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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adenoviridae / genetics
-
Animals
-
Apoptosis / physiology
-
CDC2-CDC28 Kinases*
-
Carcinoma, Non-Small-Cell Lung / genetics
-
Carcinoma, Non-Small-Cell Lung / pathology
-
Carcinoma, Non-Small-Cell Lung / therapy
-
Cell Cycle Proteins / biosynthesis
-
Cell Cycle Proteins / genetics*
-
Cell Cycle Proteins / metabolism
-
Cell Division / physiology
-
Cyclin E / metabolism
-
Cyclin-Dependent Kinase 2
-
Cyclin-Dependent Kinase Inhibitor p27
-
Cyclin-Dependent Kinases / metabolism
-
DNA Fragmentation
-
Female
-
Flow Cytometry
-
G1 Phase / physiology
-
Genetic Therapy / methods*
-
Humans
-
Lung Neoplasms / genetics
-
Lung Neoplasms / pathology
-
Lung Neoplasms / therapy*
-
Mice
-
Mice, Inbred BALB C
-
Mice, Nude
-
Phosphorylation
-
Poly(ADP-ribose) Polymerases / metabolism
-
Protein Serine-Threonine Kinases / metabolism
-
Retinoblastoma Protein / metabolism
-
S Phase / physiology
-
Transduction, Genetic
-
Tumor Cells, Cultured
-
Tumor Suppressor Proteins*
-
Xenograft Model Antitumor Assays
Substances
-
Cdkn1b protein, mouse
-
Cell Cycle Proteins
-
Cyclin E
-
Retinoblastoma Protein
-
Tumor Suppressor Proteins
-
Cyclin-Dependent Kinase Inhibitor p27
-
Poly(ADP-ribose) Polymerases
-
Protein Serine-Threonine Kinases
-
CDC2-CDC28 Kinases
-
CDK2 protein, human
-
Cdk2 protein, mouse
-
Cyclin-Dependent Kinase 2
-
Cyclin-Dependent Kinases