TARGETING TUMOR CELLS WITH GENETIC SUPPRESSOR ELEMENTS

用遗传抑制元件靶向肿瘤细胞

• 批准号: 2103104
• 负责人: IGOR B RONINSON
• 金额: $ 19.84万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-19 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2103104
• 关键词: 3T3 cells; DNA topoisomerases; HeLa cells; Retroviridae; antineoplastics; antisense nucleic acid; chemical synthesis; complementary DNA; cytotoxicity; drug design /synthesis /production; gene targeting; gene therapy; genetic library; molecular cloning; neoplastic cell; neoplastic transformation; oncogenes; transfection /expression vector

Rational development of new anticancer drugs involves the identification of potential targets in tumor cells and the development of reagents selectively acting at such targets. It is now possible to design target- specific "genetic drugs", such as antisense oligonucleotides, peptides, or vectors for gene therapy, directly on the basis of the structure of the selected target gene. Some prototype genetic drugs targeting specific oncogenes were reported to have a selective cytostatic or cytotoxic effect on neoplastically transformed relative to untransformed cells. Tumor- specific changes in the structure or expression of known oncogenes, however, do not provide all the determinants of tumor specificity for either conventional or genetic anticancer drugs. The present application proposes to undertake a general search for genetic drugs that would induce selective cytotoxicity or growth arrest in tumor cells, and to identify the cellular targets of such drugs. This search will be based on the new methodology for expression selection of genetic suppressor elements (GSEs), short cDNA fragments that encode peptides or antisense RNA sequences interfering with the function of genes from which they are derived. This methodology was developed in a bacterial model and then used in mammalian cells to isolate a series of GSEs inhibiting human topoisomerase II. In subsequent studies, GSE selection was conducted using a retroviral expression library carrying normalized (uniform abundance) fragments of total cellular cDNA from NIH 3T3 cells. These studies resulted in the isolation of several GSEs derived from previously unknown genes associated with drug resistance or neoplastic transformation. The principle of expression selection of GSEs from retroviral libraries of normalized cellular cDNA will now be combined with the use of a tightly regulated inducible promoter and negative selection techniques, to isolate GSEs of unknown nature, which are cytostatic or cytotoxic to cells in which they are expressed. GSEs that show selective inhibition of transformed relative to untransformed mouse fibroblasts, or that are cytotoxic to human HeLa carcinoma cells, will be isolated and sequenced. The activity of the isolated GSEs will be tested on cell lines derived from different types of mouse or human tumors, and on normal cells. Full- length human cDNA sequences of the genes targeted by the selected GSEs will be cloned and their expression patterns will be characterized. The cloned genes will be used individually to isolate the most efficient GSEs interfering with their functions. These GSEs will then be tested for in vitro anti tumor activity, in the form of retroviral vectors or chemically synthesized peptides or oligonucleotides. It is hoped that the proposed studies will lead to the identification of new targets and prototype genetic drugs with potential utility for the treatment of cancer.

新抗癌药的理性发展涉及鉴定 肿瘤细胞的潜在靶标和试剂的发展 有选择地在此类目标上行动。现在可以设计目标 - 特定的“遗传药物”，例如反义寡核苷酸，肽或 基因治疗的向量直接基于 选定的靶基因。一些针对特定的原型遗传药物 据报道，癌基因具有选择性的胞击或细胞毒性作用 相对于未转化的细胞，在肿瘤上转化。瘤- 已知肿瘤基因的结构或表达的特定变化， 但是，请勿提供所有肿瘤特异性的决定因素 常规或遗传抗癌药物。本申请 建议对遗传药物进行一般搜索，以诱发 肿瘤细胞中的选择性细胞毒性或生长停滞，并确定 此类药物的细胞靶标。此搜索将基于新的 遗传抑制元件的表达选择方法 （GSE），编码肽或反义RNA的短cDNA片段 序列干扰了它们的基因功能 衍生的。该方法是在细菌模型中开发的，然后使用 在哺乳动物细胞中，分离了一系列抑制人的GSE 拓扑异构酶II。在随后的研究中，使用GSE选择 带有标准化的逆转录病毒表达式文库（均匀丰度） 来自NIH 3T3细胞的总细胞cDNA的片段。这些研究 导致隔离以前未知的几个GSE 与耐药性或肿瘤转化相关的基因。这 从逆转录病毒库的GSE表达选择原理 归一化的细胞cDNA现在将与紧密的使用结合 调节的诱导启动子和负选择技术，以分离 具有未知性质的GSE，它们是细胞抑制或细胞毒性的细胞 他们是表达的。 GSE显示出选择性抑制 相对于未转换的鼠标成纤维细胞转变，或 将细胞毒性对人HeLa癌细胞进行分离和测序。 分离的GSE的活性将在得出的细胞系上进行测试 来自不同类型的小鼠或人类肿瘤，以及正常细胞。满的- 由选定GSE靶向的基因的长度人cDNA序列 将被克隆，其表达模式将被表征。这 克隆的基因将单独使用以隔离最有效的GSE 干扰他们的功能。然后将对这些GSE进行测试 体外抗肿瘤活性，以逆转录病毒载体或化学形式 合成的肽或寡核苷酸。希望提议 研究将导致确定新目标和原型 具有潜在用途的遗传药物用于治疗癌症。