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Satoru Shiraso,
Yu Katayose,
Kuniharu Yamamoto,
Masamichi Mizuma,
Shinichi Yabuuchi,
Akira Oda,
Toshiki Rikiyama,
Tohru Onogawa,
Hiroshi Yoshida,
Hiroki Hayashi,
Hideo Ohtsuka,
Fuyuhiko Motoi,
Shinichi Egawa,
Junya Kato,
Michiaki Unno
[show abstract]
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ABSTRACT: The cyclin-dependent kinase inhibitor (CDK1) p27(kip1) is a negative regulator of cell cycling and has antitumor effects. In our previous study, the recombinant adenovirus expressing wild-type p27(kip1) (Adp27-wt) induced cell cycle arrest and apoptosis, and proved that p27 is a tumor suppressor gene like p53. Another adenovirus vector expressing mutant p27(kip1) (Adp27-mt), which inhibited degradation by the ubiquitin-proteasome system, showed increased protein stability and caused a stronger induction of apoptosis. Recently, the p27(kip1) protein binding with Jab1 (Jun activating binding protein 1) was found to translocate from the nucleus into the cytosol, and then become degraded by the 26S proteasome system. The inhibition of nuclear-cytoplasmic translocation increases the protein stability of p27(kip1) and p27(kip1) with a deletion of the Jab1-binding region (p27-jab-d) is not translocated and not degraded. Therefore, a new recombinant adenovirus (Adp27-jab-d) expressing p27-jab-d was made which was able to induce greater cytotoxicity. Adp27-jab-d inhibited the growth of human cholangiocarcinoma cell line (TFK-1) cells in vitro at 3.3 times (IC(50)) lower concentration than Adp27-wt. Moreover, in a xenografted severe combined immuno-deficient (SCID) mouse model injected with TFK-1 cells in the subcutaneous tissue, treatment by intratumor injection of Adp27-jab-d once a day for 3 days after the tumor was established, inhibited tumor growth more strongly than Adp27-wt or Adp27-mt and even induced tumor regression. However, the flow cytometric TUNEL assay showed little enhancement of apoptosis. Adp27-jab-d was thought to induce not only apoptosis but also necrosis, which was due to a specific effect of the Adp27-jab-d. Thus, by enhancing the cytotoxicity through inhibiting the translocaton of p27(kip1), p27(kip1) lacking the Jab1-binding region might be useful for cancer therapy. The control protein localization might also be a new target not only for cancer treatment, but also other diseases.
Anticancer research 07/2009; 29(6):2015-24. · 1.73 Impact Factor
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Tsuyoshi Sasaki,
Yu Katayose,
Kuniharu Yamamoto,
Masamichi Mizuma, Satoru Shiraso,
Shinichi Yabuuchi,
Akira Oda,
Toshiki Rikiyama,
Masaya Oikawa,
Toru Onogawa,
Masanori Suzuki,
Choon-Taek Lee,
Michiaki Unno
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the anti-tumor effects of a novel adenovirus expressing mutant p27(kip1) (Adp27-mt), which consists of a mutation of Thr-187/Pro-188 to Met-187/Ile-188.
Using the human breast cancer cell lines, MDA-MB-231, ZR-75-1, and MCF-7, we tested Adp27-mt for cell cycle assay, growth inhibition assay, and TdT-mediated dUTP-biotin nick end labeling in a human breast cancer-grafted severe combined immunodeficiency (SCID) mouse model.
The mutant p27(kip1) induced stronger apoptosis in the breast cancer cell lines than adenovirus expressing wild-type p27(kip1) (Adp27-wt). Adp27-mt inhibits cell growth significantly; being about 5- and 3.5-fold stronger for IC50 than Adp27-wt in the breast cancer cell lines, MDA-MD-231 and ZR-75-1, respectively. In the human breast cancer-grafted SCID mouse model, Adp27-mt induced tumor regression and antitumor effects significantly better than Adp27-wt. Furthermore, Adp27-mt mainly caused G2/M arrest in the cell cycle progression, whereas Adp27-wt mediated a G1/S arrest 48 h after infection.
The mutant p27(kip1) protein induced apoptosis, and inhibited cell growth more efficiently with stronger anti-tumor effects than wild-type p27(kip1). Thus, the recombinant adenovirus expressing mutant p27(kip1) could be useful in gene therapy against breast cancer.
Surgery Today 02/2007; 37(12):1073-82. · 1.22 Impact Factor
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[show abstract]
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ABSTRACT: 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.
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).
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.
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.
Hepato-gastroenterology 51(55):68-75. · 0.66 Impact Factor
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Kuniharu Yamamoto,
Yu Katayose,
Masanori Suzuki,
Michiaki Unno,
Tsuyoshi Sasaki,
Masamichi Mizuma, Satoru Shiraso,
Hideo Ohtuka,
Kenneth H Cowan,
Prem Seth,
Seiki Matsuno
[show abstract]
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ABSTRACT: 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.
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.
This is the first report to prove that Adp27-mediated apoptosis utilizes the Fas pathway by delivering Fas ligand to the cell surface.
Hepato-gastroenterology 50(54):1847-53. · 0.66 Impact Factor
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Masamichi Mizuma,
Yu Katayose,
Kuniharu Yamamoto, Satoru Shiraso,
Tsuyoshi Sasaki,
Shinichi Yabuuchi,
Akira Oda,
Kunihiro Masuda,
Toshiki Rikiyama,
Tohru Onogawa,
Hideo Ohtsuka,
Fuyuhiko Motoi,
Shinichi Egawa,
Michiaki Unno
[show abstract]
[hide abstract]
ABSTRACT: p27Kip1 is a cyclin-dependent kinase inhibitor which has been reported to be associated with invasion, metastasis and angiogenesis in malignant tumors, but its mechanism of action remains unknown. Here, it was examined whether p27Kip1 has an inhibitory effect on cancer cell invasion and correlates with matrix metalloproteinase expression (MMPs).
The human breast cancer cell line MDA-MB-231 and MDA-MB-231 transfectedp27Kip1 MDA-MB-p27 were used for the invasion assay, Western blotting and real-time quantitative RT-PCR.
In the invasion assay, the invasion of MDA-MB-p27 was significantly less than that of the parent cell line. In Western blotting analyses, the protein level of MMP-9 was also reduced in MDA-MB-p27. Furthermore, the activity of MMP-9 in cell culture supernatants was lower in MDA-MB-p27 as compared with enzyme-linked immunosorbent assays. In real-time quantitative RT-PCR, the mRNA level of MMP-9 was lower in MDA-MB-p27 cells.
Up-regulation of p27Kip1 remarkably inhibited the invasion of the breast cancer cells, in part due to the reduced expression of MMP-9. This is the first report of p27Kip1 modulating MMP-9 and indicating that p27Kip1 might play a key role in tumor cell invasion.
Anticancer research 28(5A):2669-77. · 1.73 Impact Factor
