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

p27^<Kip1>是一种周期蛋白依赖性激酶抑制剂，在细胞周期中调节细胞从G1期进入S期的进程。在一些恶性肿瘤中，p27^<Kip1>的缺失与疾病进展和不良结果相关。我们利用pcDNA3.1-p27^<Kip1> wt、pcDNA3.1-p27^<Kip1> mt和Skp2或Jab1反义寡核苷酸（AS）在体外和体内研究了分子靶向治疗人口腔鳞状细胞癌p27^<Kip1>基因的机制。我们构建了表达突变型p27^<Kip1>基因（pcDNA3.1-p27^<Kip1> mt）的表达载体，将Thr-187/Pro-188 （ACGCCC）突变为Met-187/ il -188 (ATGATC)，不受泛素介导降解的影响，提高了p27^<Kip1>蛋白的稳定性。此外，我们使用反义Skp2或Jab1寡核苷酸抑制p27^<Kip1>蛋白的降解。电穿孔法将它们分别转染到口腔癌细胞B88和HSY中。为了估计用这种方法减少每个ca . More . More的异种移植物，我们测量了裸鼠电穿孔后的异种移植物的大小。TUNEL法观察凋亡细胞。采用免疫组化方法对p27^<Kip1>、Skp2和Jab1蛋白进行免疫染色。所有处理均抑制B88和HSY细胞的生长。生长抑制是由pcDNA3.1-p27^<Kip1> mt或Skp2 AS或Jab1 AS介导的，其特异性是由于细胞凋亡的显著诱导，其特征是细胞核断裂增加和caspase-3的激活。pcDNA3.1-p27^<Kip1> mt， Skp2 AS和Jab1 AS诱导了对异种移植肿瘤的强烈生长抑制。此外，组织学标本显示突变型p27^<Kip1>转染的肿瘤细胞凋亡比野生型或空载体增加。以同样的方式，组织学标本显示，skp2或Jab1 as处理的肿瘤中凋亡细胞死亡比每个scramble对照组增加。在实验期间，各治疗组均未观察到体重减轻，也未观察到皮肤区域出现烧伤。这些发现表明，p27^<Kip1>-mt、Skp2 AS和Jab1 AS有潜力成为一种新的强大的基因治疗工具，p27^<Kip1>蛋白的稳定性为口腔鳞状细胞癌患者提供了治疗益处。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