The impact of malignant transformation on DNA double-strand break repair.

恶性转化对DNA双链断裂修复的影响。

• 批准号: 8938189
• 负责人: Philipp Oberdoerffer
• 金额: $ 9.78万
• 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/8938189
• 关键词: 4-Hydroxy-Tamoxifen; Accounting; Acetyl Coenzyme A; Address; Affect; Breast Epithelial Cells; Cell Line; Cells; Chromatin; Chromatin Structure; Chromosome abnormality; DNA; DNA Double Strand Break; DNA Repair; Defect; Detection; Development; Disease; Disease Progression; Double Strand Break Repair; Enzymes; Epigenetic Process; Estrogen Receptors; Functional disorder; Gene Expression; Genes; Genome; Genome Stability; Genomic Instability; Genomics; Glucose; Growth; Growth Factor Receptors; Histone Acetylation; Hour; In Vitro; Lesion; Ligand Binding Domain; Ligands; Light; Link; MCF10A cells; Maintenance; Malignant - descriptor; Measures; Mediating; Metabolic; Metabolism; Molecular; Molecular Profiling; Mutation; Nonhomologous DNA End Joining; Nuclear; Nutrient; Oncogene Activation; Oncogene Proteins; Oncogenes; Outcome; Pathway interactions; Phosphotransferases; Play; Production; Regulator Genes; Reporter; Repression; Role; Signal Transduction; System; Testing; Therapeutic; Transformed Cell Line; Tumor Suppressor Proteins; Warburg Effect; aerobic glycolysis; base; cancer therapy; cell growth; cofactor; endodeoxyribonuclease SceI; glucose metabolism; histone acetyltransferase; homologous recombination; metaplastic cell transformation; neoplastic cell; novel; novel strategies; prevent; repaired; response; src-Family Kinases; tumor; tumor growth; tumorigenesis; uptake; v-src Oncogenes; 4-Hydroxy-Tamoxifen; Accounting; Acetyl Coenzyme A; Address; Affect; Breast Epithelial Cells; Cell Line; Cells; Chromatin; Chromatin Structure; Chromosome abnormality; DNA; DNA Double Strand Break; DNA Repair; Defect; Detection; Development; Disease; Disease Progression; Double Strand Break Repair; Enzymes; Epigenetic Process; Estrogen Receptors; Functional disorder; Gene Expression; Genes; Genome; Genome Stability; Genomic Instability; Genomics; Glucose; Growth; Growth Factor Receptors; Histone Acetylation; Hour; In Vitro; Lesion; Ligand Binding Domain; Ligands; Light; Link; MCF10A cells; Maintenance; Malignant - descriptor; Measures; Mediating; Metabolic; Metabolism; Molecular; Molecular Profiling; Mutation; Nonhomologous DNA End Joining; Nuclear; Nutrient; Oncogene Activation; Oncogene Proteins; Oncogenes; Outcome; Pathway interactions; Phosphotransferases; Play; Production; Regulator Genes; Reporter; Repression; Role; Signal Transduction; System; Testing; Therapeutic; Transformed Cell Line; Tumor Suppressor Proteins; Warburg Effect; aerobic glycolysis; base; cancer therapy; cell growth; cofactor; endodeoxyribonuclease SceI; glucose metabolism; histone acetyltransferase; homologous recombination; metaplastic cell transformation; neoplastic cell; novel; novel strategies; prevent; repaired; response; src-Family Kinases; tumor; tumor growth; tumorigenesis; uptake; v-src Oncogenes

BACKGROUND: Malignant transformation is characterized by enhanced uptake and utilization of glucose, which results in the persistent activation of aerobic glycolysis - a phenomenon known as the Warburg effect. Accumulating evidence suggests that the metabolic changes associated with tumor development may not simply be a consequence of increased nutrient demand, but act as causal contributors to disease development or progression. Consistent with this, altered metabolite profiles have been implicated in the epigenetic control of gene expression, including activation of oncogenes and repression of tumor suppressors, which points to an important role for metabolites as modulators of chromatin integrity in tumor cells. Notably, chromatin does not only determine gene expression and epigenetic integrity of nuclear DNA, but directly contributes to the repair of genomic lesions. The link between the tumor-associated increase in glucose metabolism, thus, presents a plausible means through which malignant transformation may alter DNA double-strand break (DSB) repair and genome stability. However, how metabolic changes promote malignant transformation remains unclear. OBJECTIVE AND RESULTS: A significant fraction of tumors is characterized by the deregulation of growth and survival genes rather than apparent defects in genome maintenance genes. Nevertheless, such tumors often carry deleterious genomic mutations and/or chromosomal aberrations. To determine if and how tumorigenesis may modulate genome maintenance, we will generate a reporter system to dissect the impact of malignant transformation on the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Our experimental approach is based on a previously described cell line, where cellular transformation can be induced in vitro via the aberrant activation of v-Src kinase, an oncogene that promotes growth factor receptor kinase signaling. Specifically, normal mammary epithelial cells (MCF10A) that contain a stably integrated v-Src gene fused to the ligand-binding domain of the estrogen receptor (ER-Src) undergo v-Src-dependent transformation within 24 to 36 hours after administration of the ER ligand 4-hydroxy-tamoxifen (4-OHT). To compare DSB repair efficiency before and after cellular transformation, MCF10A-ER-Src cells will be stably transfected with reporter constructs that allow for the detection of cells that have undergone NHEJ or HR in response to I-SceI endonuclease-mediated DSB induction. Both reporters rely on DSB repair-dependent expression of GFP, and repair efficiency can, thus, be measured as the fraction of GFP+ cells following I-SceI induction. Using this experimental system we will address the following specific aims: 1.) Determine the effect of malignant transformation on DSB repair outcome, and 2.) Characterize transformation-induced metabolic and epigenetic changes in the context of DSB repair. IMPLICATIONS: This project is expected to determine the involvement of metabolites in DSB repair, which has significant implications for the therapeutic modulation of repair outcome. Changing nutrient availability, cellular metabolism and/or metabolite abundance may eventually be utilized to either promote DNA repair and, thus, prevent damage accumulation, or to impair DSB repair, thus enhancing genotoxic therapy in cancer treatments.

背景：恶性转化的特征是增强的葡萄糖吸收和利用，这导致有氧糖酵解的持续激活 - 一种称为Warburg效应的现象。积累的证据表明，与肿瘤发育相关的代谢变化可能不仅是营养需求增加的结果，而是造成疾病发展或进展的因果关系。与此相一致的，改变的代谢产物谱与基因表达的表观遗传控制有关，包括癌基因的激活和抑制肿瘤抑制剂，这表明代谢物是作为肿瘤细胞中染色质完整性调节剂的重要作用。值得注意的是，染色质不仅确定核DNA的基因表达和表观遗传完整性，而且直接有助于修复基因组病变。因此，与葡萄糖代谢相关的增加之间的联系，因此提出了一种合理的手段，恶性转化可能会改变DNA双链断裂（DSB）修复和基因组稳定性。但是，代谢变化促进恶性转化尚不清楚。目的和结果：肿瘤的很大一部分的特征是对生长和生存基因的放松管制，而不是基因组维持基因的明显缺陷。然而，这样的肿瘤经常带有有害的基因组突变和/或染色体畸变。为了确定肿瘤发生是否可以调节基因组维持，我们将生成一个报告基因系统，以剖析恶性转化对两个主要DSB修复途径，非同源端连接（NHEJ）（NHEJ）和同源重组（HR）的影响。我们的实验方法基于先前描述的细胞系，其中可以通过异常的V-SRC激酶的激活在体外诱导细胞转化，V-SRC激酶是一种促进生长因子受体激酶信号传导的癌基因。具体而言，正常的乳腺上皮细胞（MCF10A）包含稳定整合的V-SRC基因，该基因与雌激素受体（ER-SRC）的配体结合结构域（ER-SRC）在给药后24至36小时内经历V-SRC依赖性转换，后者是在给药后24至36小时内进行的。为了比较细胞转化之前和之后的DSB修复效率，MCF10A-ER-SRC细胞将通过记者构建体稳定转染，这些构建体可以检测出对I-SCEI内核酸内核酸内核酸内核酸内切酶介导的DSB诱导的细胞。两位记者都依​​赖于DSB修复依赖性GFP的表达，因此可以将修复效率作为I-SCEI诱导后的GFP+细胞的比例来测量。使用此实验系统，我们将解决以下特定目的：1。）确定恶性转化对DSB修复结果的影响，2。）表征转换诱导的代谢和表观遗传学变化在DSB修复的背景下。含义：预计该项目将确定代谢物在DSB修复中的参与，这对修复结果的治疗调节具有重要意义。最终可能会利用营养物的可用性，细胞代谢和/或代谢物丰度来促进DNA修复，从而防止损害积累或损害DSB修复，从而增强癌症治疗中的遗传毒性治疗。