DNA repair deficient cells for analysis

用于分析的 DNA 修复缺陷细胞

基本信息

批准号：
8142928
负责人：
Jay George
金额：
$ 121.43万
依托单位：
TREVIGEN, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8142928
关键词：
Address Aging BRCA1 gene Base Excision Repairs Biological Markers Breast Cancer Cell Cancer cell line Cell Line Cell physiology Cells Chromatin Structure Clone Cells DNA DNA Damage DNA Repair DNA Repair Gene DNA Repair Pathway DNA crosslink DNA glycosylase DNA repair protein DNA-Directed DNA Polymerase Defect Development Disease Effectiveness Enzymes Epigenetic Process Etiology Excision Repair Fanconi&apos s Anemia Gene Expression Gene Expression Profile Gene Targeting Generations Genes Genome Genomic Instability Genotoxic Stress Glioma Human Human Cell Line Lentivirus Vector Maintenance Malignant Neoplasms Marketing Mediating Messenger RNA Methylation Molecular Molecular Profiling Mutagens Nonhomologous DNA End Joining Nucleotide Excision Repair Nucleotides Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Phase Phenotype Preparation Procedures Process Proteins Protocols documentation Radiation Reagent Regimen Research Sales Scientist Seeds Specificity Stress Subfamily lentivirinae Validation Work base cancer cell cell bank commercialization drug discovery expectation gene discovery gene repair homologous recombination human DNA improved inhibitor/antagonist mRNA Expression neoplastic cell novel novel strategies nuclease oncology protein expression public health relevance repaired response scale up small hairpin RNA stable cell line tool tumor

项目摘要

DESCRIPTION (provided by applicant): Successful completion of Phase I led to the development a panel of human cell lines, each deficient in one of the eleven DNA glycosylase enzymes. Depletion of target mRNA was as high as 95%, with corresponding depletion of target protein levels and enzymatic activity. To expand background diversity, the same shRNA lentiviruses were also used to develop parallel cell line panels in diferent tumor backgrounds, including glioma and breast cancer cell lines, demonstrating similar target mRNA depletion across different tumor cell backgrounds. Gene expression knockdown of the DNA glycosylases exemplify the impact of DNA repair defects on the human transcriptome. As an example of the far reaching potential for a panel of DNA repair deficient cell lines, we show that DNA glycosylase deficiency modulated both the transcriptome and epigenome, implicating some DNA glycoylases in methylation maintenance and genome expression diversity. Further, by combining both DNA glycosylase and BRCA1 knockdown, we have begun to investigate the requirement for DNA glycosylases in the effectiveness of PARP inhibitors in a BRCA1 knockdown tumor line. Phase II of the project wil utilize the successful work-flow paradigm optimized in Phase I for the development, functional characterization, cell banking and transcriptome analysis of isogenic human cel lines deficient in all known DNA repair genes. These include genes involved in Base Excision Repair, Direct Reversal of Damage, Mismatch Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Non- homologous End-Joining, the modulation of nucleotide pools, DNA polymerases, editing and processing nucleases, the Rad6 pathway, Chromatin Structure, DNA Repair genes defective in diseases and conserved DNA Damage Response genes. The studies described in Aim 1 involve the preparation of the shRNA expressing lentiviruses, transduction and generation of three different human tumor cell knockdown panels for all known DNA repair genes (>150), followed by the mRNA expression characterization (qRT-PCR) of the knockdown cell lines and optimized scale-up and step-wise characterization to prepare for cell line distribution (Cell Banking). In aim 2, the cell lines will be validated for the expected DNA repair functional deficiency by protein expression profiling and genotoxin challenge. Finally (Aim 3), whole-genome transcriptional profiles will be conducted to quantitate transcriptional reprogramming mediated by changes in endogenous DNA repair capacity and where appropriate, following specific genotoxic stress. With the expectation that DNA repair capacity impacts basic cellular functions both spontaneously and in response to genotoxic stress, alters the transcriptional and epigenetic landscape and dictates the cellular response to stress, the development of a complete panel of isogenic DNA repair deficient cell lines across multiple backgrounds will be a valuable platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations. The ready availability of this panel of cell lines will permit both academic and pharmaceutical scientists to study the molecular etiology of tumor genomic instability and to exploit it in oncology research. We envision robust market demand for the cell lines and information that relates to the global transcriptome. PUBLIC HEALTH RELEVANCE: In this Phase II proposal we plan to utilize the successful work-flow paradigm optimized in Phase I for the cell-line development and transcriptome analysis of isogenic human cells lines deficient in all known DNA repair genes. These highly characterized and annotated isogenic cell lines will form the basis for a platform for gene and drug discovery, validation of inhibitor specificity and the identification of response biomarkers and novel targets for gene/drug synthetic-lethality combinations.

描述（由申请人提供）：第一阶段的成功完成导致了一组人类细胞系的开发，每个细胞系都缺乏十一种 DNA 糖基化酶中的一种。目标 mRNA 的去除率高达 95%，目标蛋白水平和酶活性也相应降低。为了扩大背景多样性，还使用相同的 shRNA 慢病毒在不同肿瘤背景（包括神经胶质瘤和乳腺癌细胞系）中开发平行细胞系组，证明不同肿瘤细胞背景中相似的靶标 mRNA 缺失。 DNA 糖基化酶的基因表达敲低例证了 DNA 修复缺陷对人类转录组的影响。作为一组 DNA 修复缺陷细胞系的深远潜力的一个例子，我们表明 DNA 糖基化酶缺陷调节转录组和表观基因组，暗示一些 DNA 糖基化酶参与甲基化维持和基因组表达多样性。此外，通过结合 DNA 糖基化酶和 BRCA1 敲低，我们已开始研究在 BRCA1 敲低肿瘤系中 PARP 抑制剂的有效性对 DNA 糖基化酶的需求。该项目的第二阶段将利用第一阶段优化的成功工作流程范式，对缺乏所有已知 DNA 修复基因的同基因人类细胞系进行开发、功能表征、细胞库和转录组分析。这些包括参与碱基切除修复、直接逆转损伤、错配切除修复、核苷酸切除修复、同源重组、非同源末端连接、核苷酸池调节、DNA聚合酶、编辑和加工核酸酶、Rad6途径、染色质结构、疾病中缺陷的 DNA 修复基因和保守的 DNA 损伤反应基因。目标 1 中描述的研究涉及制备表达 shRNA 的慢病毒、转导和生成针对所有已知 DNA 修复基因 (>150) 的三种不同的人类肿瘤细胞敲除组，然后进行 mRNA 表达表征 (qRT-PCR)。敲低细胞系并优化放大和逐步表征，为细胞系分布做好准备（细胞库）。在目标 2 中，将通过蛋白质表达谱和基因毒素挑战来验证细胞系是否存在预期的 DNA 修复功能缺陷。最后（目标 3），将进行全基因组转录谱来定量由内源 DNA 修复能力的变化介导的转录重编程，并在适当的情况下，在特定的基因毒性应激后进行。预期 DNA 修复能力会自发地和响应基因毒性应激影响基本细胞功能，改变转录和表观遗传景观并决定细胞对应激的反应，开发了跨多个背景的完整的同基因 DNA 修复缺陷细胞系组将成为基因和药物发现、抑制剂特异性验证以及反应生物标志物和基因/药物合成致死组合的新靶点识别的有价值的平台。这组细胞系的现成可用将使学术和药物科学家能够研究肿瘤基因组不稳定性的分子病因学，并在肿瘤学研究中利用它。我们预计市场对细胞系和与全球转录组相关的信息有强劲的需求。公共健康相关性：在第二阶段提案中，我们计划利用第一阶段优化的成功工作流程范式，对缺乏所有已知 DNA 修复基因的同基因人类细胞系进行细胞系开发和转录组分析。这些高度表征和注释的同基因细胞系将构成基因和药物发现、抑制剂特异性验证以及反应生物标志物和基因/药物合成致死组合的新靶标的识别平台的基础。