Adenovirus expressing p27KIP1 induces apoptosis against cholangiocarcinoma cells by triggering Fas ligand on the cell surface.
OBJECTIVE
The prognosis of patients with cholangiocarcinoma is poor because of the difficulty of surgical curative resection. Therefore, other effective treatments must be developed especially those involving gene therapy. p27kip1, one of the cyclin-dependent kinase inhibitors, is known to limit proliferation of the cells. Our previous reports have shown that the overexpression of p27kip1 by a recombinant adenoviral vector expressing p27kip1 (Adp27) induces apoptosis. However, the mechanism of the Adp27-mediated apoptosis is not still resolved. Activation of the Fas pathway is one of the important gates for apoptosis. In this report, we examined whether p27kip1-induced apoptosis is closely related to the Fas/Fas ligand (FasL) system.


RESULTS
After infection of Adp27, flow cytometric analysis showed that Fas ligand was expressed on the cell surface of cholangiocarcinoma cell lines (TFK-1). In spite of detecting the cell surface expression of Fas ligand, overexpression of p27kip1 increased no amount of Fas ligand in mRNA by quantitative RT-PCR and protein level by Western blot. In addition, the immunocytochemical analysis showed that Fas ligand was adequately stored within the cytosol of TFK-1 cells. More interestingly, Adp27-induced apoptosis was completely inhibited by the neutralizing antibody of Fas ligand (NOK-1). This result suggests that overexpression of p27kip1 may deliver Fas ligand to the cell surface and mainly utilizes the Fas pathway as a gate of apoptosis.


CONCLUSIONS
This is the first report to prove that Adp27-mediated apoptosis utilizes the Fas pathway by delivering Fas ligand to the cell surface.
Yamamoto
Kuniharu
K
Department of Surgery, Division of Gastroenterological Surgery, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Katayose
Yu
Y
Suzuki
Masanori
M
Unno
Michiaki
M
Sasaki
Tsuyoshi
T
Mizuma
Masamichi
M
Shiraso
Satoru
S
Ohtuka
Hideo
H
Cowan
Kenneth H
KH
Seth
Prem
P
Matsuno
Seiki
S
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics*; D001650:Bile Duct Neoplasms / Q000473:pathology*; D001943:Breast Neoplasms / Q000473:pathology; D018797:Cell Cycle Proteins / Q000235:genetics*; D018281:Cholangiocarcinoma / Q000473:pathology*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D053222:Fas Ligand Protein; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D015316:Genetic Therapy; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured / Q000473:pathology*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D018797:Cell Cycle Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27