Cell and Cancer Biology Branch Confocal Microscopy Core

细胞和癌症生物学分支共焦显微镜核心

• 批准号: 8554085
• 负责人: Yvona Ward
• 金额: $ 117.81万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554085
• 关键词: 3-Dimensional; ABCG2 gene; AIDS therapy; Adhesions; Adult; Annual Reports; Apoptosis; Basic Science; Biology; CCR; CD97 gene; Cancer Biology; Cancer cell line; Carcinoma; Cells; Cellular biology; Child; Clinical; Clinical Research; Clinical Trials; Collaborations; Colorectal Neoplasms; Communities; Computer software; Confocal Microscopy; Core Facility; Couples; Cytoskeleton; DNA; Data; Death Domain; Differentiation Antigens; Educational process of instructing; Embryo; Endothelial Cells; Epithelial Cells; Equipment; Esophageal Neoplasms; Family; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Goals; HIV; Heterodimerization; Heterotrimeric GTP-Binding Proteins; Homodimerization; Housing; Image; Image Analysis; Incubators; Inflammatory; Integrin alpha5; Investigation; Label; Laboratories; Lead; Ligands; Location; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Malignant neoplasm of thyroid; Mammary gland; Manuscripts; Medical Oncology; Metastatic Neoplasm to the Bone; Metastatic malignant neoplasm to brain; Microinjections; Microscope; Microtubules; Milk Proteins; Mission; Molecular; Mus; Neoplasm Metastasis; Nuclear; Optics; Ovarian; Pediatric Oncology; Platinum Compounds; Play; Population; Postdoctoral Fellow; Preparation; Prostate; Prostatic Neoplasms; Proteins; Publications; Research; Research Personnel; Resistance; Resolution; Role; Scanning; Scientist; Signal Pathway; Signal Transduction; Signaling Molecule; Staining method; Stains; Stomach Neoplasms; Structure; Students; Techniques; Thyroid Gland; Toxic effect; Tubular formation; Tubulin; Tumor Cell Line; Y Chromosome; anticancer research; cancer cell; cancer genetics; cellular imaging; chemotherapy; computer monitor; embryonic stem cell; empowered; endoplasmic reticulum stress; fluorescence microscope; gang; lens; malignant breast neoplasm; meetings; neoplastic cell; novel therapeutic intervention; nucleoside analog; overexpression; progenitor; progression marker; receptor; research study; rho; stem; stem cell niche; therapy development; thyroid neoplasm; tumor progression; tumorigenesis

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 agentsb.Localization of p53 in cancer cell lines and colocalization of p53 with tubulin and importinc.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.

核心设施配备了安装在 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 光学器件的直立式 Axioplan 2 荧光显微镜，以及配备了用于图像采集的 axiovision 软件。此外，该设施还配备一台 VivaView 荧光培养显微镜，能够对培养物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)，包括工作人员在内的 21 名科学家科学家、博士后研究员、学士学位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研究员正在使用 DNA 核显微注射来研究死亡结构域蛋白在细胞凋亡中的作用。该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 Kathy Kelly 博士的实验室中，三名博士后研究员和一名学士学位后研究员正在使用共聚焦显微镜研究分子机制并识别原代小鼠前列腺细胞上的分化标记和信号分子，以阐明与前列腺癌转移有关的信号通路。他们还对 2 维和 3 维培养物中用进展标记物和信号分子染色的正常和转化的前列腺上皮细胞进行了表征。共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。一名研究员和一名学士学位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该核心作为他们研究的一个组成部分。以下是使用核心设施正在进行的项目的一些示例。 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新编程为胚胎干（ES）细胞的能力。共聚焦显微镜用于确认 ES 衍生细胞中 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是否具有功能。 3. 医学肿瘤学分支： a. GXXXG 的作用ABC 半转运蛋白 ABCG2 的同二聚化中的基序，赋予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 与微管蛋白和重要的是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减轻这些化合物相关毒性的疗法非常重要。除了在涉及共焦显微镜核心设施的项目上进行合作外，还为科学家们提供教学在共聚焦显微镜和显微注射的各个方面，以及维护核心设备的同时，我正在与CCBB分会主任Kathy合作研究CD97信号转导机制以及CD97在癌症进展和转移中的作用凯莉.凯西·凯利的年度报告中描述了该项目的详细信息。 CD97 是一种粘附类 G 蛋白偶联受体，在炎症细胞和多种癌症上表达。在甲状腺、食管、胃、结直肠和前列腺肿瘤中，CD97 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 α5 beta1 和 alphav beta3 的配体。我们证明，作为受体，CD97 信号传导通过异三聚体 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，内源表达 CD97 的异常过度表达似乎会导致配体依赖性信号传导。前列腺肿瘤细胞系中内源性 CD97 的消耗导致骨转移减少。我们最近发表的两篇文章证明 CD97 可能通过与溶血磷脂受体的异二聚化在前列腺癌和甲状腺癌进展中发挥积极作用。