GENETIC ELEMENTS TRANSFORMING MOUSE FIBROBLASTS

描述(由申请人提供)：这项提案是正在进行的用于识别癌症相关基因的新功能基因发现方法开发计划的延伸。在前一阶段，它主要集中在检测新的候选抑癌基因和药物敏感基因。最近方法学的进步使得现在有可能将重点放在预期的分子抗癌治疗靶点(ACTT)上，ACTT被定义为细胞因子，其抑制导致对肿瘤细胞的选择性杀伤或增敏治疗。候选ACTT搜索基于三种强大的技术的组合-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)。这些技术集成在一个技术管道中，可以识别使肿瘤细胞对致癌RAS耐受的基因事件。对ACTT的搜索将从收集代表基因的候选序列池开始，这些基因在一系列人成纤维细胞群体中上调，这些基因是在初步研究过程中确定的不同合作遗传元件从RAS介导的生长停滞中拯救出来的。我们将从所选择的序列中构建GSE文库，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。识别的基因将通过使用针对每个单独候选的siRNA构建来验证为预期的ACTT，这些候选将被测试其对肿瘤细胞的特定细胞毒性。已确定的候选ACTT与自然产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确定的ACTT来开发具有抗癌特性的小分子。这项拟议研究的成功将预示着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识别。

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.