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Published on: July 30, 2018
An adenovirus expressing mutant p27 showed more potent antitumor effects than adenovirus-p27 wild type
1Department of Internal Medicine, Seoul National University College of Medicine, Lung Institute of Medical Research Center, Seoul National University, Seoul 110-744, Korea.
Abstract:
The main inhibitory action of p27, a cyclin-dependent kinase inhibitor (CDKI), arises from its binding with the cyclin E/cyclin-dependent kinase 2 (Cdk2) complex that results in G(1)-S arrest. Degradation of p27 is mediated by phosphorylation of Thr-187 of p27, which follows ubiquitination. In this study, we generated two adenoviruses expressing wild-type p27 (ad-p27wt) and mutant p27 (ad-p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), which was produced with the belief that mutant p27 would bind cyclin E/CDK2 more stably and show more potent antitumor effects. Ad-p27wt and ad-p27mt expressed p27 proteins that were indistinguishable by anti-p27 antibody. A pulse chase experiment showed that p27mt was more resistant to degradation than p27wt. In human lung cancer cell lines, ad-p27mt showed stronger growth inhibition than ad-p27wt. Both types of ad-p27 induced G(1)-S arrest and apoptosis; however, ad-p27mt induced stronger G(1)-S arrest and apoptosis. Intratumoral injection of ad-p27mt induced partial regression of established tumors and inhibited the growth of human lung cancer xenografts more strongly than ad-p27wt. From these results, we conclude that ad-p27mt has the potential to become a novel and powerful gene therapy tool.
Insights
A novel gene therapy tool, ad-p27mt, shows enhanced antitumor effects by resisting degradation and inducing stronger G1-S arrest and apoptosis in lung cancer cells compared to wild-type p27.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- p27 is a cyclin-dependent kinase inhibitor (CDKI) crucial for G1-S cell cycle arrest.
- p27 degradation, mediated by Thr-187 phosphorylation and ubiquitination, limits its inhibitory action.
- Targeting p27 degradation is a potential strategy for enhancing anti-cancer therapies.
Purpose of the Study:
- To develop and evaluate a mutant p27 (ad-p27mt) with enhanced stability and antitumor efficacy.
- To compare the efficacy of ad-p27mt with wild-type p27 (ad-p27wt) in inhibiting lung cancer growth.
- To assess the potential of ad-p27mt as a gene therapy tool.
Main Methods:
- Generation of adenoviruses expressing wild-type (ad-p27wt) and mutant (ad-p27mt) p27.
- Mutation of Thr-187/Pro-188 to Met-187/Ile-188 to enhance p27 stability.
- Pulse-chase experiments to assess protein degradation rates.
- In vitro studies on human lung cancer cell lines.
- In vivo studies using human lung cancer xenografts.
Main Results:
- ad-p27mt exhibited increased resistance to degradation compared to ad-p27wt.
- ad-p27mt demonstrated stronger inhibition of lung cancer cell growth in vitro.
- Both ad-p27wt and ad-p27mt induced G1-S arrest and apoptosis, with ad-p27mt showing a more potent effect.
- Intratumoral injection of ad-p27mt led to partial tumor regression and superior inhibition of xenograft growth.
Conclusions:
- The mutated p27 protein (p27mt) is more stable and exhibits enhanced anti-cancer activity.
- ad-p27mt is a promising gene therapy agent with superior efficacy over ad-p27wt.
- ad-p27mt holds potential as a novel and powerful tool for lung cancer gene therapy.
