Adenovirus expressing mutant p27kip1 enhanced apoptosis against cholangiocarcinoma than adenovirus-p27kip1 wild type.
OBJECTIVE
The prognosis of cholangiocarcinoma is extremely poor despite the aggressive multidisciplinary cancer therapies that have been used clinically. Thus, it is imperative to develop new and effective treatments, such as gene therapy in order to treat this disease. p53 is the most common target for cancer gene therapy treatment. However, cholangiocarcinoma has a low frequency of p53 mutation, which makes this protein a poor candidate for gene therapy in this disease and another suitable gene therapy target must be found. p27kip1 is a universal cyclin-dependent kinase (CDK) inhibitor that blocks cell cycle progression and inhibits proliferation. Our previous reports have demonstrated the role of p27kip1 in the induction of apoptosis using a recombinant adenoviral vector expressing p27kip1 (Adp27) in several different human cancer cells. p27kip1 is regulated by two mechanisms composed of a ubiquitin-proteasome system and proteolytic processing system that requires the phosphorylation of p27kip1 on Thr-187 by the cyclinE/Cdk2 complex followed by proteolytic degradation.


METHODS
In this study, we focused our aim on the degradation of p27kip1 protein mediated by a recombinant adenoviral expressing a mutant p27kip1 (Adp27-mt), which has a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC).


RESULTS
We observed that the mutated p27kip1 markedly inhibited ubiquitination and the subsequent degradation of p27kip1 when compared to wild type p27kip1. Consequently, we found that mutated p27kip1 induced a stronger induction of apoptosis and cell growth inhibition than wild type p27kip1 in cholangiocarcinoma cell lines TFK-1 and HuCCT-1. Furthermore, we demonstrated that Adp27-mt mainly caused G2/M arrest in the cell cycle progression and a decreased cyclinB1 and Cdc2 protein where as Adp27-wt mediated a G1/S arrest at 48 hours after infection.


CONCLUSIONS
In this study, we showed that adenoviral vector expression of mutant p27kip1 protein inhibited degradation by the ubiquitin-proteasome system and strongly induced apoptosis and cell growth inhibition compared to wild type p27kip1. Thus recombinant adenovirus expressing mutant p27kip1 may be potentially useful for gene therapy against cholangiocarcinoma.
Sasaki
Tsuyoshi
T
Department of Gastroenterological Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan.
Katayose
Yu
Y
Suzuki
Masanori
M
Yamamoto
Kuniharu
K
Shiraso
Satoru
S
Mizuma
Masamichi
M
Unno
Michiaki
M
Takeuchi
Heigo
H
Lee
Choon-Taek
CT
Matsuno
Seiki
S
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis; D001650:Bile Duct Neoplasms / Q000628:therapy*; D001653:Bile Ducts, Intrahepatic; D018797:Cell Cycle Proteins / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000628:therapy*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D015316:Genetic Therapy; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D007763:Lac Operon / Q000235:genetics; D011233:Precipitin Tests; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D018797:Cell Cycle Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases