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通路信号的异常调节，从而导致Hedgehog靶基因转录的变化。这种异常转录的突变可能通过肿瘤抑制基因PTC的失活或原癌基因Smoothens(SMO)的激活而起作用。与在基底细胞癌中发现的相同的突变，转染了E1A和SMO的组合的REF52细胞形成转化灶，来自这些灶的细胞在软琼脂中以锚定独立的方式生长，并且当被注射到裸鼠皮下时作为快速生长的肿瘤(谢等，自然391：90-92,1998)。与野生型不同，突变的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满足了高效、基于机制的化学预防药物筛选的标准。本研究的目的是利用野生型和突变型SMO的REF培养物的表型和基因组图谱来筛选大量的化学预防药物。体外筛选转化的细胞应利用以下终点建立化学预防药物的有效性和作用机制：在24孔Linbro托盘中，在没有和存在代表所有类别化学预防药物的20种化合物的情况下，对转导E1a mut-SMO的REF52细胞进行非锚定生长的调节。为了确定对Hedgehog(HH)途径异常具有特定疗效的化合物，将E1Aras基因导入REF52细胞。

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.