Site Specific Alkylation of the HER2/neu Promoter

描述(申请人提供)：HER-2/neu癌基因似乎在 在许多类型的人类癌症的发生和发展中起着重要作用， 包括大约25%的非小细胞肺癌(NSCLC)， 美国男性和女性癌症死亡的主要原因 这个项目的目标是找到新的方法来特异性地抑制HER-2/neu 开发三链形成寡核苷酸(TFO)-烷基化子的表达 与HER-2/neu启动子结合 通过三链DNA的形成并导致特定的共价DNA修饰 鸟嘌呤碱基的DNA烷基化试剂。这项建议的具体目的 旨在解决成功开发 基于TFO的DNA结合药物。与TFOS偶联的位点特定的烷基化反应 氮芥将在HER-2/neu启动子中得到证明。这些数据 将通过分子模拟来合理化，这些模型将被用于 进一步完善TFO-烷化剂共轭化合物的设计(具体目标1)。 这项工作现在大部分已经完成，未来的工作将集中在 新型小沟槽DNA结合剂的设计和实验研究 核酸酶抗性寡核苷酸骨架修饰的研究。这个 这些化合物抑制转录启动的能力将被研究。 通过在报告质粒中形成三螺旋，瞬时地将其导入 NSCLC细胞系，以及NSCLC细胞的识别和修复能力 三链导向的DNA烷基化将在转染后进行表征 (具体目标2)。我们的初步数据表明，一个TFO与一个 氮芥末的3‘和5’端都可以直接指向两个鸟嘌呤加合物 邻近三重螺旋的两端，以抵抗DNA修复和抑制 HER-2/neu启动子在NSCLC细胞中的活性。一种基于腺病毒载体的ODN载体 系统将被开发，腺病毒介导ODN摄取的能力 当腺病毒-ODN复合体被发现时，核定位将被确定 由半稳定的化学连接物形成(特定目标3)。的能力 三链形成ODN与内源性HER-2/neu基因结合并定向 将通过Southern印迹和连接来研究特定部位的DNA烷基化 TFO-烷化剂处理非小细胞肺癌细胞后的介导型聚合酶链式反应 共轭化合物(特定目标4)。这些化合物的治疗潜力 抑制HER-2/neu的表达并改变HER-2/neu的恶性和侵袭性表型 NSCLC细胞将在组织培养中进行评估(特定目标5)。这个 HER-2/neu特异性抗基因化合物的开发将提供很大的 关于HER-2/neu基因在启动和修复中的作用的大量信息 NSCLC的进展并可能导致HER-2/neu的新治疗方法 表现为非小细胞肺癌等癌症。

DESCRIPTION (PROVIDED BY APPLICANT): The HER-2/neu oncogene appears to play an important role in the initiation and progression of many types of human cancer, including approximately 25 percent of non-small cell lung cancer (NSCLC), the leading cause of cancer death in both men and women in the U.S. The overall goal of this project is to find novel ways to specifically inhibit HER-2/neu expression by developing triplex forming oligonucleotide (TFO)-alkylator conjugates what will bind in a site-specific manner to the HER-2/neu promoter by triplex DNA formation and lead to covalent DNA modification at specific guanine bases by the DNA alkylating agent. The specific aims of this proposal are designed to address the major obstacles to the successful development of a TFO-based DNA binding drug. Site-specific alkylation with TFOs conjugated to nitrogen mustards will be demonstrated in the HER-2/neu promoter. These data will be rationalized by molecular modeling, and these models will be used to further refine the design of the TFO-alkylator conjugates (Specific Aim 1). Much of this work has now been accomplished, and future work will focus on the design of novel conjugates with a minor groove DNA binding agent and the investigation of nuclease resistant oligonucleotide backbone modifications. The ability of these compounds to inhibit transcription initiation will be studied by triple helix formation in a reporter plasmid transiently transfected into NSCLC cell lines, and the ability of NSCLC cells to recognize and repair triplex directed DNA alkylation will be characterized after transfection (Specific Aim 2). Our preliminary data demonstrates that a TFO conjugated to a nitrogen mustard at both the 3' and 5' ends can direct two guanine adducts adjacent to both ends of the triple helix to resist DNA repair and suppress HER-2/neu promoter activity in NSCLC cells. An adenovirus based ODN delivery system will be developed, and the ability of adenoviruses to mediate ODN uptake and nuclear localization will be determined when adenovirus-ODN complexes are formed by a semi-stable chemical linker (Specific Aim 3). The ability of triplex forming ODNs to bind to the endogenous HER-2/neu gene and direct site-specific DNA alkylation will be studied by Southern blot and ligation mediated-PCR after the treatment of NSCLC cells with the TFO-alkylator conjugates (Specific Aim 4). The therapeutic potential of these compounds to inhibit HER-2/neu expression and alter the malignant and invasive phenotype of NSCLC cells will be evaluated in tissue culture (Specific Aim 5). The development of a HER-2/neu specific anti-gene compound will provide a great deal of information about the role of the HER-2/neu gene in the initiation and progression of NSCLC and may lead to novel treatment approaches for HER-2/neu expressing cancers such as NSCLC.