Akihiro Ohyama

Establishment and characterization of the rhabdomyosarcoma cell line designated NUTOS derived from the human tongue sarcoma: Special reference to the susceptibility of anti-cancer drugs

Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco’s modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 μg of streptomycin, 50 U/mL of penicillin and 0.25 μg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, α-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.

Novel amelanotic and melanotic cell lines NM78-AM and NM78-MM derived from a human oral malignant melanoma

Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 °C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.

Establishment and characterization of a cell line (IGSK-3) secreting human chorionic gonadotropin, adrenocorticotropic hormone and parathyroid hormone-related protein derived from primary poorly differentiated adenocarcinoma of the stomach

We recently established human chorionic gonadotropin-, adrenocorticotropic hormone- and parathyroid hormone-related protein-secreting cell line derived from primary poorly differentiated adenocarcinoma of the stomach. The cell line was designated as IGSK-3. Inverted-phase contrast microscopy revealed that the IGSK-3 cells consist of two morphological subtypes. One type has visible nucleoli and clear nuclei, but nucleoli and nuclear membrane of the othertype are invisible. The population-doubling time was about 43 h. An analysis of conditioned medium by IGSK-3 cells cultured for 4 days revealed the IGSK-3 cells secrete human chorionic gonadotropin-β (0.5 ng/mL), adrenocorticotropic hormone (5.5 pg/mL), parathyroid hormone-related protein (3.4 pmol/mL) and epidermal growth factor (14.2 pg/mL). Histopathological diagnosis of the graft of IGSK-3 cells revealed that IGSK-3 cells built a poorly differentiated adenocarcinoma which resembled the original tumor. In addition, the IGSK-3 cell line was immunocytochemically positive for human chorionic gonadotropin-β and epidermal growth factor receptor, and negative for vascular endothelial growth factor.

Prolactin selectively transported to cerebrospinal fluid from blood under hypoxic/ischemic conditions

Aim The aim of this study was to determine and to verify the correlation between the amount of prolactin (PRL) levels in the blood and in the cerebrospinal fluid (CSF) by various causes of death as an indicator for acute hypoxia in autopsy cases. It is to confirm the cause of the change in prolactin level in CSF by in vitro system. Materials and methods In autopsy materials, the PRL levels in blood from the right heart ventricle and in the CSF were measured by chemiluminescent enzyme immunoassay, and changes in the percentage of PRL-positive cells in the pituitary gland were examined using an immunohistochemical method. Furthermore, an inverted culture method was used as an in vitro model of the blood-CSF barrier using epithelial cells of the human choroid plexus (HIBCPP cell line) and SDR-P-1D5 or MSH-P3 (PRL-secreting cell line derived from miniature swine hypophysis) under normoxic or hypoxic (5% oxygen) conditions, and as an index of cell activity, we used Vascular Endothelial Growth Factor (VEGF). Results and discussion Serum PRL levels were not significantly different between hypoxia/ischemia cases and other causes of death. However, PRL levels in CSF were three times higher in cases of hypoxia/ischemia than in those of the other causes of death. In the cultured cell under the hypoxia condition, PRL and VEGF showed a high concentration at 10 min. We established a brain-CSF barrier model to clarify the mechanism of PRL transport to CSF from blood, the PRL concentrations from blood to CSF increased under hypoxic conditions from 5 min. These results suggested that PRL moves in CSF through choroidal epithelium from blood within a short time. PRL is hypothesized to protect the hypoxic/ischemic brain, and this may be because of the increased transportation of the choroid plexus epithelial cells.