Ken Ishitani

p54nrb acts as a transcriptional coactivator for activation function 1 of the human androgen receptor

The androgen receptor (AR) has two transactivation functions that have been mapped to the N- and C-terminal domains and designated as activation function-1 (AF-1) and AF-2, respectively. While the molecular basis for AF-2 function has been well studied, little is known about AF-1 coregulators. Therefore, we attempted to identify AF-1-interacting proteins from HEK293 cells by biochemical purification followed by mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Purified AF-1 region-interacting proteins were found to contain nuclear RNA-binding protein p54(nrb), polypyrimidine tract-binding protein-associated splicing factor (PSF), paraspeckle protein 1 (PSP1), and PSP2, which are assumed to be involved in pre-mRNA processing. p54(nrb) interacted with AR via the A/B domain in a ligand-dependent manner. Reflecting the physical interaction between p54(nrb) and the AR A/B domain, AR AF-1 function was potentiated by p54(nrb). Our results suggest that p54(nrb) functions as a coactivator of AR that potentiates transcription, and presumably splicing as well.

Relation between climacteric symptoms and ovarian hypofunction in middle-aged and older Japanese women.

Objective: To gain insight into the characteristics and current status of climacteric symptoms reported by middle-aged and older women in Japan, we surveyed women presenting at our menopause clinic. Design: The participants included 1,069 women, ranging in age from 40 to less than 60 years (mean age, 50.2 y). Climacteric (indefinite) symptoms were objectively assessed with the use of the Keio questionnaire, which grades the severity of 40 types of symptoms classified into 20 subgroups. The total scores obtained for the 40 symptoms were used to calculate symptom prevalence and severity. To evaluate ovarian function, concentrations of estradiol and follicle-stimulating hormone (FSH) in sera were measured. Results: The most frequent symptom was general fatigue, reported by 88.2% of the women. Shoulder stiffness was the symptom rated to be severe by the highest percentage of women (38.1%). The prevalence and severity of hot flushes (and sweats) were slightly higher in perimenopausal and early postmenopausal women than in premenopausal and late postmenopausal women. The prevalence and severity of hot flushes and sweats were higher in women with estradiol < 25 pg/mL and FSH > 40 mIU/mL than in those with estradiol ≥ 25 pg/mL and FSH ≤ 40 mIU/mL. Conclusion: General fatigue and shoulder stiffness, symptoms with low hormone dependence, are the two most frequent climacteric symptoms in our clinic. Hot flushes and sweats, symptoms with high hormone dependence, are also common symptoms.

Pentosidine Accumulation in Human Oocytes and Their Correlation to Age-Related Apoptosis

Age-related atresia of ovarian follicles is characterized by apoptosis of the constituent cells. Recent studies have indicated that dysfunction of the proteasome and endoplasmic reticulum and subsequent apoptosis in the presence of oxidative stress have relevance to aging. The aim of this study was to assess the involvement of these processes in age-related follicular atresia. Formalin-fixed, paraffin-embedded sections of ovaries obtained at surgery from 74 women (age: 21–54 y) were examined with the terminal deoxynucleotidyl transferase-mediated, dUTP-biotin nick-end labeling (TUNEL) method and an immunohistochemical technique. Primary antibodies used in immunohistochemistry were against pentosidine, ubiquitin and caspase 12. Histological localization of these substances in oocytes was observed by light microscopy, and labeling indices of these cells were evaluated by regression analysis. Positive signals for pentosidine, ubiquitin, caspase 12, and TUNEL were detectable in oocytes of the primordial, primary and their atretic follicles. Regression analysis revealed an age-related increase in the labeling indices for pentosidine, ubiquitin, caspase 12, and TUNEL. These results suggest that pentosidine accumulation in human oocytes is related to apoptosis and increases with age. Further studies will be necessary to clarify the involvement of pentosidine accumulation, proteasome inhibition, and endoplasmic reticulum stress in age-related apoptosis of oocytes in human ovaries.

Reconstruction of functional endometrium-like tissue in vitro and in vivo using cell sheet engineering.

Uterus is a female specific reproductive organ and plays critical roles in allowing embryo to grow. Therefore, the endometrial disorders lead to female infertility. Hence, the regeneration of endometrium allowing fertilized ovum to implant might be valuable in the field of fertility treatment. Recently, cell sheet engineering using a temperature-responsive culture dish has advanced in regenerative medicine. With this technology, endometrial cells were harvested as a contiguous cell sheet by reducing temperature. Firstly, mouse endometrial cell sheets were re-cultured for 3 days to evaluate the function. Histological analyses revealed that endometrial epithelial cell-specific cytokeratin 18 and female-specific hormone receptors, estrogen receptor β and progesterone receptor, were expressed. Furthermore, endometrial epithelial cells constructed epithelial layer at the apical side. Then, endometrial cell sheets from green-fluorescent-protein rat cells were transplanted onto the buttock muscle of nude rat for evaluating the function in vivo. Histological analyses showed that endometrial cell sheets reconstructed endometrium-like tissue, which was found to form uterus-specific endometrial glands having hormonal receptor to estrogen. In this study, endometrial cell sheets were speculated to contribute to the regeneration of functional endometrium as a new therapy.

Variations in Circulating Osteoprotegerin and Soluble RANKL during Diurnal and Menstrual Cycles in Young Women

Background/Aims:Physiological bone turnover shows diurnal variations and changes within the menstrual cycle. The aim of this study was to assess the variability of osteoprotegerin (OPG) and soluble RANKL (sRANKL) serum levels during diurnal and menstrual cycles. Method: Blood was collected from 15 young women at 6-hour intervals between 08.00 and 20.00 h during the follicular phase. Moreover, to compare the follicular and luteal phases, blood was also collected at 14.00 h during the luteal phase. Serum bone-specific alkaline phosphatase (BAP), N-telopeptide of type I collagen (NTX), OPG and free sRANKL were measured. Results: No diurnal variations in BAP, OPG, sRANKL and sRANKL/OPG ratio were detected. NTX was significantly higher in the morning than in the afternoon and at night (p = 0.02). There were no menstrual variations in either. Conclusions: The consistent absence of diurnal variations in circulating OPG and sRANKL levels may reflect the absence of diurnal variation in their expression in the bone microenvironment. In this case, the nocturnal rise and the fall in bone resorption in the luteal phase should be accounted for by other factors than RANKL/OPG-mediated factors. Timing of sampling is unlikely to influence the results of circulating OPG and sRANKL measurement.

Novel virtual cytological analysis for the detection of endometrial cancer cells using autoscan fluoromicroscopy

The current medical examinations for detecting endometrial cancer can sometimes be stressful and inconvenient for examinees and examiners. Therefore, we attempted to develop an autoscan‐virtual cytology system for detecting endometrial cancer without relying on judgment by the human eye. Exfoliated cells from the uterus were retrieved using a tampon inserted for 3 h. More than 100 monoclonal antibodies (mAb) developed by us were screened in three steps of immunohistochemistry to find mAb sets that would enable the cancer and normal endometrium to be perfectly distinguished. The exfoliated cells provided by 30 endometrial cancer patients and a total of 37 samples of 14 non‐malignant volunteers including the menstrual cycle were analyzed using imaging cytometry. All samples contained epithelial cells and dysplasia cells, but the pathologist could not definitively diagnose all of them as endometrial cancer cells because most cells had degenerated. Twenty‐two of 28 endometrial cancer tissues (79%) were positive with four mAb sets, CRELD1, GRK5, SLC25A27 and STC2, and 22 of 22 normal endometriums (100%) were negative. Our newly developed autoscan‐virtual cytology for exfoliated endometrial cells showed overall sensitivity for endometrial cancer patients and overall specificity for volunteers of 50% (15/30) and 95% (35/37), respectively. Our autoscan‐virtual cytology combined with cancer‐specific mAb and imaging cytometry could be useful for endometrial cancer detection. Autoscan‐virtual cytology for endometrial cancer deserves further evaluation for future endometrial cancer screening. (Cancer Sci 2011; 102: 1068–1075)

Ovarian cystadenofibroma with solid nodular components masqueraded as ovarian cancer

IntroductionThe histological features and image findings on computed tomography (CT) and magnetic resonance imaging (MRI) for ovarian cystadenofibroma mimicking malignant ovarian tumor are reported.CaseA 62-year-old woman was diagnosed as an ovarian cancer on abdominal CT. However, it was diagnosed as a cystadenofibroma on magnetic resonance T2-weighted images that showed the solid components of the tumor with very low intensity. Although ovarian cystadenofibroma is a relatively rare benign tumor, its ultrasonographic feature of cystic lesions with solid components resembles that of malignant ovarian tumor.ConclusionMRI was considered to be useful modality for ovarian cystadenofibroma to be effectively diagnosed preoperatively.

Possible Application of Ascites-infiltrating Gamma-delta T Cells for Adoptive Immunotherapy

Background/Aim: Malignant ascites contain many tumour-infiltrating lymphocytes. γδ T cells with antitumour activity have attracted attention as effector cells in cancer immunotherapy. Vδ2+ T cells were cultured from peripheral blood mononuclear cells (PBMCs) and ascites-infiltrating lymphocytes (AILs) to compare the differences in response to 2-methyl-3-butenyl-1-pyrophosphate (2M3B1-PP) and zoledronate (Zol) as antigens in vitro. Materials and Methods: To expand Vδ2+ T cells from PBMCs and AILs from 29 patients with cancer, these cells were cultured and subjected to analysis. Results: The proliferation rate of Vδ2+ T cells was higher in both PBMCs and AILs when cultured with Zol than with 2M3B1-PP. Although Vδ2+ T cells show a higher rate of expansion in AILs compared to PBMCs, the number of mixed tumour cells in ascites was decreased when cultured with Zol. Conclusion: Vδ2+ T cells in AILs are cytotoxic to tumour cells in ascites and may be considered in adoptive immunotherapy.

Liquid-Based Endometrial Cytology Using SurePath™ Is Not Inferior to Suction Endometrial Tissue Biopsy in Clinical Performance for Detecting Endometrial Cancer Including Atypical Endometrial Hyperplasia

Objective: We evaluated the clinical performance of liquid-based endometrial cytology (SurePath™) for detecting endometrial malignancies by comparison with the performance of suction endometrial tissue biopsy. Study Design: From November 2011 to May 2013, we consecutively collected 1,118 liquid-based endometrial cytology specimens and 674 suction endometrial tissue biopsy specimens. Results: The rate of nonpositive final histology in nonpositive liquid-based endometrial cytology (98.2%) was higher than the rate of nonpositive final histology in nonpositive suction endometrial tissue biopsy (97.0%). None of the clinical performance values of liquid-based endometrial cytology for detecting the endometrial malignancies were statistically inferior to those of the suction endometrial tissue biopsy. When the positivity threshold was more than “atypical endometrial cells of undetermined significance,” the rate of positive liquid-based endometrial cytology from cases with a positive final histology (84.5%) was higher than the rate of positive suction endometrial tissue biopsy from cases with a positive final histology (69.8%). However, there were still no significant differences among all the performance values. Conclusions: Our liquid-based endometrial cytology would be more appropriate in various clinical situations as the initial detection tool for endometrial malignancies, rather than suction endometrial tissue biopsy. In addition, it could be used in screening for endometrial malignancies on a broader scale.

Gorlin syndrome with an ovarian leiomyoma associated with a PTCH1 second hit

We describe a Gorlin syndrome (GS) case with two different second hit mutations of PTCH1, one in a keratocystic odontogenic tumor (KCOT) and the other in an ovarian leiomyoma. GS is a rare genetic condition manifesting as multiple basal cell nevi associated with other features such as medulloblastomas, skeletal abnormalities, and ovarian fibromas. A 21‐year‐old Japanese woman with a history of two KCOTs was diagnosed with GS according to clinical criteria. A PTCH1 mutation, c.1427del T, was detected in peripheral blood. A novel PTCH1 mutation, c.264_265insAATA, had been found in the maxillary KCOT as a second hit mutation. More recently, the ovarian tumor was detected during a gynecological examination. Laparoscopic adnexectomy was performed, and the pathological diagnosis of the ovarian tumor was leiomyoma. Interestingly, another novel mutation, loss of heterozygosity spanning from 9q22.32 to 9q31.2, including PTCH1 and 89 other genes, was detected in this ovarian tumor, providing evidence of a second hit mutation. This is the first report describing a GS‐associated ovarian tumor carrying a second hit in the PTCH1 region. We anticipate that accumulation of more cases will clarify the importance of second hit mutations in ovarian tumor formation in GS.