Investigating Regulatory Networks that Control Cell Proliferation and Apoptosis

研究控制细胞增殖和凋亡的调控网络

• 批准号: 8552594
• 负责人: KURT KOHN
• 金额: $ 73.21万
• 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/8552594
• 关键词: Affect; Apoptosis; Binding; Bioinformatics; Biological; Biological Assay; Breast Cancer Cell; Cancer cell line; Categories; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Death; Cell Death Signaling Process; Cell Line; Cell Proliferation; Cell division; Cells; Cellular biology; Characteristics; Chemotherapy-Oncologic Procedure; Code; Colon; Communities; Computer software; Consensus; Cytotoxic agent; DNA; DNA Damage; DNA Repair; DNA biosynthesis; Data; Data Set; Databases; Development; Electronics; Engineering; Enterobacteria phage MS2; Epithelial; Epithelial Cells; Event; Exhibits; Extensible Markup Language; Extracellular Matrix; Fluorescence; Gene Cluster; Gene Expression; Gene Expression Profile; Gene Proteins; Genes; Genomics; Glioblastoma; Growth Factor Receptors; Human; Indium; Individual; Infiltration; Intervention; Investigation; Ionizing radiation; Laboratories; Lead; Learning; Life; Link; Literature; Lymphoid Cell; MDM2 gene; MS2 coat protein; Malignant Neoplasms; Maps; Measures; Messenger RNA; Methods; Mitosis; Molecular; Molecular Diagnosis; Molecular Profiling; Neoplasm Metastasis; Noise; Normal Cell; Online Systems; Ontology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Plug-in; Post-Translational Protein Processing; Procedures; Process; Production; Protein Binding; Protein p53; Publishing; Radiation; Radiation Induced DNA Damage; Regulation; Research; Research Project Grants; Role; Sampling; Signal Transduction; Simulate; Software Tools; Structure; Surveys; System; Systems Biology; TP53 gene; Tertiary Protein Structure; Testing; The Cancer Genome Atlas; Tight Junctions; Time; Tissue Sample; Transcript; Tumor Cell Line; Tumor Tissue; Validation; Work; adduct; base; cancer cell; cell killing; cell motility; cell type; data mining; design; gene function; grasp; inhibitor/antagonist; killings; melanoma; microbial alkaline proteinase inhibitor; neoplastic cell; network models; novel strategies; overexpression; prevent; repaired; research study; response; selective expression; simulation; software development; stem; therapy design; tool; tumor

A. Using MIMs in coordination with gene expression datasets to unravel cell regulatory networks.This project is based on the premise that genes that are co-regulated in a variety of cell types are likely to be functionally related. We developed that concept utilizing data mining software developed in our Laboratory (?CellMiner?) to analyze gene expression data in the NCI-60 human tumor cell lines.We have also now published the CellMiner software tools and made them generally available:Reinhold, W.C., Sunshine, M., Liu, H., Varma, S., Kohn, K.W., Morris, J., Doroshow, J., and Pommier, Y. (2012). CellMiner: A Web-Based Suite of Genomic and Pharmacologic Tools to Explore Transcript and Drug Patterns in the NCI-60 Cell Line Set. Cancer Res 72, 3499-3511.Utilizing those facilities, we investigated control networks involved in cell migration/invasion and molecular interactions specific to cells having epithelial character, as follows.A1. Molecular interaction network model of cell migration/invasion processes.We began with a set of 70 genes derived by a new clustering procedure designed by our collaborator, Barry Zeeberg (see reference below) that links expression in the NCI-60 panel of human tumor cells with functions assigned by the Gene Ontology (GO) database.Zeeberg, B.R., Reinhold, W., Snajder, R., Thallinger, G.G., Weinstein, J.N., Kohn, K.W., and Pommier, Y. (2012). Functional categories associated with clusters of genes that are co-expressed across the NCI-60 cancer cell lines. PLoS One 7, e30317.After adding other NCI-60 expression-correlated genes to one of Zeeberg?s gene clusers, we derived a set of 32 genes whose expressions were mutually correlated with each other. We found that most of those genes had functions related to interactions with extracellular matrix. Utilizing molecular interaction maps (MIMs), we published a coherent picture of this regulatory network: Kohn, K.W., Zeeberg, B.R., Reinhold, W.C., Sunshine, M., Luna, A., and Pommier, Y. (2012). Gene expression profiles of the NCI-60 human tumor cell lines define molecular interaction networks governing cell migration processes. PLoS One 7, e35716.A2. Epithelial gene expression signatures from the NCI-60 human tumor cell lines.In a new approach to the characterization of cells having epithelial character, we focused on the expression of genes for the proteins involved in tight-junctions, structures that are required to maintain polarity of epithelial cells. From our NCI-60 gene expression data, we identified a subset of 12 cell lines whose selective expression of a given gene serves as a consensus pattern or signature for that gene having specific functions in tumor cells of epithelial character. We collected a 75 genes that most closely fit that consensus pattern and investigated their known functions from evidence in the scientific literature. Most of the genes had known epithelial functions, although many did not, usually due to very little published information about them. Since the latter genes had expression patterns in the NCI-60 that closely fit the epithelial consensus, their further investigation from that point of view is likely to be fruitful. We are preparing molecular interaction maps to provide an integrated view of the functional features characteristic of epithelial-type cancer cells. This will help to distinguish cancer cell types amenable to different therapies.B. Enhancing the utility of our Molecular Interaction Map (MIM) notation. In order to make the MIM notation readily accessible to the scientific community and make it easier to learn, as well as to provide tools for developers of MIM applications, we have developed the following software:B1. PathVisio-MIMWe developed and published a plug-in to PathVisio for creating and editing MIMs and we now use this tool for creating and editing MIMs:Luna, A., Sunshine, M.L., van Iersel, M.P., Aladjem, M.I., and Kohn, K.W. (2011). PathVisio-MIM: PathVisio plugin for creating and editing Molecular Interaction Maps (MIMs). Bioinformatics 27, 2165-2166. B2. Tools for MIM developersIn order to provide tools for MIM software development based on our MIM notation rules (Kohn et al., Mol. Biol. Cell 17: 1-13, 2006), we published the following Research Article:Luna, A., Karac, E.I., Sunshine, M., Chang, L., Nussinov, R., Aladjem, M.I., and Kohn, K.W. (2011). A formal MIM specification and tools for the common exchange of MIM diagrams: an XML-Based format, an API, and a validation method. BMC Bioinformatics 12:167.B3. PathVisio ValidatorWe collaborated with developers of PathVisio and SBGN (Systems Biology Graphics Notation) to develop a validation framework in PathVisio that can be used with rule sets for MIM or other diagram notations, and we published the following Systems Biology note:Chandan, K., van Iersel, M.P., Aladjem, M.I., Kohn, K.W., and Luna, A. (2012). PathVisio-Validator: a rule-based validation plugin for graphical pathway notations. Bioinformatics 28, 889-890.The Validator will help users check whether their diagrams conform to a standard rule set, such as for MIM, so as to make the diagrams unambiguous and coded in a manner suitable for exchange with other users. C. Functions of MdmX in Fine-Tuning the Response of p53 to DNA DamageThe regulation of the response of p53 to DNA damage is key to the ability of cancer cells to survive following treatment with cytotoxic drugs or radiation. In cells having the normal functioning gene, p53 induced by such treatments promotes the cell?s DNA repair capabilities and causes cell cycle delays to allow more time for repair to take place before the cell begins mitosis, the critical cell division event. The p53 induction however must be tightly controlled to avoid overshooting p53 production, which can cause cells to die by apoptosis. Understanding how this control works is important for designing therapy to protect normal cells while selectively killing cancer cells. We think that one mechanism for this control involves the gene MdmX, also known as MDM4. MdmX interacts with and alters the activites of both p53 and MDM2, the major inhibitor and destroyer of p53. Therefore we are investigating the role of MdmX in the p53 response to DNA damage.Based on a molecular interaction map (MIM) of the p53-Mdm2-MdmX control network, we simulated a network model and surveyed parameter space to disclose regions exhibiting different types of p53 responses:Kim S, Aladjem MI, McFadden GB, Kohn KW. Predicted functions of MdmX in fine-tuning the response of p53 to DNA damage. PLoS Comput Biol. 6(2):e1000665, 2010.A major finding was that MdmX/MDM4 can dampen the p53 oscillations that occur after DNA damage. That action may prevent high transient p53 activity at could lead to inappropriate cell death. We have now engineered cell lines to test this hypothesis at the single-cell level and more generally to investigate the role of MdmX in the response of p53 to ionizing radiation-induced DNA damage. We measure p53 gene activity by assay of the mRNA in living cells using a bacteriophage MS2 system. The induced p53 gene was engineered to have an added sequence of several MS2 coat protein-binding stem-loops in the mRNA. The MS2 coat protein binding to this mRNA is measure by fluorescence of a GFP adduct on the transduced MS2 coat protein. We have successfully implemented this state-of-the-art system and are now working to optimize the signal-to-noise ratio.

A.使用MIMs与基因表达数据集协调来揭示细胞调控网络。这个项目是基于一个前提，即在多种细胞类型中共同调节的基因可能是功能相关的。我们利用我们实验室开发的数据挖掘软件（CellMiner）来分析NCI-60人类肿瘤细胞系的基因表达数据。我们现在还发布了CellMiner软件工具，并使它们普遍可用：Reinhold， w.c., Sunshine， M., Liu， H., Varma， S., Kohn, k.w., Morris， J., Doroshow， J.和Pommier， Y.（2012）。CellMiner：一套基于网络的基因组学和药理学工具，用于探索NCI-60细胞系的转录和药物模式。巨蟹座Res 72, 3499-3511。利用这些设施，我们研究了涉及细胞迁移/侵袭和具有上皮特征的细胞特异性分子相互作用的控制网络。细胞迁移/侵袭过程的分子相互作用网络模型。我们从一组70个基因开始，这些基因是由我们的合作者Barry Zeeberg（见下文）设计的一种新的聚类程序衍生的，该程序将NCI-60人类肿瘤细胞小组中的表达与基因本体（GO）数据库指定的功能联系起来。Zeeberg, b.r., Reinhold， W., Snajder， R., Thallinger, g.g., Weinstein, j.n., Kohn, k.w.和Pommier， Y.（2012）。与NCI-60癌细胞系共表达的基因簇相关的功能类别。《科学通报》7，e30317。将其他NCI-60表达相关基因加入Zeeberg？S基因簇，我们得到了一组32个基因，它们的表达是相互相关的。我们发现这些基因中的大多数具有与细胞外基质相互作用相关的功能。利用分子相互作用图（MIMs），我们发表了这一调控网络的连贯图片：Kohn， k.w., Zeeberg, b.r., Reinhold， W.C, Sunshine， M., Luna, a .和Pommier， Y.（2012）。NCI-60人类肿瘤细胞系的基因表达谱定义了控制细胞迁移过程的分子相互作用网络。科学通报，7(5):356 - 356。NCI-60人肿瘤细胞系上皮基因表达特征。在一种表征具有上皮细胞特征的新方法中，我们关注了与紧密连接有关的蛋白质基因的表达，紧密连接是维持上皮细胞极性所需的结构。从我们的NCI-60基因表达数据中，我们确定了12个细胞系的一个子集，其特定基因的选择性表达作为该基因在上皮性肿瘤细胞中具有特定功能的共识模式或标志。我们收集了75个最符合这种共识模式的基因，并从科学文献的证据中调查了它们已知的功能。大多数基因具有已知的上皮功能，尽管许多基因不具有，通常是因为关于它们的公开信息很少。由于后一种基因在NCI-60中的表达模式与上皮一致，因此从这一观点出发，他们的进一步研究可能会取得丰硕成果。我们正在准备分子相互作用图，以提供上皮型癌细胞功能特征的综合视图。这将有助于区分适合不同治疗方法的癌细胞类型。增强分子相互作用图（MIM）表示法的实用性。为了使MIM表示法易于科学界使用和学习，并为MIM应用程序的开发人员提供工具，我们开发了以下软件：B1。PathVisio- mimi我们开发并发布了一个用于创建和编辑mimi的插件，我们现在使用这个工具来创建和编辑mimi:Luna， a ., Sunshine, m.l., van Iersel， M.P, Aladjem， M.I和Kohn， K.W.（2011）。用于创建和编辑分子相互作用图（MIMs）的PathVisio插件。生物信息学27,2165-2166。B2。为了提供基于MIM符号规则的MIM软件开发工具（Kohn et al., Mol. Biol.）。Cell 17: 1-13, 2006)，我们发表了以下研究文章：Luna， A., Karac, e ., Sunshine， M., Chang， L., Nussinov， R., Aladjem， M.，和Kohn， K.W.（2011）。用于通用交换MIM图的正式MIM规范和工具：基于xml的格式、API和验证方法。生物信息学[c]: 17 - 17。我们与PathVisio和SBGN（系统生物学图形符号）的开发人员合作，在PathVisio中开发了一个验证框架，可以与MIM或其他图表符号的规则集一起使用，并且我们发布了以下系统生物学注释：Chandan， K., van Iersel, m.p., Aladjem, m.i., Kohn， K.W.和Luna， a .（2012）。PathVisio-Validator：一个基于规则的图形路径符号验证插件。生物信息学28,889-890。Validator将帮助用户检查他们的图是否符合标准规则集，例如对于MIM，以便使图明确并以适合与其他用户交换的方式进行编码。C. MdmX在调控p53对DNA损伤的反应中的作用调控p53对DNA损伤的反应是细胞毒性药物或放射治疗后癌细胞存活能力的关键。在具有正常功能基因的细胞中，这种治疗方法诱导的p53会促进细胞凋亡。在细胞开始有丝分裂（关键的细胞分裂事件）之前，它会导致细胞周期延迟，以便有更多的时间进行修复。然而，p53的诱导必须严格控制，以避免过度产生p53，这可能导致细胞凋亡死亡。了解这种控制是如何起作用的，这对于设计保护正常细胞同时选择性杀死癌细胞的治疗方法非常重要。我们认为这种控制的一种机制涉及MdmX基因，也被称为MDM4。MdmX与p53和MDM2相互作用并改变其活性，MDM2是p53的主要抑制剂和破坏者。因此，我们正在研究MdmX在p53对DNA损伤的反应中的作用。基于p53- mdm2 -MdmX控制网络的分子相互作用图（MIM），我们模拟了一个网络模型，并调查了参数空间，揭示了表现出不同类型p53反应的区域：Kim S， Aladjem MI, McFadden GB, Kohn KW.预测了MdmX在微调p53对DNA损伤反应中的功能。计算机工程学报，6(2):663 - 668,2010。一个重要的发现是MdmX/MDM4可以抑制DNA损伤后发生的p53振荡。这一作用可能会阻止可能导致不适当细胞死亡的p53高瞬时活性。我们现在已经设计了细胞系，在单细胞水平上测试这一假设，并更广泛地研究MdmX在p53对电离辐射诱导的DNA损伤的反应中的作用。我们通过使用噬菌体MS2系统测定活细胞中的mRNA来测量p53基因的活性。诱导的p53基因在mRNA中增加了几个MS2外壳蛋白结合茎环的序列。通过在转导的MS2外壳蛋白上的GFP加合物的荧光测量与该mRNA结合的MS2外壳蛋白。我们已经成功地实现了这个最先进的系统，现在正在努力优化信噪比。