p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. is expected to be a promising novel cancer therapy. and . Thus, the combination of p53 and RNAi was shown to be a potential novel cancer therapy. To discover factors that modulate p53 pathways, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. In this study, we screened a genome-wide lentiviral shRNA library ADL5859 HCl in cancer cells resistant to p53-related apoptosis. We identified specific shRNAs that were significantly decreased in p53-infected cells but not in control cells. These results indicated that specific shRNAs evoked the apoptotic response following p53 transduction or functional restoration of p53 in cancer cells resistant to p53-induced apoptosis, suggesting that the combination of p53 restoration and treatment with specific shRNAs may be an effective cancer therapy. RESULTS Genome-wide shRNA screening in p53-resistant cancer cells To identify shRNAs that can relieve resistance to p53-induced apoptosis in cancer cells, we screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 cells and pancreatic cancer Panc-1 cells, as summarized in Figure ?Figure1A1A and briefly described below. The Huh-7 and Panc-1 cell lines have mutated p53 and a weak apoptotic response following wild-type p53 transduction. The human shRNA library, comprising approximately 200,000 shRNA sequences targeting 47,400 human mRNA transcripts, was packaged into lentivirus particles and pooled. First, we infected the cell lines with the pooled lentiviral shRNA library. Seventy-two hours after lentiviral infection, the cells were infected with adenovirus expressing p53. Forty-eight hours after adenoviral infection, total mRNA was extracted, and mRNA, including shRNA sequences Nrp1 derived from lentiviral vectors, was amplified by PCR. In this shRNA library, each lentiviral vector shRNA sequence corresponds to a probe sequence found on an Affymetrix microarray (GeneChip). Therefore, the shRNA populations can be quantified by cDNA microarray to determine the expression levels of each shRNA; as a reference, the shRNA population in each cell line infected with adenovirus expressing LacZ was also analyzed. Figure 1 Schematic representation of the array-based shRNA library screening process Identification of shRNA-decreased populations in p53-transduced cells We identified the shRNAs that were significantly decreased in p53-infected cells compared with control cells (Fig. ?(Fig.2A).2A). Several shRNAs evoked the apoptotic response following p53 transduction in cancer cells resistant to p53-induced apoptosis. In Huh-7 cells, 547 shRNAs were decreased more than 4-fold in cells with p53 transduction compared with control cells. In Panc-1 cells, 1418 shRNA were decreased more than 16-fold in cells with p53 transduction compared with control cells (Fig. ?(Fig.2A,2A, areas encircled by dashed line). Furthermore, 28 shRNAs were commonly decreased in both cell lines (Fig. ?(Fig.2B,2B, Supplementary Table S1). Among these shRNAs, shRNA-58335 in the presence of p53 was markedly decreased (66-fold in Huh-7 cells and 436-fold in Panc-1 cells). Therefore, we further analyzed shRNA-58335. Figure 2 Analysis of the shRNA library screen in p53-transduced cells Enhancement of p53-induced apoptosis by shRNA-58335 Next, we stably infected lentivirus expressing shRNA-58335 or a control sequence into Huh-7 cells and quantified p53-induced apoptosis by evaluating the sub-G1 population. In these cells, p53 transduction induced a strong apoptotic response in shRNA-58335-infected cells compared with control cells (Fig. 3A, B). Additionally, treatment of adriamycin significantly enhanced the apoptotic response (Fig. 3A, B). Increased caspase-3 cleavage, which serves as another indicator of apoptosis, was also observed by western ADL5859 HCl blotting (Fig. ?(Fig.3C).3C). In colorectal cancer SW480 cells, which also have mutated p53 and a weak apoptotic response following p53 transduction, shRNA-58335 sensitized the p53-induced apoptotic response (Fig. ?(Fig.3D).3D). These results indicated that shRNA-58335 improved the apoptotic response after p53 transduction in other cancer cell lines. Figure 3 Effect of shRNA-58335 on p53-induced apoptosis Inhibition of tumor growth by p53 transduction with shRNA-58335 was correlated with ADL5859 HCl a therapeutic effect gene by shRNA-58335 was confirmed, although the.