GENETIC ELEMENTS TRANSFORMING MOUSE FIBROBLASTS

描述（由申请人提供）：该提案是正在进行的用于鉴定癌症相关基因的新功能基因发现方法开发项目的延伸。前期主要是检测新的候选肿瘤抑制基因和药敏基因。最近方法学的进步使得现在有可能关注潜在的分子抗癌治疗靶点（ACTTs），这些靶点被定义为细胞因子，其抑制导致选择性杀死或对肿瘤细胞的治疗增敏。候选ACTT搜索是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）。这些技术整合在一个技术管道中，允许鉴定使肿瘤细胞耐受致癌Ras的遗传事件。对actt的研究将从收集一组候选序列开始，这些序列代表了在一系列人类成纤维细胞群体中从ras介导的生长停滞中获救的基因，这些基因是由初步研究过程中确定的不同协同遗传元件组成的。从选择的序列中构建GSE库，并使用SSA技术筛选能够杀死ras转化但不能杀死正常成纤维细胞的GSE。鉴定出的基因将通过siRNA结构对每个候选基因进行前瞻性actt验证，并测试其对肿瘤细胞的特异性细胞毒性。已鉴定的候选ACTT与自然发生的肿瘤细胞的相关性将被表征，该计划将进一步扩展到通过抑制已鉴定的ACTT来开发具有抗癌特性的小分子。这项研究的成功将预示着一种强大的靶标发现工具的发展，这种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.