Laboratory of Genitourinary Cancer Pathogenesis Microscopy Core Facility

核心设施包括安装在Axio Observer Z1电动荧光显微镜上的蔡司LSM 510 META扫描模块，配备高分辨率镜头和Nomarski DIC光学器件的立式Axioplan 2荧光显微镜，以及配备Axio Observer Z1荧光显微镜，配备用于图像采集的axiovision软件的计算机和显示器。此外，该设施还设有VivaView荧光培养显微镜，能够对培养数天或数周内生长的细胞进行成像。来自NCI 15个不同实验室（大约40个用户）的研究人员目前正在使用共聚焦核心设备。使用该设施的科学家的研究重点包括卵巢癌，乳腺癌，前列腺癌，甲状腺癌和肺癌的进展和转移，用于儿科肿瘤学的化疗以及艾滋病治疗。在细胞和癌症生物学分部（CCBB）， 21名科学家，包括专职科学家、博士后研究员、博士后研究员和暑期学生，在他们的研究工作中经常使用核心设施。刘正刚博士实验室的博士后正在利用DNA核显微注射研究死亡结构域蛋白在细胞凋亡中的作用。共聚焦显微镜被这个小组用来研究内质网应激的分子机制和参与细胞凋亡的蛋白质的细胞定位。在Kathy Kelly博士的实验室里，三名博士后和一名学士研究生正在使用共聚焦显微镜研究小鼠原发性前列腺细胞的分子机制，并识别分化标记和信号分子，以阐明前列腺癌转移的信号通路。他们还在二维和三维培养中用进展标记和信号分子染色正常和转化的前列腺上皮细胞。共聚焦显微镜对于观察这些成分在三维培养中生长的球形和管状结构中的定位是必不可少的。一名研究员和一名本科毕业后的研究员正在使用共聚焦显微镜对荧光标记的前列腺癌细胞的脑转移进行成像。来自其他几个分支机构或实验室的科学家目前正在使用该核心作为他们研究的一个组成部分。下面是一些使用核心设施的正在进行的项目的示例。1.癌症生物学和遗传学实验室：用于艾滋病治疗的核苷类似物的研究。2.乳腺生物学和肿瘤发生：乳腺干细胞生态位及其将祖细胞重编程为胚胎干细胞（ES）细胞的能力的研究。利用共聚焦显微镜确认es来源细胞中y染色体的存在，并通过乳蛋白免疫组织化学确定这些细胞在其当前位置具有功能。3.肿瘤医学分支：a. GXXXG基序在ABC半转运体ABCG2同二聚化中的作用，这赋予了对几种重要临床药物的抗性；b. p53在癌细胞系中的定位以及p53与微管蛋白和输入蛋白的共定位；c.铂化合物对微管细胞骨架的影响。这些是与临床试验数据相关的基本研究，这些研究的信息可能对开发减轻这些化合物相关毒性的治疗方法很重要。除了参与共聚焦显微镜核心设施的项目，教授科学家共聚焦显微镜和显微注射的各个方面，以及维护核心设备外，我还与CCBB分院院长Kathy Kelly合作，开展CD97信号转导机制和CD97在癌症进展和转移中的作用的研究。这个项目的细节在Kathy Kelly的年度报告中有描述。CD97是一种粘附类G蛋白偶联受体，在炎症细胞和几种癌细胞上表达。在甲状腺、食管、胃、结直肠和前列腺肿瘤中，CD97的表达与恶性程度平行升高。我们已经证明CD97既可以作为肿瘤细胞的细胞自主受体，也可以作为内皮细胞上整合素α 5 β a1和α v β a3的配体。我们发现，作为受体，CD97信号通过G-alpha 12/13异三聚体G蛋白家族偶联，导致Rho活性增加。对前列腺和甲状腺肿瘤细胞系的分析表明，内源性表达CD97的异常过表达似乎导致了不依赖配体的信号传导。前列腺肿瘤细胞系中内源性CD97的缺失导致骨转移减少。我们最近发表的两篇文章表明，CD97可能通过与溶血磷脂受体的异源二聚化作用在前列腺癌和甲状腺癌的进展中发挥积极作用。

The Core Facility houses a Zeiss LSM 510 META scanning module mounted on an Axio Observer Z1 motorized fluorescent microscope, an upright Axioplan 2 fluorescent microscope equipped with high resolution lenses and Nomarski DIC optics, and an Axio Observer Z1 fluorescent microscope, computer and monitor equipped with axiovision software for image acquisition. In addition, The facility houses a VivaView fluorescent incubator microscope capable of imaging cells growing in culture over several days or weeks. Researchers from fifteen different laboratories (approximately 40 users) in the NCI are currently using the Confocal Core Facility. The research focus of the scientists using the facility includes ovarian, breast, prostate, thyroid, and lung cancer progression and metastasis, chemotherapy used in pediatric oncology, and AIDS therapy. In the Cell and Cancer Biology Branch (CCBB), 21 scientists including staff scientists, postdoctoral fellows, post baccalaureate fellows, and summer students routinely use the core facility in their research efforts. Postdoctoral fellows in Dr. Zheng-Gang Lius laboratory are using nuclear microinjection of DNA to investigate the role of death domain proteins in apoptosis. Confocal microscopy is being used by this group to investigate the molecular mechanisms of endoplasmic reticulum stress and cellular localization of proteins involved in apoptosis. In Dr. Kathy Kelly's laboratory three postdoctoral fellows and one post baccalaureate fellow are using confocal microscopy to study molecular mechanisms and identify differentiation markers and signaling molecules on primary murine prostate cells in order to elucidate the signaling pathways involved in prostate cancer metastasis. They are also characterizing normal and transformed prostate epithelial cells stained with progression markers and signaling molecules in 2 and 3 dimensional cultures. Confocal microscopy is essential for observing the localization of these components in the spherical and tubular structures that grow in 3D culture. A research fellow and a post baccalaureate fellow are using confocal microscopy for imaging brain metastasis from fluorescently labeled prostate cancer cells. Scientists from several other branches or laboratories are currently using the core as an integral part of their research. Below are some examples of ongoing projects using the core facility. 1.Lab of Cancer Biology and Genetics: Study of nucleoside analogs used in AIDS therapy. 2.Mammary Biology and Tumorigenesis: Investigation of the mammary stem cell niche and its ability to reprogram progenitor populations to the embryonic stem (ES) cell. Confocal microscopy is being used to confirm the presence of the Y-chromosome in the ES-derived cells and to establish that these cells are functional in their current location by milk protein immunohistochemistry. 3.Medical Oncology Branch: a.The role of the GXXXG motif in the homodimerization of the ABC half-transporter, ABCG2, which confers resistance to several important clinical agents b.Localization of p53 in cancer cell lines and colocalization of p53 with tubulin and importin c.Effect of platinum compounds on the microtubule cytoskeleton. These are essential studies that will be correlated to clinical trial data and information from these studies may prove to be important in the development of therapies to alleviate the toxicity associated with these compounds. Apart from collaborating on projects involving the confocal microscopy core facility, teaching scientists various aspects of confocal microscopy and microinjection, and maintaining the equipment in the core, I am conducting research on the mechanisms of CD97 signal transduction and the role of CD97 in cancer progression and metastasis in collaboration with the branch Chief of CCBB, Kathy Kelly. The details of this project are described in Kathy Kelly's annual report. CD97, an adhesion class G protein coupled receptor, is expressed on inflammatory cells and several carcinomas. CD97 expression increases in parallel with malignant grade in thyroid, esophageal, gastric, colorectal, and prostate tumors. We have demonstrated that CD97 acts both as a cell-autonomous receptor on tumor cells and as a ligand for integrins alpha5 beta1 and alphav beta3 on endothelial cells. We showed that in its capacity as a receptor, CD97 signaling couples through the G-alpha 12/13 family of heterotrimeric G proteins resulting in increased Rho activity. An analysis of prostate and thyroid tumor cell lines has shown that abnormal overexpression of endogenously-expressed CD97 seems to lead to ligand-independent signaling. Depletion of endogenous CD97 in prostate tumor cell lines resulted in decreased metastasis to bone. We have two recent publications demonstarting that CD97 plays an active role in prostate and thyroid cancer progression probably via heterodimerization with the lysophosphatidic receptor.