Development of molecular targeting therapy against p27^<Kip1> in oral squamous cell carcinoma.

P27^&lt；Kip1&gt；是一种细胞周期蛋白依赖性的激酶抑制物，在细胞周期中调节细胞从G1期进入S期。在几种恶性肿瘤中，p27；&lt；Kip1和gt；的缺失与疾病的进展和不良结局有关。我们在体内外用pcDNA3.1-p27；kip1&gt；wt、pcDNA3.1-p27^&lt；kip1&gt；mt和Skp2或Jab1反义寡核苷酸(AS)研究了p27^&lt；Kip1&gt；基因靶向治疗口腔鳞癌的机制。我们构建了表达突变型p27^&lt；Kip1&gt；基因的表达载体，将Thr-187/Pro-188(ACGCCC)突变为Met-187/Ile-188(ATGATC)，不受泛素介导的降解影响，提高了p27^&lt；Kip1&gt；蛋白的稳定性。此外，我们还使用反义Skp2或Jab1寡核苷酸来抑制p27^&lt；Kip1&gt；蛋白的降解。我们通过电穿孔的方法将其导入口腔癌细胞B88和HSY。估算每个ca…的减少量通过这种方法，我们测量了电穿孔后的裸鼠移植瘤的大小。用原位末端标记法检测细胞凋亡率。免疫组织化学方法检测p27^&lt；Kip1&Gt；、Skp2和Jab1蛋白的表达，所有药物均能抑制B88和HSY细胞的生长。这种生长抑制作用是由pcDNA3.1-p27^&lt；Kip1&gt；Mt或Skp2 AS或Jab1 AS介导的，这是由于显著诱导了以核碎裂增加和caspase-3激活为特征的细胞凋亡。PcDNA3.1-p27；Kip1&gt；Mt、Skp2As和Jab1As对移植瘤有较强的生长抑制作用。此外，组织学标本显示，与野生型或空载体相比，突变型p27^&lt；Kip1&gt；转染型肿瘤细胞的凋亡率增加。同样，组织学标本显示，在Skp2或Jab1 AS处理的肿瘤中，凋亡细胞死亡比每一种混杂对照都增加。在实验期间，各治疗组均未观察到体重减轻，也未观察到包括烧伤在内的皮肤区域。这些结果表明，p27；；kip1；-mT、Skp2 as和jab1 as有可能成为一种新的、强大的基因治疗工具，p27；；kip1&gt；蛋白的稳定性为口腔鳞状细胞癌患者提供了治疗优势。较少

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 … More 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