Sohn T A, Su G H, Ryu B, Yeo C J, Kern S E
Departments of Surgery, Pathology, and Oncology, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.
Ann Surg. 2001 May;233(5):696-703. doi: 10.1097/00000658-200105000-00014.
To screen a library of small chemicals for compounds that activate the DPC4 signal transduction pathway in a human pancreatic cancer cell line.
Various tumor-suppressor genes are mutated in all human cancers. Specifically, DPC4 (deleted in pancreatic carcinoma, locus 4 or MADH4/SMAD4) is a tumor-suppressor gene mutated in approximately 50% of human pancreatic adenocarcinomas. DPC4 plays an important role in the well-studied transforming growth factor-beta (TGFbeta) signaling pathway. It would be useful to identify therapies that augment or restore the downstream functions of this critical signal transduction pathway, in hopes that such therapy would have a rational role in anticancer therapy.
Using a commercially available plasmid vector with a luciferase reporter gene already incorporated, a DPC4-specific reporter construct was genetically engineered. This was done by inserting six copies of the palindromic Smad binding element (6SBE), which is a DNA binding element specific for DPC4, in front of the minimal promoter in the plasmid. This construct was then stably integrated into the genome of a human pancreatic cancer cell line (PANC-1) that has wild-type DPC4. Several stably transfected clones were tested for basal luciferase expression and inducibility with TGFbeta, which is known to activate the DPC4 signal transduction pathway. A single transfected clone was chosen for the drug screen based on basal luciferase (reporter) expression and TGFbeta inducibility. A systematic screen of the chemical library was then performed, using luciferase activity to detect DPC4 activity and induction of the signaling pathway.
A high-throughput system based on this stably integrated reporter system was used to screen a library of 16,320 random compounds to identify agents that conferred robust augmentation of the DPC4 signal transduction pathway. Of the 16,320 compounds screened, 11 were associated with a 2- to 5-fold induction of luciferase activity, and one with a 12-fold activation. The latter compound was shown to be a novel histone deacetylase inhibitor and was further characterized.
These results confirm the feasibility of a specific high-throughput reporter system to screen a large compound library in human cells efficiently. The screening identified several compounds capable of augmenting DPC4-specific luciferase reporter activity, and a specific mechanism for one compound was identified. The discovery of such agents will aid our understanding of complex tumor-suppressive signaling pathways and may identify other potential therapeutic targets within this critical signaling pathway. In addition, random drug screening provides an unbiased method for identifying drugs or lead compounds for potential therapeutic use.
在人胰腺癌细胞系中筛选一个小分子化学文库，寻找能激活DPC4信号转导通路的化合物。
各种肿瘤抑制基因在所有人类癌症中均有突变。具体而言，DPC4（在胰腺癌中缺失，4号位点或MADH4/SMAD4）是一种肿瘤抑制基因，在约50%的人胰腺腺癌中发生突变。DPC4在研究充分的转化生长因子-β（TGFβ）信号通路中发挥重要作用。鉴定增强或恢复这一关键信号转导通路下游功能的疗法将很有意义，希望这种疗法在抗癌治疗中能发挥合理作用。
利用已整合荧光素酶报告基因的市售质粒载体，通过基因工程构建了DPC4特异性报告基因构建体。具体做法是在质粒的最小启动子前插入6个回文Smad结合元件（6SBE）拷贝，6SBE是DPC4特异性的DNA结合元件。然后将该构建体稳定整合到具有野生型DPC4的人胰腺癌细胞系（PANC-1）的基因组中。对几个稳定转染的克隆进行基础荧光素酶表达检测以及对已知能激活DPC4信号转导通路的TGFβ的诱导性检测。基于基础荧光素酶（报告基因）表达和TGFβ诱导性选择一个转染克隆进行药物筛选。然后使用荧光素酶活性检测DPC4活性和信号通路的诱导情况，对化学文库进行系统筛选。
基于这种稳定整合的报告系统的高通量系统用于筛选一个包含16320种随机化合物的文库，以鉴定能强力增强DPC4信号转导通路的试剂。在筛选的16320种化合物中，11种与荧光素酶活性2至5倍的诱导相关，1种与12倍的激活相关。后一种化合物被证明是一种新型组蛋白去乙酰化酶抑制剂，并对其进行了进一步表征。
这些结果证实了一种特异性高通量报告系统在人细胞中高效筛选大型化合物文库的可行性。筛选鉴定出了几种能够增强DPC4特异性荧光素酶报告基因活性的化合物，并确定了一种化合物的具体作用机制。此类试剂的发现将有助于我们理解复杂的肿瘤抑制信号通路，并可能在这一关键信号通路中识别出其他潜在治疗靶点。此外，随机药物筛选为鉴定潜在治疗用途的药物或先导化合物提供了一种无偏倚的方法。
