A Facile and Effective Screening Method for p21^WAF1 Promoter Activators from Microbial Metabolites

Abstract

We have developed a novel p21^WAF1 promoter activator screening system based on rapid and facile luciferase activity assay of a model cell system (H1299/tsp53-luc cells), a stable luciferase expression cell line established by transfecting H1299/tsp53 cells with a reporter gene construct pWWP-Luc-BSD. This plasmid was constructed by subcloning the 2.4kb p21^WAF1 promoter and a 2.6kb of luciferase cDNA fragment activated by the p21^WAF1 promoter into a pMAM2-BSD expression vector containing the blasticidin S deaminase gene (BSD). A BSD-resistant clone H1299/tsp53-luc#4, showing the highest response to p53 activation (by temperature shift from 37°C to 32°C) by luciferase production, was used for screening microbial culture broths. Among approximately 1200 screened samples, trichostatin A related compounds and a new compound, lucilactaene, were isolated. This provides an effective and facile screening system for p21^WAF1 promoter activators which should be of considerable value in the rapid identification of new anticancer agents.