GENETIC ELEMENTS TRANSFORMING MOUSE FIBROBLASTS

描述（由申请人提供）：该提案是正在进行的开发用于鉴定癌症相关基因的新功能基因发现方法的项目的扩展。在前一阶段，主要集中在检测新的候选肿瘤抑制基因和药物敏感基因。最近的方法学进展使得现在有可能关注被定义为细胞因子的前瞻性分子抗癌治疗靶点（ACTT），其抑制导致选择性杀死肿瘤细胞或对肿瘤细胞的治疗敏感。候选ACTT搜索基于三种强大技术的组合-微阵列基因表达分析，遗传抑制元件（GSE）方法和新开发的选择减法（SSA）。这些技术被整合在一个技术管道中，该技术管道允许鉴定使肿瘤细胞耐受致癌Ras的遗传事件。ACTT的搜索将从收集代表基因的候选序列库开始，这些基因在一系列人成纤维细胞群体中被初步研究过程中鉴定的不同协同遗传元件从Ras介导的生长停滞中拯救出来。将从所选序列构建GSE文库，并使用SSA技术筛选能够杀死Ras转化但不杀死正常成纤维细胞的GSE。通过使用siRNA构建体针对将测试其对肿瘤细胞的特异性细胞毒性的每个个体候选物，将鉴定的基因验证为前瞻性ACTT。将表征鉴定的候选ACTT与天然存在的肿瘤细胞的相关性，并且该计划将进一步扩展到通过抑制鉴定的ACTT发挥抗癌作用的小分子的开发。这项研究的成功将表明一种强大的靶点发现工具的开发，该工具将适用于其他类型癌症的ACTT鉴定。

DESCRIPTION (provided by applicant): This proposal is an extension of ongoing program of development of new functional gene discovery methodologies for identification of cancer-related genes. In the previous phase, it was primarily focused on detection of new candidate tumor suppressor and drug sensitivity genes. Recent methodological advancements made it possible now to focus on prospective molecular anticancer treatment targets (ACTTs) defined as cellular factors, the repression of which leads to selective killing or sensitization to treatment of tumor cells. Candidate ACTT search is based on a combination of three powerful techniques - microarray gene expression analysis, genetic suppressor element (GSE) methodology and newly developed selection-subtraction approach (SSA). These techniques are integrated in one technological pipeline that allows identification of genetic events making tumor cells tolerant to oncogenic Ras. The search for ACTTs will start from collection of a pool of candidate sequences representing genes that become upregulated in a series of populations of human fibroblasts rescued from Ras-mediated growth arrest by different cooperating genetic elements identified in the course of preliminary studies. GSE library will be constructed from the selected sequences and screened, using SSA technique, for the GSEs capable of killing Ras-transformed but not normal fibroblasts. Identified genes will be validated as prospective ACTTs by using siRNA constructs against each individual candidate that will be tested for their specific cytotoxicity for tumor cells. Relevance of identified candidate ACTTs to naturally occurring tumor cells will be characterized and the program will be further extended towards development of small molecules with anticancer properties acting by suppression of identified ACTT. The success of the proposed study will indicate development of a powerful target discovery tool that will be applicable for ACTT identification in other types of cancer.