| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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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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