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Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells.

Idogawa M, Ohashi T, Sugisaka J, Sasaki Y, Suzuki H, Tokino T - Oncotarget (2014)

Bottom Line: We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis.We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells.Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan. Contributed equally to this work.

ABSTRACT
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. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

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Effect of shRNA-58335 on p53-induced apoptosis(A) Huh-7 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Twenty-four hours after infection, the cells were treated with adriamycin (0.5 μg/ml) or not. Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. Error bars indicate the S.E. * indicates a p value < 0.05 by a t-test. (B) Representative flow cytometry data in Huh-7 cells infected with Ad-p53. The percentage of cells in the sub-G1 phase is indicated. (C) Under the same conditions used in (A), total cell lysates were extracted and analyzed by Western blot with the indicated antibodies. (D) SW480 cells were infected with lentivirus expressing shRNA-58335 (shRNA: +) or a control sequence (shRNA: -). These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Forty-eight hours after infection, total cell lysates were extracted and analyzed by Western blot with the indicated antibodies.
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Figure 3: Effect of shRNA-58335 on p53-induced apoptosis(A) Huh-7 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Twenty-four hours after infection, the cells were treated with adriamycin (0.5 μg/ml) or not. Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. Error bars indicate the S.E. * indicates a p value < 0.05 by a t-test. (B) Representative flow cytometry data in Huh-7 cells infected with Ad-p53. The percentage of cells in the sub-G1 phase is indicated. (C) Under the same conditions used in (A), total cell lysates were extracted and analyzed by Western blot with the indicated antibodies. (D) SW480 cells were infected with lentivirus expressing shRNA-58335 (shRNA: +) or a control sequence (shRNA: -). These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Forty-eight hours after infection, total cell lysates were extracted and analyzed by Western blot with the indicated antibodies.

Mentions: 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 blotting (Fig. 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. 3D). These results indicated that shRNA-58335 improved the apoptotic response after p53 transduction in other cancer cell lines.


Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells.

Idogawa M, Ohashi T, Sugisaka J, Sasaki Y, Suzuki H, Tokino T - Oncotarget (2014)

Effect of shRNA-58335 on p53-induced apoptosis(A) Huh-7 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Twenty-four hours after infection, the cells were treated with adriamycin (0.5 μg/ml) or not. Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. Error bars indicate the S.E. * indicates a p value < 0.05 by a t-test. (B) Representative flow cytometry data in Huh-7 cells infected with Ad-p53. The percentage of cells in the sub-G1 phase is indicated. (C) Under the same conditions used in (A), total cell lysates were extracted and analyzed by Western blot with the indicated antibodies. (D) SW480 cells were infected with lentivirus expressing shRNA-58335 (shRNA: +) or a control sequence (shRNA: -). These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Forty-eight hours after infection, total cell lysates were extracted and analyzed by Western blot with the indicated antibodies.
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Figure 3: Effect of shRNA-58335 on p53-induced apoptosis(A) Huh-7 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Twenty-four hours after infection, the cells were treated with adriamycin (0.5 μg/ml) or not. Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. Error bars indicate the S.E. * indicates a p value < 0.05 by a t-test. (B) Representative flow cytometry data in Huh-7 cells infected with Ad-p53. The percentage of cells in the sub-G1 phase is indicated. (C) Under the same conditions used in (A), total cell lysates were extracted and analyzed by Western blot with the indicated antibodies. (D) SW480 cells were infected with lentivirus expressing shRNA-58335 (shRNA: +) or a control sequence (shRNA: -). These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Forty-eight hours after infection, total cell lysates were extracted and analyzed by Western blot with the indicated antibodies.
Mentions: 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 blotting (Fig. 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. 3D). These results indicated that shRNA-58335 improved the apoptotic response after p53 transduction in other cancer cell lines.

Bottom Line: We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis.We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells.Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan. Contributed equally to this work.

ABSTRACT
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. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.

Show MeSH
Related in: MedlinePlus