cells are at least partly attributed to an enriched SP fraction as well as increased MMP-1 expression. These results will be of assistance in the establishment of molecular target therapy to CSCs in endometrial cancer.

Progestin inhibits the growth of normal and cancerous endometria via the progesterone receptor (PR), but the distinct functions and signalings of PR subtypes have not been fully understood. The aim of the present study was to dissect the key pathways of progestin to inhibit endometrial epithelial growth. Immortalized endometrial epithelial cells (EM-E6/E7/TERT) with stable PRA or PRB expression were established and used for the experiments. In vitro growth inhibition by progestin was mainly observed in EM-E6/E7/TERT cells with PRB rather than those with PRA. RT-PCR assay confirmed that FOXO1, a key gene for progestin action, was up-regulated by progestin in a PRB-dependent manner. cDNA microarray analysis identified IGFBP-1, which contains FOXO1 binding sites on its promoter, to be induced by medroxyprogesterone acetate (MPA) in EM-E6/E7/TERT cells with PRB but not with PRA. siRNA knockdown of FOXO1 disturbed the induction of IGFBP-1 by MPA, while IGFBP-1 knockdown showed no effect on MPA-induced FOXO1 expression, indicating that FOXO1 is an upstream regulator of IGFBP-1. Luciferase reporter assays showed that MPA activated the IGFBP-1 promoter, which was cancelled by FOXO1 knockdown. Chromatin immunoprecipitation assay confirmed the in vivo binding of FOXO1 to the core promoter of IGFBP-1. IGFBP-1 knockdown significantly attenuated the growth inhibitory effects of MPA. The FOXO1/IGFBP-1 axis is essential for PRB-dependent growth inhibition of endometrial epithelial cells, offering a potential therapeutic clue to enhance the progestin effect.

Endometriosis is a common gynaecological disorder associated with dysmenorrhoea, pelvic pain and subfertility and is a leading cause of disability and loss of productivity in women of reproductive age (Olive and Schwartz, 1993). Numerous studies have attempted to dissect the biology of endometriosis. These studies mainly use in vitro culture of stromal cells rather than culture of epithelial cells from endometriotic tissues, because the former cells are more easily and stably cultured for much longer periods than the latter cells (Noble et al, 1997; Gurates et al, 2002). In fact, it is difficult to culture endometriotic epithelial cells in vitro, because these cells lose their proliferative capacity during ongoing cultivation of primary cultures over several days. The inability of endometriotic epithelial cells to survive in vitro is an obstacle to gaining a better understanding of the biology of this disease. In particular, malignant change of endometriosis, especially of ovarian endometrioma, for which epithelial cells are exclusively responsible, has lately attracted considerable clinical attention (Kurman and Craig, 1972; McMeekin et al, 1995; Ness, 2003; Oral et al, 2003). There is therefore an urgent need to establish a stable system for the culture of endometriotic epithelial cells that can be used for research not only into the biology of endometriosis but also into its carcinogenesis. There are two major barriers within epithelial cells that inhibit cell division under usual culture conditions: premature senescence and telomere-dependent senescence (Kiyono et al, 1998). The former is observed during early passage in primary culture and is caused by the activation of Rb that leads to cell cycle arrest, whereas the latter is found at a later stage of culture and is caused by telomere shortening after a considerable number of cell divisions. Inhibition of both Rb function and telomere shortening is therefore required for long-term culture of epithelial cells. In previous studies, we successfully established a stable system for the culture of primary endometrial epithelial cells, in which the human papillomavirus type 16 E6/E7 genes and the human telomerase reverse transcriptase (hTERT) were introduced to inhibit Rb functions and to activate telomerase, respectively (Kyo et al, 2003). These immortal cells were not transformed but retained the original characteristics of endometrial epithelial cells, such as steroid responsiveness. Subsequent studies have demonstrated that overexpression of cyclin D1 or cdk4, instead of HPV E6/E7, effectively inhibited Rb activity and might be an alternative method of overcoming premature senescence in primary epithelial cells of other origins (Ramirez et al, 2004; Sasaki et al, 2009). In the present study, we sought to generate a stable culture of epithelial cells isolated from the ovarian endometriomas by the introduction of various genetic elements. These cells were successfully immortalised without generation of transformed phenotypes and were responsive to progestin and oestrogen. These cells are thus potentially useful as an experimental model for analysis of the mechanisms of steroid hormone functions as well as of carcinogenesis arising from ovarian endometrioma.

The use of molecular target therapy has not been established for endometrial cancer. The present study investigated the potential therapeutic strategy of targeting CD117-positive cancer cells as a novel molecular target therapy. FACS-sorted CD117+ cells isolated from endometrial cancer cell lines (Ishikawa or MFE280 cells) exhibited higher proliferative capacity in vitro and colony forming activity on soft agar, and decreased sensitivity to cisplatin, compared to CD117- cells. Immunohistochemical analyses with surgical specimens of endometrial cancers showed that high CD117 expression was tightly linked to advanced FIGO stages, myometrial invasion and histological grade, and was significantly associated with poor overall survival and relapse-free survival (Kaplan-Meier analysis; p Citation Format: Mitsuhiro Nakamura, Satoru Kyo, Xiuzhi Zhang, Masahiro Takakura, Yasunari Mizumoto, Yukiko Bono, Toshiyuki Sasagawa, Hiroshi Fujiwara. Imatinib sensitizes endometrial cancer cells to cisplatin by targeting CD117-positive growth-competent cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 205. doi:10.1158/1538-7445.AM2014-205

The cerebellar Purkinje neuron (PN) serves as an important model in studies of neuronal development in the mammalian central nervous system. Dissociated PN preparations maintained in an in-vitro environment with simplified cellular and biochemical conditions can facilitate molecular analyses of neuronal development. Here we describe a reliable method to prepare dissociated cultures of mouse cerebellar neurons maintained with a serum-free, Dulbecco's modified Eagle's medium/F-12 nutrient-based medium, which facilitates PN survival and dendritic differentiation. The survival of mouse PNs in this culture was maximized when cerebellar cells were (1) taken from prenatal animals, (2) dissociated with papain, and (3) seeded at a density of 5 000 000 cells/ml or higher. Dissociated PNs prepared by our method from mice of embryonic day 18 (E 18) reproduced several morphological and electrophysiological changes seen in intact postnatal rodents with similar time-courses. Therefore, our culture method offers a useful model for investigating molecular mechanisms underlying postnatal neuronal development.

Emerging evidence has shown that a small subpopulation of cancer cells plays critical roles in tumorigenesis, establishing the cancer stem cells (CSCs) theory. CSCs have properties of tumorigenesis, self-renewal and multilineage differentiation. Previous our study demonstrated that CD133+ enodometrial cancer cells showed tumorigenic and self-renewal ability, suggesting that CD133 was a potential CSC marker in endometrial cancer. In this study, we focused on multilineage differentiation and investigated an ability of endothelial differentiation in endometrial CSCs. Immunohistochemistry of subcutaneous xenografts showed that human endometrial tumors contained human vessels labeled by human-specific anti-CD31 and vWF antibodies. Endothelial tube formation assay (in vitro angiogenesis) revealed that isolated CD133+ endometrial cancer cells could show tube formation while CD133- cells could not. CD133+ cells expressed CD31 and VEGFR-1 higher than CD133- cells. In some cell types of endometrial cancer cells, flow cytometric analysis showed expression of CD31 was high ratio under hypoxia compared to normoxia. Furthermore, in vitro angiogenesis assay demonstrated hypoxic condition could promote and keep tube formation in CD133+ cells, but not CD133- cells. These results suggest that CSCs have potential of endothelial differentiation and hypoxia enhances endothelial differentiation of CSCs. These findings shed light on the establishment of new target therapy for endometrial cancer. Citation Format: Mitsuhiro Nakamura, Xiuzhi Zhang, Masahiro Takakura, Yoshiko Maida, Yasunari Mizumoto, Yukiko Bono, Toshiyuki Sasagawa, Satoru Kyo. Hypoxia enhances tumor vascularization by endothelial differentiation of endometrial cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2013-4892

Evidence for existence of cancer stem cells (CSCs) has been reported in several malignant tumors. CSCs have properties of self-renewal, tumorigenesis and multilineage differentiation. Many studies about CSCs have focused on self-renewal, tumorigenesis. However, it is unclear about multilineage differentiation. Our previous study showed CD133 was a CSC marker and a critical prognostic marker in endometrial cancer. In that study, we demonstrated that CD133+ cells were capable of generating both CD133+ and CD133- cells, whereas CD133 cells did not generate CD133+ cells, indicating that CD133+ CSCs have a differential ability. The present study investigated endothelial differentiation of endometrial cancer cells. We have established endometrial cancer cells from normal human endometrial epithelial cells by introduction of HPV16 E6/E7, hTERT and K-ras genes (EM-E6/E7/hTERT/K-ras cells). Histological findings of tumors formed from EM-E6/E7/hTERT/K-ras cells showed that tumors were composed of solid and vascular regions. Vascular regions were stained human-specific anti-CD31 antibody, suggesting that EM-E6/E7/hTERT/K-Ras cells are capable of differentiating to endothelial cells. Subcutaneous xenografts from clinical samples in endometrial cancer also demonstrated that human endometrial tumors contained human vessels labeled by human-specific anti-CD31 antibody. Endothelial tube formation assay (in vitro angiogenesis) revealed that isolated CD133+ endometrial cancer cells could show tube formation while CD133- cells could not, indicating that CD133+ endometrial cancer cells had an ability of endothelial differentiation. Furthermore, western blot analysis showed high expression of VEGFR1 in CD133+ cells. We demonstrated a new function of CSCs in endometrial cancer cells. These findings shed light on the establishment of new target therapy to endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5199. doi:1538-7445.AM2012-5199

Background; Cancer stem cells (CSCs) have been known to be the major source of tumor propagation and might be an attractive therapeutic target. Our previous study showed that CD133+ Ishikawa and MFE 280 endometrial cancer cells have the ability of tumorigenicity, self-renewal and differentiation capacity in vitro and in vivo, while CD133- cells did not. Furthermore, CD133+ cells showed more resistance to cisplatin- or paclitaxel-induced cytotoxicity than CD133- cells. These results indicate that CD133 is the CSC maker in endometrial cancer cells. In the present study, we sought to analyze gene profile and identify molecular targets in endometrial cancer stem cells. Methods; We examined microarray analysis to investigate the molecules essential for the CSCs characteristics by 3D-Gene chip (TORAY). Results; Microarray analysis of total 25,000 genes showed that about 500 genes were highly expressed in CD133+ cells, compared to CD133- cells. The ALDH1 gene was up-regulated in CD133+ cells, in accordance with the previous reported expression of ALDH1 on breast and colorectal CSCs. We also found out the elevated expression of ABC transporter genes, ABCG2, ABCG3, ABCG4, ABCG8, ABCC8 and ABCC9. It is well known that cells expressing those genes are resistant to chemotherapeutic agents and associated with side population (SP). Fluorescence activated cell sorter analysis demonstrated that the ratio of SP fraction in CD133+ Ishikawa cells is slightly higher than in CD133- Ishikawa cells (0.8% vs 0.6%). Isolated SP/CD133+ cells ells showed more extensive proliferation and tumorigenicity, compared with non-SP/CD133+, SP/CD133- and non-SP/CD133- cells. Finally, we focused on high expression of MMP14 (MT1-MMP) gene in CD133+ cells. Invasion assay revealed that CD133+ Ishikawa and MFE 280 cells have more invasive property than CD133- Ishikawa and MFE 280 cells. The inhibition of MT1-MMP gene by siRNA significantly reduced the invasive ability of CD133+ cells. These results suggest that MT1-MMP is one of the critical molecules that generate biological features of CSC and may be a potential therapeutic target in endometrial cancer. Conclusions; We demonstrated that SP/CD133+ cells might be more defined CSCs in endometrial cancer and identified important molecules essential for characterizing endometrial CSCs in which MT1-MMP plays critical roles in generating biological features of CSCs in this tumor type. These findings shed light on establishment of molecular target therapy in endometrial cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3411. doi:10.1158/1538-7445.AM2011-3411