| Co-Investigator(Kenkyū-buntansha) |
HATA Kazuko Kitasato Univ., Sch. Med.,, 医学部, 助手 (10146441)
HATA Hiroki Kitasato Univ., Sch. Med., Assistant Professor, 医学部, 講師 (30146451)
SHIMODA Takao Kitasato Univ., Sch. Med., Assistant Professor, 医学部, 講師 (60162731)
KATO Yoshiki Kitasato Univ., Sch. Med., Assistant Professor, 医学部, 講師 (70146439)
JOUBOU Toshiko Kitasato Univ., Sch. Med., Assistant Professor, 医学部, 講師 (80110873)
森澤 孝行 北里大学, 医学部, 講師 (40133300)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1990: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
(1) Materials for experimental model : Twenty cases of normal endometrium, had been taken from operation material with uterine fibromas, were primarily cultured for the experiments. These materials were useful for observations of hormonal actions in vitro, but were not successful for immunocytochemical assay. Nine cell lines, had been established from the cases of endometrial cancer, were used in this research.
(2) Receptor assay : Seventy cases of normal endometrium taken from the patient with normal menstrual cycle, and 31 endometrial cancer tissues from operation materials, were stained with immunocytochemical methods for the detection of estrogen and progesterone receptors, using the monoclonal antibodies against to estrogen and progesterone receptors. The receptor contents and localization of 9 endometrial cancer cells also detected by both biochemical and immunocytochemical procedures. The receptor contents of normal endometrium detecting by immunocytochemical staining were change
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d by estrogen and progesterone status during menstrual cycle. The incidence of receptor positive cases in endometrial cancer tissues were decreased corresponding to the loss of histological differentiation. The positive rates of both estrogen and progesterone receptors of G1, G2 and G3 endometrial adenocarcinoma tissues were 8/11 (73%), 3/13 (23%) and 0/4 (0%), respectively. The Ishikawa cells, derived from a well differentiated endometrial adenocarcinoma possess estrogen and progesterone receptors are showen to respond to estrogens and progestins by changing a variety of parameters. The localization of estrogen and progesterone receptors are found in the nuclei by immunocytochemical stainings, confirming the correctness of Gorski and Greene's one step theory involving the action mechanisms of steroid hormones. The amounts progesterone binding of Ishikawa cells measured by whole cell binding assay for 3H-R5020 were increased by the addition of estradiol to the culture media as dose and time dependent manner. Optimal concentration of estradiol were 10nM.
(3) Various parameters for estradiol and progestin action.
(1) Cell proliferation and cycle : The cell proliferation of Ishikawa cells were stimulated by the addition of estradiol at 10nM under low serum condition. The proportionality of the S-phase cells detection by the stainings for antiーBrdU monoclonal antobodiesimmunocytochemically also clearly increased by the estradiol at 10nM.
(2) 17 beta estradiol dehydrogenase (E2DH) activity : The activity of E2DH as a parameter of progestin action of normal endometrium which contains the functional progesterone receptor were found in Inshikawa endometrial cancer cells. These activity of Ishikawa cells were lower compared with normal endometrium but were stimulated by the simultaneous addition of estradiol and progestin to the culture media.
(3) Glycogen metabolism : These parameter of progestin action of normal endometrium were also investigated in endometrial cancer cells. The activity of glycogen synthetase (GS) and glycogen content of Ishikawa cells were stimulated by the additon of progestins (P4, MPA, R5020 and Org 2058). These effects of progestins were completely abolished by RU486 which is competitive reagent on progesterone receptor. Progestins and RU486 may be thought to act via not progesterone receptor but glucocorticoid receptor. Two glucocorticoids (Cortisol, Dexamethasone), had been also added to the culture media d no effect on glycogen metabolism, confirming the progesterone receptors of Ishkawa cells were functional. On the contrast, the activity of glycogen phosphorylase of Ishikawa cells were decreased by progestins.
(4) Autocrine and paracrine actions of local growth factors
EGF (Epidermal growth factor) is well known as local growth factor which may be able to stimulate the proliferation of a various types of cells. It has been reported that the receptors of EGF (EGFR) of normal endometrium are changed by estradiol and progesterone status, suggesting to be parameters of steroid hormones. In this study, EGFR of endometrial cancer cells were detected by immunocytoctemical and biochemical methods. The localization of EGFR, stained by anti-EGFR monoclonal antibodies were found on cell membrane and internalized immediately after the addition of EGF to culture media. The amounts of EGF bindings of Ishikawa cells and HEC-50 cells, measured by competitive assay of 125I-EGF with unlabeled EGF, were 2,500 and 11,000 sites/cell, respectively. The proliferations of Ishikawa cells were simulated by the addition of EGF to culture media at dose dependent manner under serum-free conditions. Less
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