性ホルモンリセプタ―のシグナル傳達を指標としたホ���モン樣化學物質スクリ―ニング法の開發 : In vivo and in vitro assay and screening of chemicals involved in cellular signaling through sex hormone receptors

다국어 초록 (Multilingual Abstract)

Biological discovery has moved at an accelerated pace in recent years, with considerable focus on the transition from in vitro to in vivo models. As such, there has been a greater need to adapt biological knowledges for feasible cellular or animal levels of new assays based on biochemical processes. Considerable efforts have been directed in recent years toward the development of cellular, noninvasive, high-resolution, small animal in vivo imaging technologies.
The author presents in his thesis the achievements during his doctoral course, that is mainly 1) a new in vitro screening assay for endocrine-disrupting chemicals (EDCs) using yellow fluorescence protein (YFP)-labeled estrogen receptor (ER), a hormone-dependent binding between ER and estrogen responsive element (ERE), 2) a new in vivo screening assay for EDCs using translocation of androgen receptor (AR), split-luciferase and reconstitution system in a cell line, 3) a new noninvasive quantitative imaging method in a small animal of endo- or exogenous hormones, 4) a brain imaging method using a bioluminescence and its hormone-dependent photon emission and a subsequent determination of the influence of EDCs on brain. The specific contents are as followings.
In the in vivo approach, the author validates a new hormone-dependent imaging method, which is for analysis of the hormonal activities of androgens in cells and living mice based on a split-Renilla luciferase system and androgen-dependent translocation of androgen receptor (AR) into the cellular nucleus. For this approach, we expressed a fusion protein containing AR with C-terminal fragments of DnaE, a self-catalyzed protein-splicing intein, and Renilla luciferase origined from Sea Pansy (Renilla Renformis), which localizes in the cytosol of mammalian COS-7 cells. The counter part, N-terminal fragments of DnaE and Renilla luciferase, was expressed with a three-repeat nuclear localization signal (NLS, 8AA; (DPKKKRKV)3), and anchored in the nucleus. Androgen-dependently, the AR-fused C-terminal protein (cytosol) is translocated into the nucleus and makes an interaction with the waiting N-terminus part of the counter fusion protein (Nucleus). Through the protein splicing, Renilla luciferase is reconstituted in the cellular nucleus and emits visible light ranged from 400 nm to 650 nm in the presence of its specific substrate, coelenterazine. The author extensively validates the quantifying ability to image androgen-dependent bioluminescence in living mice, in which transiently transfected COS-7 cells were implanted in each interesting site, e.g., brain. A highly sensitive cooled charge-coupled device (CCD) camera was used for imaging bioluminescence in living mice through a photon counting. Cells transiently expressing the N- and C-terminal fragments of Renilla luciferase were imaged while implanted in the brain of living mice after the brain-injection with its specific substrate, coelenterazine. This specific visualization method for a cellular molecular event, i.e., a hormone-dependent translocation of AR into cellular nucleus, in living subjects will allow to study cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals with near-simultaneously monitoring of interesting inner-cellular molecular events.
In the in vitro approach, the author describes a new in vitro screening assay, which is for the determination of endocrine disrupting chemicals (EDCs), such as synthetic estrogens, with an array-type DNA glass slide having characteristics of 1) a high sample throughput, 2) a compact size allowing a small sample volume, and 3) a sensitive determination based on the estrogen-dependent binding of the human estrogen receptor α (hERα) with its estrogen responsive element (ERE; Vit. A2 gene promoter). We devised a glass slide on which a thin agarose gel was mounted. Avidin was then covalently immobilized on each well of the glass slide after the gel was activated by a NaIO4 solution. Also, the biotinylated ERE as a DNA probe was immobilized on the gel layer through avidin-biotin binding. After the estrogen-dependent binding of a yellow fluorescent protein-fused hERα (YFP-hERα) to ERE on the gel layer, the fluorescence intensity of YFP-hERα quantitatively extracted into the gel was directly determined with a fluorescence microplate reader. Pre-incubation of YFP-hERα with estrogen at 37℃ for 30 min enhanced the estrogen-dependent hERα―ERE binding. The determined hormonal activities of estrogens on the interaction of YFP-hERα with ERE were as follows in their decreasing order: diethylstilbestrol (DES) > 17ß-estradiol (E2) ≒ ethynylestradiol (EE2) > 4-hydroxy tamoxifene (OHT) > clomiphene (Clo). The present method provides a sensitive estrogen-dependent dose-response curve down to ~10-13 M in the case of DES.
Through these in vivo and in vitro studies, the author intended to validate a new breakthrough for making generally applicable analytical methods to quantitatively visualize a specific protein-protein interaction in microplates, cells, living mice, or even in an interesting inner organ such as brain of living mice with near-simultaneous manner.

목차 (Table of Contents)

• Abstracts = 3
• Table of Contents = 7
• Chapter 1 General Introduction = 1
• 1.1 Sex hormone receptors and their roles in body = 2
• 1.1.1 Androgen receptor (AR) as a member of the superfamily of nuclear receptor = 2
• 1.1.2 The signal pathway of androgen receptor and the AR-related diseases = 8
• 1.1.3 Estrogen receptor (ER) and its general importance = 16
• 1.1.4 The signal pathway of estrogen receptor and the related diseases = 16
• 1.2 Endocrine disrupting chemicals (EDCs) and their influences on the signaling pathway of sex hormone receptors = 19
• 1.2.1 Historical background of the finding of EDCs = 19
• 1.2.2 Chemical structural view of representative natural hormones and EDCs = 20
• 1.2.3 Consideration of the possible effects of endocrine disruptors to wild-life and human health = 25
• 1.2.4 Requirements for an assay of EDCs = 25
• 1.2.5 Methods for screening hormonal activity of synthetic chemicals = 26
• 1.3 Protein splicing as a tool of the quantification of protein-protein interactions in a cell = 28
• 1.3.1 History of the discovery of bioluminescence = 28
• 1.3.2 Characteristics of Renilla luciferase = 29
• 1.3.3 Protein splicing and its mechanism = 30
• 1.4 Medical imaging methods and recent progress = 33
• 1.4.1 Historical background of the medical imaging = 33
• 1.4.2 Advantages and drawbacks of molecular imaging strategies = 34
• 1.4.3 Medical imaging techniques; Comparison = 36
• 1.4.4 Determination principles of each medical imaging technique = 39
• 1.5 Reporter gene imaging in living subjects = 44
• 1.5.1 General requirements for performing molecular imaging in living subjects = 45
• 1.5.2 Gene marking of cells = 49
• References = 54
• Chapter 2 High-throughput sensing and non-invasive imaging of protein nuclear transport by using intein-mediated reconstitution of split Renilla luciferase = 61
• 2.1 Introduction = 63
• 2.2 Experimental =65
• 2.2.1 Construction of plasmids = 65
• 2.2.2 Cell culture and transfection = 65
• 2.2.3 Western blot analysis = 66
• 2.2.4 Immunocytochemistry = 66
• 2.2.5 Cell-based in vitro assay = 66
• 2.2.6 In vivo imaging of living mice = 67
• 2.3 Results = 69
• 2.3.1 Basic concept = 69
• 2.3.2 Quantitative in vitro sensing of AR translocation into the nucleus = 72
• 2.3.3 In vivo imaging of AR translocation in living mice = 79
• 2.4 Discussion = 87
• 2.5 Conclusion = 89
• References = 90
• Chapter 3. A Screening Method for Estrogens Using an Array-Type DNA Glass Slide = 92
• 3.1 Introduction = 94
• 3.2 Experimental = 97
• 3.2.1 Equipment and reagents = 97
• 3.2.2 Preparation of DNA probe-modified glass slides = 98
• 3.2.3 Construction of plasmid (HisTag-YFP-hERα), fusion protein expression, and purification = 99
• 3.2.4 Quantification of the immobilized avidin on a glass slide = 100
• 3.2.5 Determination of the optimal YFP-hERα protein concentration for glass slide assay = 101
• 3.2.6 Glass slide assay and data analysis = 101
• 3.3 Results and Discussion = 104
• 3.3.1 Quantification of the avidin immobilized on an agarose layer of a glass slide = 104
• 3.3.2 Optimization of the fusion protein (YFP-hERα) concentrations for the EDC screening assay = 104
• 3.3.3 Determination of hERα―ERE binding induced by estrogens, E2, EE2, DES, OHT, and Clo = 105
• 3.3.4 Sensitivity and detection limit of the glass slide-based EDC screening assay = 107
• 3.4 Conclusion = 111
• References = 112
• Chapter 4 General Conclusion = 114
• 4.1 Environmental problems, current trials to solve them, and requirements for the determination of endocrine-disrupting chemicals (EDCs) = 115
• 4.2 A new in vitro approach of this thesis to to fulfill the requirements for constructing a feasible EDC assay = 116
• 4.3 Future expectation of the EDC assays = 117
• 4.4 Characteristics of current imaging technologies and a new current of molecular imaging technologies = 118
• 4.5 An approach of this thesis to develop a new noninvasive quantitative imaging method in mice = 118
• 4.6 Future outlook in molecular imaging = 119
• References = 121