Cell and Cancer Biology Branch Confocal Microscopy Core

核心设施包括安装在Axio观察者Z1电动荧光显微镜上的蔡司LSM 510元扫描模块，配备高分辨率镜头和Nomarski DIC光学元件的立式Axioplan 2荧光显微镜，以及配备Axiovision图像采集软件的Axio观察者Z1荧光显微镜、计算机和监视器。来自NCI的15个不同实验室(约40名用户)的研究人员目前正在使用共焦核心设施。使用该设备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，儿科肿瘤学中使用的化疗，以及艾滋病治疗。在细胞和癌症生物学分部(CCBB)，包括工作人员科学家、博士后研究员、学士研究生和暑期学生在内的21名科学家经常在他们的研究工作中使用核心设施。刘正刚博士实验室的博士后研究员正在使用核显微注射DNA来研究死亡结构域蛋白在细胞凋亡中的作用。这个小组正在使用共聚焦显微镜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。在凯西·凯利博士的实验室里，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分子机制，并在原代小鼠前列腺细胞上寻找分化标志物和信号分子，以阐明前列腺癌转移所涉及的信号通路。他们还在二维和三维培养中表征了正常和转化的前列腺上皮细胞，这些细胞染色有进展标记和信号分子。共聚焦显微镜对于观察这些成分在3D培养中生长的球形和管状结构中的定位是必不可少的。一名研究人员和一名毕业后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。Niederhubers博士实验室的一名工作人员、博士后研究员和学士后研究员正在使用共聚焦显微镜研究MAGE11对缺氧诱导因子Prolyl羟基酶PHD2的抑制和对缺氧反应的激活。来自其他六个分支或实验室的科学家目前正在使用该核心作为他们研究的组成部分。以下是使用核心设施的一些正在进行的项目的例子。2.人类癌变实验室：嘌呤支架HSP90抑制剂对卵巢癌细胞迁移、侵袭和形态的影响3.肿瘤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4.乳腺生物学和肿瘤发生：研究乳腺干细胞的生态位及其将祖细胞群体重新编程为胚胎干细胞的能力。共聚焦显微镜被用来确认胚胎干细胞中Y染色体的存在，并通过牛奶蛋白免疫组织化学来确定这些细胞在当前位置是有功能的。5.医学肿瘤学分支：A.GXXXG基序在ABC半转运体ABCG2同质二聚化中的作用，ABCG2使几种重要的临床药物产生耐药；P53在癌细胞系中的定位以及P53与微管蛋白和进口蛋白的共存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，来自这些研究的信息可能被证明在开发减轻与这些化合物相关的毒性的治疗方法方面很重要。除了在涉及共焦显微镜核心设施的项目上合作，向科学家传授共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分支机构负责人凯西·凯利合作开展CD97信号转导机制和CD97在癌症进展和转移中的作用研究。凯西·凯利的年报中描述了该项目的细节。CD97是一种黏附G类蛋白偶联受体，表达于炎症细胞和多种肿瘤细胞上。CD97在甲状腺、食道、胃、结直肠和前列腺肿瘤中的表达随着恶性程度的增加而增加。我们已经证明，CD97既可以作为肿瘤细胞上的细胞自主受体，也可以作为内皮细胞上整合素α5β1和αvβ3的配体。最近我们发现，作为受体，CD97信号通过G-α12/13异源三聚体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，内源性表达的CD97的异常过表达似乎导致了非配体依赖的信号转导。前列腺癌细胞内源性CD97缺失可减少骨转移。目前，我正在研究CD97作为一个进展因子在人类异种移植和小鼠癌症模型系统中的作用。

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. 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. A Staff Scientist, postdoctoral fellows, and post baccalaureate fellows in Dr. Niederhubers laboratory are using confocal microscopy to study the inhibition of hypoxia-inducible factor prolyl hydroxylase, PHD2 and activation of hypoxic response by MAGE11. Scientists from six 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. 2.Lab of Human Carcinogenesis: Effect of purine-scaffold HSP90 inhibitor on migration, invasion, and morphology of ovarian cancer cells 3.Lab of Cancer Biology and Genetics: Study of nucleoside analogs used in AIDS therapy. 4.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. 5.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. Recently we have shown 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. Currently, I am investigating the role of CD97, as a progression factor in human xenograft and mouse model systems of cancer.