Cell and Cancer Biology Branch Confocal Microscopy Core

核心设施包括安装在Axio Observer Z1电动荧光显微镜上的Zeiss LSM 510 Meta扫描模块，配备高分辨率镜头和Nomarski DIC光学器件的直立Axioplan 2荧光显微镜，以及配备axiovision软件用于图像采集的Axio Observer Z1荧光显微镜，计算机和监视器。此外，该设施还配备了VivaView荧光培养箱显微镜，能够对培养中生长数天或数周的细胞进行成像。来自NCI 15个不同实验室（约40名用户）的研究人员目前正在使用共聚焦核心设施。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，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支（CCBB），21名科学家，包括工作人员的科学家，博士后研究员，学士后研究员和暑期学生经常使用的核心设施在他们的研究工作。刘正刚博士实验室的博士后研究员正在利用细胞核DNA显微注射技术研究死亡结构域蛋白在细胞凋亡中的作用。共聚焦显微镜正在被这个小组用来研究内质网应激的分子机制和参与凋亡的蛋白质的细胞定位。在Kathy Kelly博士的实验室中，三名博士后研究员和一名学士后研究员正在使用共聚焦显微镜研究分子机制，并确定原代小鼠前列腺细胞上的分化标志物和信号分子，以阐明前列腺癌转移中涉及的信号通路。他们还在二维和三维培养中表征用进展标志物和信号分子染色的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3D培养中生长的球形和管状结构中的定位至关重要。一位研究员和一位学士后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。来自其他几个分支或实验室的科学家目前正在使用核心作为其研究的一个组成部分。以下是正在进行的使用核心设施的项目的一些例子。1.癌症生物学和遗传学实验室：研究用于艾滋病治疗的核苷类似物。2.乳腺生物学和肿瘤发生：研究乳腺干细胞生态位及其将祖细胞群重编程为胚胎干细胞（ES）的能力。共聚焦显微镜被用来确认Y染色体的ES-衍生的细胞中的存在，并建立这些细胞在其当前位置的牛奶蛋白免疫组织化学功能。3.肿瘤内科分支：a. GXXXG基序在ABC半转运蛋白ABCG 2的同源二聚化中的作用，ABCG 2赋予对几种重要临床药物的抗性B.癌细胞系中p53的定位和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。这些都是与临床试验数据相关的重要研究，这些研究的信息可能对开发缓解这些化合物相关毒性的疗法很重要。除了在涉及共聚焦显微镜核心设施的项目上进行合作，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，我正在与CCBB的分支负责人Kathy Kelly合作，研究CD 97信号转导机制以及CD 97在癌症进展和转移中的作用。这个项目的细节在凯西·凯利的年度报告中有描述。CD 97是一种粘附G类蛋白偶联受体，在炎性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，CD 97表达与恶性程度平行增加。我们已经证明，CD 97作为肿瘤细胞上的细胞自主受体和作为内皮细胞上整合素α 5 β 1和α v β 3的配体。我们表明，在其作为受体的能力中，CD 97信号传导通过异源三聚体G蛋白的G-α 12/13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CD 97的异常过表达似乎导致配体非依赖性信号传导。前列腺肿瘤细胞系中内源性CD 97的耗尽导致骨转移减少。我们有两个最近的出版物证明，CD 97可能通过与溶血磷脂受体的异源二聚化在前列腺癌和甲状腺癌的进展中起着积极的作用。

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. 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.