Discovery Tools for Chemotherapy Resistance to Cell Death.

发现化疗抵抗细胞死亡的工具。

• 批准号: 8201177
• 负责人: Jay George
• 金额: $ 21.69万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-15 至 2013-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201177
• 关键词: Address; Antineoplastic Agents; Apoptosis; Apoptotic; Autophagocytosis; BAX gene; Biochemical; Camptothecin; Cancer cell line; Cell Death; Cell Death Process; Cell Line; Cell Separation; Cell Survival; Cells; Cellular Stress; Characteristics; Clone Cells; Code; Communities; Coupled; Defect; Development; Epigenetic Process; Evaluation; Event; Gene Mutation; Gene Proteins; Gene Targeting; Genes; Genetic; Genotoxic Stress; Growth; Growth Factor; HCT116 Cells; Homeostasis; Human; Human Cell Line; Image; Image Analysis; Inflammation; Lentivirus Vector; Libraries; Life; Link; Luciferases; Maintenance; Malignant Neoplasms; Messenger RNA; Necrosis; Neoplastic Cell Transformation; Oncogenic; Pathway interactions; Pharmacologic Substance; Phase; Positioning Attribute; Process; Proteins; Protocols documentation; Regimen; Regulation; Reporter; Research; Research Personnel; Resistance; Role; Series; Signal Transduction; Staurosporine; Stress; Subfamily lentivirinae; Sulindac; TNF gene; TNFRSF10A gene; TNFSF10 gene; Therapeutic; Time; Tissues; Tumor Angiogenesis; Tumor Cell Line; Validation; Withdrawal; Xenograft Model; Xenograft procedure; base; cancer cell; caspase-3; caspase-8; caspase-9; cell bank; cellular imaging; chemotherapeutic agent; chemotherapy; colon cancer cell line; cytokine; design; drug testing; functional loss; glioma cell line; mRNA Expression; neoplastic cell; novel; protein expression; response; small hairpin RNA; stable cell line; suicidal; temozolomide; tool; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): Apoptosis is an evolutionarily conserved cell death process that involves over 100 gene products. In response to cellular stress or to maintain tissue homeostasis, the apoptotic machinery initiates and carries out a series of biochemical events leading to cell death in the absence of inflammation characteristic of necrosis. Apoptosis is essential to remove damaged or dangerous cells, and defects in apoptosis contribute both to tumorigenesis and resistance to anti-cancer chemotherapeutic regimens. The complexity of the apoptotic response to chemotherapy coupled with functional crosstalk between apoptosis and the cell survival process of autophagy presents a significant challenge in our understanding of the cellular resistance to chemotherapy. To help characterize the cellular response to different classes of chemotherapeutic agents, particularly in tumor cells with defects in apoptosis, we propose to develop a set of isogenic human cell lines as discovery tools for characterizing the apoptosis genes involved in chemotherapy resistance. In this Phase I feasibility project, we will prepare and characterize shRNA expressing lentiviruses specific for six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways (DR4, Caspase-8, PUMA, BAX, Caspase-9 and Caspase-3). These lentiviruses will be used for the development of stable cell lines with specific gene knockdown in both the glioma cell line LN428 and the colon cancer cell line HCT-116, followed by mRNA expression (qRT-PCR) characterization of each of the knockdown cells and single-cell clones. This will be coupled with analysis to validate apoptosis deficiency via protein expression loss, functional analysis of multiple apoptotic and autophagy endpoints and selective response to apoptosis inducing agents (Temozolomide, Camptothecin, staurosporine and Sulindac). The optimum shRNA for each will then inform for the development of cell lines with the specific gene knockdown together with (i) a far-red fluorescent reporter (FP635) for selection, (ii) a luciferase reporter amenable to real-time imaging of apoptosis and (iii) expression of LC3-EGFP for a direct analysis of autophagy induction, linked via T2A sequences in a single gene cassette. These cells will function as valuable tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In addition, these novel cell lines are designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models. The development of such isogenic human cells specific for an additional 100 genes coding for apoptosis proteins will be the topic of the second phase of this proposal. PUBLIC HEALTH RELEVANCE: We describe the creation of isogenic human cell lines as discovery tools for the identification of key apoptotic targets in chemoresistance and the discovery of agents designed to overcome gene-specific defects in apoptosis. In this Phase I project, we will demonstrate the feasibility of this approach by developing isogenic LN428 and HCT-116 cell lines functionally deficient in one of six human proteins that are key nodes in either the extrinsic or intrinsic apoptotic pathways. Finally, these cell lines will be modified by co-expression of fluorescent markers for utility as valuable tools for discovery of agents designed to be amenable to high-throughput drug testing or analysis using cell-based and xenograft models.

描述(申请人提供)：细胞凋亡是一种进化保守的细胞死亡过程，涉及100多种基因产物。为了响应细胞应激或维持组织内环境的稳定，细胞凋亡机制启动并执行一系列生化事件，导致细胞死亡，而不是以坏死为特征的炎症。细胞凋亡对于清除受损或危险的细胞是必不可少的，而细胞凋亡的缺陷导致肿瘤的发生和对抗癌化疗方案的抵抗。化疗后细胞凋亡反应的复杂性，再加上细胞凋亡和自噬的细胞存活过程之间的功能串扰，给我们理解细胞对化疗的耐药性带来了巨大的挑战。为了帮助描述细胞对不同类型化疗药物的反应，特别是在具有凋亡缺陷的肿瘤细胞中，我们建议开发一组等基因的人类细胞系作为发现工具来表征与化疗耐药有关的凋亡基因。在这个第一阶段的可行性项目中，我们将制备和鉴定针对六种人类蛋白(DR4、Caspase-8、PUMA、Bax、Caspase-9和Caspase-3)的表达shRNA的慢病毒。这些慢病毒将用于发展胶质瘤细胞系LN428和结肠癌细胞系HCT-116中具有特定基因敲除的稳定细胞系，随后将对每个敲除细胞和单细胞克隆进行mRNA表达(qRT-PCR)鉴定。这将与通过蛋白质表达缺失、多个凋亡和自噬终点的功能分析以及对凋亡诱导剂(替莫唑胺、喜树碱、星形孢子素和舒林酸)的选择性反应来验证凋亡缺陷的分析相结合。然后，每一个的最佳shRNA将与(I)用于选择的远红荧光报告(FP635)，(Ii)能够实时成像凋亡的荧光素酶报告，以及(Iii)用于直接分析自噬诱导的LC3-EGFP的表达一起，为具有特定基因敲除的细胞系的发展提供信息，通过单基因盒中的T2a序列连接。这些细胞将作为有价值的工具，用于识别化疗耐药中的关键凋亡靶点，并发现旨在克服细胞凋亡中基因特异性缺陷的药物。此外，这些新的细胞系被设计成能够使用基于细胞的模型和异种移植模型进行高通量药物测试或分析。开发这种针对另外100个编码凋亡蛋白的基因的同源人类细胞将是这项提议的第二阶段的主题。 公共卫生相关性：我们描述了创建等基因人类细胞系作为发现工具，以识别化疗耐药中的关键凋亡靶点，并发现旨在克服细胞凋亡中基因特异性缺陷的药物。在这个第一阶段的项目中，我们将通过建立等基因的LN428和HCT-116细胞株来证明这种方法的可行性，这些细胞系在六种人类蛋白质中的一种存在功能缺陷，这六种蛋白质是外在或内在凋亡途径中的关键节点。最后，这些细胞系将通过共表达荧光标记进行修饰，作为有用的工具，用于发现可用于高通量药物测试或使用基于细胞和异种移植模型的分析的试剂。