Action Mechanisms and Responsiveness for Steroid Hormones in Human normal Endometrium and Endometrial Cancer.

类固醇激素在人类正常子宫内膜和子宫内膜癌中的作用机制和反应性。

• 批准号: 63570797
• 负责人: KURAMOTO Hiroyuki
• 金额: $ 1.28万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570797/
• 关键词: Normal endometrium and endometrial cancer; Action of steroid hormones; Receptors of steroid hormones; Antiーsteroid hormone monoclonal antibodies; Glycogen metabolism; Anti-BrdU monoclonal antibodies; 17 beta-estradiol dehydrogenase; EGFR; 子宮内膜増殖症

(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 … More 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 factorsEGF (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

(1)实验模型材料：20例正常子宫内膜，取自子宫纤维瘤手术材料，原代培养。这些材料是有用的观察激素的行动在体外，但不成功的免疫细胞化学测定。本研究使用了从子宫内膜癌病例中建立的9个细胞系。(2)受体测定：应用抗雌激素受体（ER）和孕激素受体（PR）单克隆抗体，对70例正常月经周期患者的正常子宫内膜和31例手术切除的子宫内膜癌组织进行了ER和PR的免疫细胞化学染色。用生化和免疫细胞化学方法检测了9例子宫内膜癌细胞的受体含量和定位。用免疫细胞化学染色法检测正常子宫内膜受体含量变化 ...更多信息 d受月经周期雌孕激素状态影响。随着子宫内膜癌组织分化程度的降低，受体阳性率也相应降低。G1、G2和G3期子宫内膜腺癌组织中雌激素受体和孕激素受体的阳性率分别为8/11（73%）、3/13（23%）和0/4（0%）。石川细胞来源于分化良好的子宫内膜腺癌，具有雌激素和孕激素受体，显示通过改变多种参数对雌激素和孕激素产生反应。免疫细胞化学染色显示雌激素和孕激素受体定位于细胞核，证实了Gorski和格林关于甾体激素作用机制的一步学说的正确性。用全细胞结合法测定石川细胞对3 H-R 5020的孕酮结合量，雌二醇可增加细胞孕酮结合量，并呈剂量和时间依赖性。雌二醇的最适浓度为10 nM。(3)雌二醇和孕酮作用的各种参数。(1)细胞增殖和细胞周期：在低血清条件下，加入10 nM雌二醇刺激石川细胞增殖。10 nM雌二醇也明显增加了抗人BrdU单克隆抗体免疫细胞化学染色检测S期细胞的比例。(2)17 β雌二醇脱氢酶（E2 DH）活性：在Inshikawa子宫内膜癌细胞中发现E2 DH活性作为含有功能性孕酮受体的正常子宫内膜的孕酮作用的参数。这些活动的石川细胞低于正常子宫内膜相比，但刺激同时加入雌二醇和resistin的培养基。(3)糖原代谢：在子宫内膜癌细胞中也研究了正常子宫内膜的这些参数。孕激素（P4、MPA、R5020和R52058）刺激石川细胞糖原合成酶（GS）活性和糖原含量。孕激素的上述作用可被孕激素受体竞争剂RU 486完全阻断。孕激素和RU 486可能被认为不是通过孕激素受体而是通过糖皮质激素受体起作用。两种糖皮质激素（皮质醇、地塞米松）也加入到培养基中，对糖原代谢没有影响，证实Ishkawa细胞的孕酮受体是功能性的。而孕激素可降低石川细胞糖原磷酸化酶活性。(4)局部生长因子的自分泌和旁分泌作用EGF（表皮生长因子）是众所周知的局部生长因子，它可能能够刺激各种类型细胞的增殖。有研究表明，正常子宫内膜的表皮生长因子受体（EGFR）可随雌、孕激素水平的变化而改变，提示其可能是甾体激素的参数。本研究采用免疫细胞化学和生物化学方法检测子宫内膜癌细胞EGFR的表达。抗EGFR单克隆抗体染色显示EGFR定位于细胞膜上，并在加入EGF后立即内化。通过125 I-EGF与未标记EGF的竞争性测定法测定的石川细胞和HEC-50细胞的EGF结合量分别为2，500和11，000位点/细胞。在无血清条件下，以剂量依赖性方式向培养基中添加EGF来模拟石川细胞的增殖。少

项目成果

秦 宏樹,他: "子宮内膜癌のステロイドホルモン感受性とその作用機序" Human cell. 2. 265-271 (1989)

Hiroki Hata 等人：“子宫内膜癌的类固醇激素敏感性及其作用机制”人类细胞。2. 265-271 (1989)

蔵本 博行: "エストロジェンと子宮体癌" 産婦人科の世界. 42. 1073-1077 (1990)

Hiroyuki Kuramoto：“雌激素和子宫癌”妇产科世界 42. 1073-1077 (1990)。

蔵本 博行,他: "子宮体癌ホルモン療法の作用機序" 癌と化学療法. 16. 1851-1857 (1989)

Hiroyuki Kuramoto 等人：“子宫内膜癌激素治疗的作用机制”《癌症与化疗》16。1851-1857 (1989)。

蔵本 博行,他: "婦人科癌発症のNatural history 子宮体癌" 臨床婦人科産科. 44. 209-213 (1990)

Hiroyuki Kuramoto 等人：“妇科癌症的自然史：子宫癌”《临床妇产科》44. 209-213 (1990)。

Anzai, Y., et al.: "Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line)." Cancer Research. 49. 2362-2365 (1989)

Anzai, Y. 等人：“4-羟基他莫昔芬对人子宫内膜腺癌细胞（石川系）增殖的刺激作用。”