Discovery Tools for Chemotherapy Resistance to Cell Death.

描述（由申请人提供）：细胞凋亡是一种进化上保守的细胞死亡过程，涉及超过100种基因产物。为了响应细胞应激或维持组织稳态，凋亡机制启动并进行一系列生物化学事件，导致在没有坏死的炎症特征的情况下细胞死亡。细胞凋亡是去除受损或危险细胞所必需的，并且细胞凋亡中的缺陷有助于肿瘤发生和对抗癌化疗方案的抗性。对化疗的凋亡反应的复杂性加上凋亡和自噬的细胞存活过程之间的功能串扰，在我们理解细胞对化疗的抗性方面提出了重大挑战。为了帮助表征不同类别的化疗药物的细胞反应，特别是在肿瘤细胞的凋亡缺陷，我们建议开发一套等基因的人类细胞系作为发现工具，用于表征参与化疗耐药的凋亡基因。在这个I期可行性项目中，我们将制备和表征对六种人类蛋白质具有特异性的shRNA表达慢病毒，这些蛋白质是外在或内在凋亡途径中的关键节点（DR4，Caspase-8，BAX，Caspase-9和Caspase-3）。这些慢病毒将用于开发在神经胶质瘤细胞系LN 428和结肠癌细胞系HCT-116中具有特异性基因敲低的稳定细胞系，然后对每个敲低细胞和单细胞克隆进行mRNA表达（qRT-PCR）表征。这将与分析相结合，以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、星形孢菌素和舒林酸）的选择性应答来验证凋亡缺陷。然后，每种最佳的shRNA将为开发具有特定基因敲低的细胞系提供信息，以及（i）用于选择的远红荧光报告物（FP635），（ii）适于细胞凋亡实时成像的荧光素酶报告物和（iii）用于直接分析自噬诱导的LC3-EGFP的表达，其通过单个基因盒中的T2A序列连接。这些细胞将作为有价值的工具，用于识别化疗耐药性中的关键凋亡靶点，并发现旨在克服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适合于使用基于细胞和异种移植模型的高通量药物测试或分析。开发这种对另外100个编码凋亡蛋白的基因具有特异性的同基因人类细胞将是本提案第二阶段的主题。 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，用于识别化疗耐药中的关键凋亡靶点，并发现旨在克服凋亡中基因特异性缺陷的药物。在这个I期项目中，我们将证明这种方法的可行性，通过开发功能缺陷的六种人类蛋白质之一的同基因LN 428和HCT-116细胞系，这些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物进行修饰，以用作发现被设计成适合于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的试剂的有价值的工具。

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.