2004 Fiscal Year Final Research Report Summary
Development of molecular targeting therapy against p27^<Kip1> in oral squamous cell carcinoma.
| Project/Area Number |
15592116
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Surgical dentistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
HARADA Koji University of Tokushima, Graduate School, Institute of Health Bioscience, Therapeutic Regulation for Oral Tumor, Assistant lecturer, 大学院・ヘルスバイオサイエンス研究部, 助手 (60253217)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Wild type p27^<Kip1> / Mutant type p27^<Kip1> / Skp2 / Jab1 / Expression vector / Antisense oligonucleotide / Molecular targeting therapy |
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| Research Abstract |
p27^<Kip1> is a cyclin-dependent kinase inhibitor which regulates the progression of cell from the G1 into S phase in a cell cycle. Loss of p27^<Kip1> is associated with disease progression and an unfavorable outcome in several malignancies. We have now investigated the mechanism of molecular targeting therapy p27^<Kip1> gene in human oral squamous cell carcinoma using pcDNA3.1-p27^<Kip1> wt, pcDNA3.1-p27^<Kip1> mt and Skp2 or Jab1 antisense oligonucleotides (AS) in vitro and in vivo. We constructed an expression vector expressing mutant type p27^<Kip1> gene (pcDNA3.1-p27^<Kip1> mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), which is not influenced by ubiquitin-mediated degradation for increasing stability of p27^<Kip1> protein. In addition, we used the antisense Skp2 or Jab1 oligonucleotides for suppressing the degradation of p27^<Kip1> protein. We transfected them into oral cancer cells, B88 and HSY by electroporation. To estimate the reduction of each ca
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ncer xenograft by this method, we measured the size of xenografts in nude mice after electroporation with them. Apoptotic cells were investigated TUNEL method. Immunostaining of p27^<Kip1>, Skp2 and Jab1 protein was performed by immunohistochemistry.
All of treatments inhibited the growth of B88 and HSY cells. The growth inhibition was mediated by pcDNA3.1-p27^<Kip1> mt or Skp2 AS or Jab1 AS specifically due to a significant induction of apoptosis characterized by an increase in fragmentation of nuclei and activation of caspase-3. pcDNA3.1-p27^<Kip1> mt, Skp2 AS and Jab1 AS induced a strong growth inhibition of xenograft tumors. Moreover, histological specimens revealed apoptotic cell death was increased in mutant type p27^<Kip1>-transfected tumors than wild type or empty vector only. In the same way, histological specimens revealed apoptotic cell death was increased in skp2 or Jab1 AS-treated tumors than each scramble control. During the experimental period, no loss of body weight was observed in each treatment group, and that no skin region including a burn also was observed.
These findings suggest that p27^<Kip1>-mt, Skp2 AS and Jab1 AS have the potential to become a novel and powerful gene therapy tool, and stability of p27^<Kip1> protein offer therapeutic benefits in patients with oral squamous cell carcinoma cells. Less
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