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

NF-kB1-p50 对 DNA 烷基化损伤的反应

基本信息

批准号：
8082754
负责人：
Bakhtiar Yamini
金额：
$ 30.26万
依托单位：
UNIVERSITY OF CHICAGO
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：
2009-08-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8082754
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 Health 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 research study response ribosyltransferase small hairpin RNA temozolomide transcription factor tumor

项目摘要

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SN1-甲基化剂如替莫唑胺（TMZ）（一种用于治疗恶性神经胶质瘤和其他人类肿瘤的化疗剂）诱导的主要细胞毒性损伤。未修复的 O6-MeG 加合物会导致错配修复 (MMR) 导向的细胞凋亡，或者如果耐受的话，可能会导致突变的诱导。在研究 TMZ 的作用机制时，我们最近报道 TMZ 通过 DNA 结合的损伤特异性减弱来抑制核因子-:B (NF-:B) 的活性。我们工作的总体目标是研究 NF-:B 在化疗甲基化药物反应中的作用，作为改善其抗肿瘤作用和改善其致癌作用的基础。初步研究表明，p50 (NF-:B1) 亚基是 TMZ 抑制 NF-:B 所必需的，并且 p50 的缺失使细胞对 TMZ 诱导的杀伤具有高度抵抗力。基于上述数据，我们假设对 O6-MeG 诱导剂的 DNA 损伤反应是由 p50 介导的。下面概述的实验将确定 p50 是否作为 O6-MeG 诱导的 DNA 损伤反应途径特异的效应子，以及 p50 表达水平是否在调节 TMZ 的化疗和致癌作用中发挥重要作用。在目标 1 中，将进行实验来检查 p50 介导 TMZ 诱导的 NF-:B DNA 结合抑制的机制。研究将首先在 Chk1/p50 相互作用处查看 p50 上游，然后在 :B 位点 DNA 序列处查看 p50 下游在目标 2 中，实验将检查 p50 介导的 NF-:B 抑制是否是对 O6 的特异性反应- 麦格损伤。 O6-MeG 损伤将使用寡核苷酸双链体进行分离，并在体外和完整细胞中进行研究。我们将首先观察 p50 的上游，以检查 NF-:B 的抑制是否是对 O6-MeG 损伤的特定反应，还是 DNA 错配识别的一般结果。接下来，实验将检查 O6-MeG 诱导的 p50 介导的 NF-:B 抑制是否足以引发涉及细胞凋亡的下游损伤反应。在目标 3 中，为了确定 p50 的缺失是否与 TMZ 治疗神经胶质瘤具有临床相关性，动物实验将检查 p50 的缺失是否会使神经胶质瘤异种移植物对 TMZ 的抗肿瘤作用产生抵抗。在目标 4 中，为了检查 p50 的缺失是否会导致化疗诱导的癌变，将进行实验以确定 p50 的缺失是否会使细胞在 TMZ 治疗后容易发生突变和恶性转化。公共卫生相关性：项目叙述烷化化疗药物的有效性取决于肿瘤细胞内特定分子的表达。在该提案中，将研究特定中间体核因子-:B被烷化剂杀死的能力，试图确定该分子是否在患者对烷化化疗的总体反应中发挥作用。