Yasukazu Sagawa

The correlation of TERT expression with c-myc expression in cervical cancer

Recently putative catalytic telomerase subunit was identified as telomerase reverse transcriptase (TERT). Several reports showed that TERT expression correlated with telomerase activity. It has also been found that c-myc can induce telomerase activation through TERT expression. We examined expression of TERT and c-myc and their correlation in cervical cancers by reverse transcription-polymerase chain reaction (RT-PCR). It was found that TERT and c-myc expression was observed mostly in malignancies and expression of c-myc was concordant for positivity and negativity with TERT. These results support recent studies that c-myc expression is closely associated with TERT and telomerase activity. c-myc up-regulation may play an important role in activation of TERT and telomerase.

Establishment of three cisplatin-resistant endometrial cancer cell lines using two methods of cisplatin exposure

AbstractUsing the endometrial cancer cell line EI established in our department, we attempted to establish cisplatin (CDDP)-resistant cell lines by incremental exposure and high concentration exposure methods. Three CDDP-resistant cell lines were isolated, which could be distinguished by morphological differences. 1.Upon acquiring CDDP resistance, the cells tended to become small and grow in a floating state. This tendency was especially marked when using incremental exposure method. Using the incremental exposure method, a cell line obtained by isolating and culturing only adherent cells was designated EICR-Ia, and a cell line established by culturing only floating cells was designated EICR-If. A cell line obtained by the high concentration exposure method was designated EICR-II.2.Upon acquiring CDDP resistance, tumor markers such as TPA and LDH increased, while proliferative capability of the cells was lowered.3.The invasion capability was diminished in EICR-If cells, but was increased in EICR-Ia and EICR-II cells.4.Following exposure to CDDP, the intracellular platinum concentrations were markedly elevated in EI and EICR-If cells, whereas the increase was mild in EICR-Ia and EICR-II cells and the concentration was lower than that in parent EI cells.5.Studies of drug resistance gene expression revealed increased expression of MDR1, GSTπ, and Topo-II in EICR-If cells; increased expression of GSTπ in EICR-II cells; but no expression of any of the genes in EICR-Ia cells.6.Analyses of cancer- and apoptosis-related genes showed increased expressions of Bcl-2, c-Myc, p53, and ICE in EICR-If cells.7.Upon acquiring CDDP resistance, sensitivity to mitomycin and adriamycin decreased, but sensitivity to etoposide and 5-fluorouracil increased. The findings indicate that the mechanisms of CDDP resistance are different in the three cell lines.

Establishment of a new human cell line (EN) with TP53 mutation derived from endometrial carcinoma

We present a new cell line, EN, established from an invasive endometrioid adenocarcinoma of the uterine corpus in 50-year-old patient. The cells show rapid growth in culture with a doubling time of 24.4 hours and high migration activity. Monolayer-cultured cells were polygonal in shape and showed a tendency to pile up without contact inhibition. Subcutaneous transplantation of the EN cells into nude mice formed solid tumors that were histologically diagnosed as adenocarcinoma, whereas no metastasis was observed. Cultured EN cells produced tissue polypeptide antigen. Genetic and molecular analyses revealed high telomerase activity and estrogen receptor beta but not alpha expression. Using the polymerase chain reaction-single strand conformation polymorphism technique, we have screened EN cells for TP53 mutation in exons 5-8. A mobility shift was observed in this cell line in exon 8. A nucleotide insertion (CGT-->CAGT) was detected at codon 273, which resulted in a creation of a stop codon at codon 308. This cell line thus appears to represent the development of a more malignant clone with divergent receptor function and growth behavior, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.

Telomerase activity in human trophoblast

Summary Recent reports have suggested that many proliferating normal cells express telomerase activity, although telomerase activity was previously considered to be specific for cancer cells. This fact draws attention to the investigation of telomerase activity in trophoblast cells which have characteristics of proliferation, differentiation and invasion. Sub-populations of trophoblast cells were separated by a modification of Klimans method and magnetic cell sorting with antibodies of integrins. Telomerase activity was assayed semi-quantitatively using fluorescence-based TRAP. Cytological detection of telomerase activity was also examined by the newly developed in situ TRAP procedure and the expression of human TERT was detected by RT-PCR. Telomerase activity was detected in 42% of normal placental tissues, and demonstrated decreased activity with gestational age. Telomerase activities was closely correlated with expression of TERT. In sub-populations of CT separated from placenta from early and term gestation, telomerase activity was predominantly detected in columnar CT from early placenta. Fluorescence signals after in situ TRAP were observed in the trophoblastic cell layers in early placenta as well as isolated CT, but not in term placenta. These results suggest that telomerase activity correlates with the regulation of trophoblastic cell proliferation and in situ TRAP is useful for the detection of telomerase positive cells.

Pathological Characterization of Ovarian Cancer Patients Who Underwent Debulking Surgery in Combination With Diaphragmatic Surgery: A Cross-Sectional Study.

AbstractDespite exhaustive efforts to detect early-stage ovarian cancers, greater than two-thirds of patients are diagnosed at an advanced stage. Although diaphragmatic metastasis is not rare in advanced ovarian cancer patients and often precludes optimal cytoreductive surgery, little is known about the mechanisms and predictive factors of metastasis to the diaphragm. Thus, as an initial step toward investigating such factors, the present study was conducted to characterize the pathological status of ovarian cancer patients who underwent debulking surgery in combination with diaphragmatic surgery.This is a retrospective and cross-sectional study of patients who underwent debulking surgery in combination with diaphragmatic surgery at our institution between January 2005 and July 2015. Clinicopathological data were reviewed by board-certified gynecologists, pathologists, and cytopathologists. The rates of various pathological findings were investigated and compared by Fisher exact test between 2 groups: 1 group that was pathologically positive for diaphragmatic metastasis (group A) and another group that was pathologically negative for diaphragmatic metastasis (group B).Forty-six patients were included: 41 patients pathologically positive and 5 pathologically negative for diaphragmatic metastasis. The rates of metastasis to the lymph node (95.8% vs 20%, P = 0.001) and metastasis to the peritoneum except for the diaphragm (97.6% vs 60.0%, P = 0.028) were significantly increased in group A compared with group B. However, no significant differences between the 2 groups were found for rates of histological subtypes (high-grade serous or non-high-grade serous), the presence of ascites, the presence of malignant ascites, exposure of cancer cells on the ovarian surface, blood vascular invasion in the primary lesion, and lymphovascular invasion in the primary lesion.Our study demonstrated that metastasis to the lymph node and nondiaphragmatic metastasis to the peritoneum are significantly associated with metastasis to the diaphragmatic peritoneum, indicating that these factors may be pathological predictors of diaphragmatic metastasis in patients with ovarian cancer. However, as the data available are not sufficient to demonstrate the predictive power of these factors, a further comprehensive, large-scale study should be performed.