MECHANISM BASED SCREENING OF CHEMOPREVENTIVE AGENTS

基底细胞癌是人类最常见的癌症，普遍存在hedgehog途径信号的异常调控，从而导致hedgehog靶基因的转录发生变化。这种异常转录背后的突变可能通过肿瘤抑制基因PTC失活或原癌基因Smoothened （SMO）的激活而起作用。携带与基底细胞癌相同突变的E1A和SMO组合转染的REF52细胞产生转化灶，这些灶的细胞在软琼脂中不依赖锚定生长，皮下注射到裸鼠体内时，肿瘤快速生长（Xie et al., Nature 391:90- 92,1998）。与野生型不同，突变型SMO可以与腺病毒E1A合作转化大鼠胚胎成纤维细胞（REF52），这一原型细胞系统似乎符合有效的、基于机制的化学预防药物筛选标准。本研究的目的是通过对转染野生型或突变型SMO的REF培养物进行表型和基因组分析，筛选大量的化学预防药物。利用以下终点，在体外筛选转化细胞应使用，以确定化学预防剂的功效和作用机制：在24孔Linbro托盘中，在代表所有化学预防剂的20种化合物缺失和存在的情况下，对转染了E1A+mut-SMO的REF52细胞进行了锚定独立生长的调节。为了鉴定对hedgehog （HH）通路异常有效的化合物，我们在REF52细胞中转染了E1A+ras。

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.