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（由申请人提供）：凋亡是一种涉及100多种基因产物的进化保守细胞死亡过程。为了响应细胞应激或维持组织稳态，凋亡机制启动并进行了一系列生化事件，导致在没有坏死的炎症特征的情况下导致细胞死亡。凋亡对于去除受损或危险的细胞至关重要，凋亡缺陷既有助于肿瘤发生和抗抗癌化学治疗方案。凋亡对化学疗法的凋亡反应的复杂性以及凋亡之间的功能性串扰和自噬的细胞存活过程在我们理解细胞对化学疗法的耐药性方面构成了重大挑战。为了帮助表征对不同类别的化学治疗剂的细胞反应，尤其是在凋亡缺陷的肿瘤细胞中，我们建议开发一组同基因的人类细胞系，作为用于表征与化学疗法抗性抗凋亡基因的发现工具。在此阶段I的可行性项目中，我们将准备并表征shRNA表达慢病毒的shRNA，该植物病毒特异性针对六种人蛋白，这些蛋白是外在或内在凋亡途径中的关键淋巴结（DR4，Caspase-8，PUMA，BAX，BAX，CASPASE，CASPASE-9和CASPASE-3）。这些慢病毒将用于在神经胶质瘤细胞系LN428和结肠癌细胞系HCT-116中使用特定基因敲低的稳定细胞系的发展，然后对每个敲低细胞和单细胞克隆的mRNA表达（QRT-PCR）表征。这将与分析相结合，以通过蛋白质表达丧失，多个凋亡和自噬终点的功能分析以及对凋亡诱导剂的选择性反应（Temozolomide，Camptothecin，Staurosporine和Sulindac）的选择性反应。 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.这些细胞将充当鉴定化学耐药性关键凋亡靶标的有价值的工具，并发现旨在克服凋亡中基因特异性缺陷的药物的发现。此外，这些新型细胞系设计为使用基于细胞和异种移植模型的高通量药物测试或分析。该提案第二阶段的主题是针对另外100个编码凋亡蛋白的100个基因的特异性人类细胞的开发。 公共卫生相关性：我们将异源性人类细胞系的创建描述为鉴定化学上关键凋亡靶标的发现工具，并发现旨在克服凋亡中基因特异性缺陷的药物的发现。在此I阶段项目中，我们将通过开发六种人蛋白质之一的一种在功能上缺陷的等源性LN428和HCT-116细胞系来证明这种方法的可行性，这是外部或内在凋亡途径中的关键节点之一。最后，这些细胞系将通过将荧光标记物共表达为效用的荧光标记，以作为发现旨在使用基于细胞和异种移植模型的高通量药物测试或分析的有价值的工具。