MECHANISM BASED SCREENING OF CHEMOPREVENTIVE AGENTS

基于机制的化学预防剂筛选

• 批准号: 6096053
• 负责人: Martin Lipkin
• 金额: $ 48.06万
• 依托单位: STRANG CANCER PREVENTION CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-30 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6096053
• 关键词: athymic mouse; biological signal transduction; cancer prevention; chemoprevention; drug screening /evaluation; fibroblasts; gene expression; gene mutation; neoplastic cell; nucleic acid sequence; tissue /cell culture; transfection

Basal cell carcinomas, the commonest human cancer, uniformly have abnormal regulation of hedgehog pathway signaling with consequent changes in transcription of hedgehog target genes. Mutations underlying this aberrant transcription may act through inactivation of the tumor suppressor gene PTC or activation of the protooncogene Smoothened (SMO). REF52 cells transfected with a combination of E1A and SMO carrying the same mutations as found in basal cell carcinomas develop transformed foci, and the cells from these foci grow with anchorage- independence in soft agar and as rapidly-growing tumors when injected subcutaneously into nude mice( Xie et al., Nature 391:90-92, 1998). This prototypic cell system where the mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblasts (REF52) appears to fulfill the criteria for an efficient, mechanism based, screen of chemopreventive agents. The objective of this study is to screen a large number of chemopreventive agents using phenotypic and genomic profiling of REF cultures transfected with wild type or mutated SMO. Transformed cells in an in vitro screen shall be used to establish efficacy and mechanism of action of chemopreventive agents utilizing the following endpoints: Modulation of anchorage independent growth is being carried out on REF52 cells transfected with E1A+mut-SMO in a 24-well Linbro tray in the absence and presence of 20 compounds representing all classes of chemopreventive agents. To identify compounds specifically with efficacy against hedgehog (HH) pathway abnormalities, REF52 cells transfected with E1A+ras.

基底细胞癌是最常见的人类癌症，其一致地具有hedgehog通路信号传导的异常调节，从而导致hedgehog靶基因转录的变化。 这种异常转录的基础突变可能通过肿瘤抑制基因PTC的失活或原癌基因Smoothened（SMO）的激活起作用。 用携带与基底细胞癌中发现的相同突变的E1 A和SMO的组合转染的REF 52细胞产生转化灶，并且来自这些灶的细胞在软琼脂中以锚定非依赖性生长，并且当皮下注射到裸鼠中时作为快速生长的肿瘤生长（Xie等人，Nature 391：90-92，1998）。 与野生型不同，突变型SMO可以与腺病毒E1 A合作转化大鼠胚胎成纤维细胞（REF 52）的这种原型细胞系统似乎满足有效的、基于机制的化学预防剂筛选的标准。本研究的目的是使用野生型或突变SMO转染的REF培养物的表型和基因组分析来筛选大量的化学预防剂。体外筛选中的转化细胞应用于利用以下终点确定化学预防剂的功效和作用机制：在不存在和存在代表所有类别化学预防剂的20种化合物的情况下，在24孔Linbro托盘中对用E1 A +mut-SMO转染的REF 52细胞进行锚定非依赖性生长的调节。为了鉴定对hedgehog（HH）通路异常具有特异性功效的化合物，用E1 A +ras转染REF 52细胞。