NF-kB1-p50 in the response to DNA alkylation damage

NF-kB1-p50 对 DNA 烷基化损伤的反应

• 批准号: 7736451
• 负责人: Bakhtiar Yamini
• 金额: $ 31.2万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7736451
• 关键词: Alkylating Agents; Animal Experiments; Animal Model; Animals; Apoptosis; Apoptotic; Attenuated; Cell Cycle; Cell Survival; Cells; Checkpoint kinase 1; Complement Factor B; Cytosine; DNA; DNA Alkylation; DNA Binding; DNA Damage; DNA Sequence; Data; Effectiveness; Elements; Gene Expression; Genomic Instability; Glioma; Goals; Guanine; Human; Hypoxanthines; In Vitro; Induced Mutation; Induction of Apoptosis; Lead; Lesion; Link; Malignant - descriptor; Malignant Glioma; Mediating; Mismatch Repair; Mutation; Nuclear; Nude Mice; Oligonucleotides; Pathway interactions; Patients; Play; Process; Proteins; Replication Error; Reporting; Resistance; Role; Signal Transduction; Site; System; TNFRSF5 gene; Thymine; Transducers; Translating; Work; Xenograft procedure; adduct; attenuation; base; carcinogenesis; cell killing; chemotherapeutic agent; chemotherapy; clinically relevant; cytotoxic; cytotoxicity; detector; improved; in vivo; killings; neoplastic cell; public health relevance; research study; response; ribosyltransferase; small hairpin RNA; temozolomide; transcription factor; tumor

DESCRIPTION (provided by applicant): O6-methylguanine (O6-MeG) is the primary cytotoxic lesion induced by SN1-methylating agents such as temozolomide (TMZ), a chemotherapeutic agent used in the treatment of malignant glioma and other human tumors. Unrepaired O6-MeG adducts cause mismatch repair (MMR)-directed apoptosis or, if tolerated, can lead to the induction of mutations. In investigating the mechanism of action of TMZ, we recently reported that TMZ inhibits the activity of nuclear factor-:B (NF-:B) through damage specific attenuation of DNA-binding. The overall goal of our work is to examine the role of NF-:B in the response to chemotherapeutic methylating agents as a basis to improving their anti-tumor action and ameliorating their carcinogenic effects. Preliminary studies demonstrate that the p50 (NF-:B1) subunit is required for inhibition of NF-:B by TMZ and that deletion of p50 renders cells highly resistant to TMZ-induced killing. Based on the above data, we hypothesize that the DNA damage response to O6-MeG-inducing agents is mediated by p50. The experiments outlined below will determine whether p50 acts as an effector specific to the O6-MeG-induced DNA damage response pathway and whether p50 expression level is important in regulating the chemotherapeutic and carcinogenic actions of TMZ. In Aim 1, experiments will be performed to examine the mechanism by which p50 mediates TMZ-induced inhibition of NF-:B DNA-binding. Studies will first look upstream up p50 at the Chk1/p50 interaction and then look downstream of p50 at the :B-site DNA sequence In Aim 2, experiments will examine whether p50-mediated inhibition of NF-:B is a specific response to O6- MeG damage. The O6-MeG lesion will be isolated using oligonucleotide duplexes and studies performed both in vitro and in intact cells. We will first look upstream of p50 to examine if inhibition of NF-:B is a specific response to O6-MeG damage or a general consequence of DNA mismatch recognition. Next, experiments will examine if O6-MeG-induced inhibition of NF-:B, mediated by p50, is sufficient to elicit the downstream damage response involving apoptosis. In Aim 3, to determine if loss of p50 may be clinically relevant in the treatment of glioma with TMZ, animal experiments will examine if depletion of p50 renders glioma xenografts resistant to the anti-tumor effect of TMZ. In Aim 4, to examine if loss of p50 predisposes to chemotherapeutic-induced carcinogenesis, experiments will be performed to determine whether deletion of p50 renders cells prone to mutation and malignant transformation following TMZ treatment. PUBLIC HEALTH RELEVANCE: Project Narrative The effectiveness of alkylating chemotherapeutic agents is dependent on the expression of specific molecules within the tumor cells. In this proposal, the ability of a specific intermediate, nuclear factor-:B, to enable killing by alkylating agents will be investigated in an attempt to determine whether this molecule plays a role in the overall response of patients to alkylating chemotherapy.

描述（由申请人提供）：O6-甲基鸟嘌呤（O6-MEG）是由SN1-甲基化剂（例如替莫唑胺（TMZ））诱导的主要细胞毒性病变，例如替莫唑胺（TMZ），这是一种用于治疗恶性胶质瘤和其他人类肿瘤的化学治疗剂。未修复的O6-MEG加合物会导致不匹配修复（MMR）指导的细胞凋亡，或者，如果耐受性，则可能导致突变的诱导。在研究TMZ的作用机理时，我们最近报道了TMZ通过DNA结合的损伤特异性衰减抑制了核因子：B（NF-：B）的活性。我们工作的总体目标是研究NF-：B在对化学治疗性甲基化剂反应中的作用，以此作为改善其抗肿瘤作用并改善其致癌作用的基础。初步研究表明，P50（NF-：B1）亚基是由TMZ抑制NF-：B所需的，并且P50的缺失使细胞对TMZ诱导的杀戮具有高度抗性。基于上述数据，我们假设对O6-MEG诱导剂的DNA损伤响应是由P50介导的。下面概述的实验将确定P50是否充当O6-MEG诱导的DNA损伤响应途径的效应子，以及P50表达水平在调节TMZ的化学治疗和致癌作用方面是否重要。在AIM 1中，将进行实验以检查P50介导TMZ诱导的NF-：B DNA结合的机制。研究将首先在CHK1/P50相互作用的上游查找P50，然后在AIM 2中的：B点DNA序列下方查看P50的下游，实验将检查P50介导的NF-：B：B是否是对O6- MEG损伤的特定响应。将使用寡核苷酸双链体分离O6-MEG病变，并在体外和完整细胞中进行研究。我们将首先查找P50的上游，以检查NF-：B的抑制是对O6-MEG损伤的特定反应还是DNA不匹配识别的总体后果。接下来，实验将检查由P50介导的O6-MEG诱导的NF-：B的抑制，足以引起涉及凋亡的下游损伤响应。在AIM 3中，为了确定P50的丧失在使用TMZ治疗神经胶质瘤方面是否与临床相关，动物实验将检查P50的耗竭是否会使胶质瘤异种移植具有抗TMZ抗肿瘤作用的抗性。在AIM 4中，要检查P50的损失是否易于化学治疗诱导的致癌作用，将进行实验，以确定P50的缺失是否使TMZ处理后容易突变和恶性转化的细胞是否使细胞删除。公共卫生相关性：项目叙述烷基化疗剂的有效性取决于肿瘤细胞内特定分子的表达。在此提案中，将研究特定中间体的核因子-：B的能力，以烷基化剂杀死烷基化剂，以确定该分子是否在患者对烷基化疗的总体反应中起作用。