Development of gene therapy for advanced gastrointestinal cancer utilizing novel mutant p53 gene with effective nuclear translocation

为探讨用丙氨酸取代Ser376模拟P53去磷酸化状态的突变型P53基因(P53/S376A)的可行性，分别构建了表达新突变型P53基因的腺病毒载体(ADV)和表达野生型P53基因(WtP53)的腺病毒载体(ADV)。首先，我们研究了P53基因被AdCMVp/P53/S376A或AdCMV/wtP53感染的Hep3B细胞中P53基因的转位情况。免疫化学发光法和Western blotting分析表明，P53/S376A在感染后6h即进入细胞核，明显早于野生型P53。其次，检测突变型P53对肿瘤生长的抑制作用。与野生型P53相比，腺病毒转染型突变型P53能显著抑制肿瘤生长。在此基础上，进一步探讨突变型P53对小鼠皮下移植瘤的抗肿瘤作用。瘤内注射Adv表达的突变型P53与野生型P53相比，肿瘤消退明显，生存期延长。因此，利用这种新的突变型p53基因可以增强p53基因治疗癌症的抗肿瘤作用，并使肿瘤特异性启动子能够适用于克服启动子活性低的问题。

To investigate the feasibility of novel mutant p53 gene (p53/S376A), which mimics the dephosphorylated state p53 by substituting Ser376 with Ala, we constructed adenoviral vectors (AdV) expressing novel mutant p53, or wild type p53 (wtp53) under control of cytomegalovirus immediate early promoter/enhancer (CMVp), respectively resulting in AdCMVp/p53/S376A, AdCMV/wtp53. First we investigated the translocation of p53 after transduction into Hep3B cells, of which p53 status is null/null, infected by AdCMVp/p53/S376A or AdCMV/wtp53. Immunochemiluminescent and western blotting analysis showed that p53/S376A was translocated into nuclear 6 hours after infection clearly earlier translocation to nuclear than that of wild type p53. Next, the effect of the tumor growth suppression by mutant p53 was examined in vitro. The adenovirally-transfered mutant p53 could significantly suppress the tumor growth in comparison with that of wt p53. Extended these results, we then explored the antitumor effect of mutant p53 on subcutaneous xenograft mice model. Intratumoral administration of the AdV expressing mutant p53 showed significant tumor regression and prolongation of survival compared with that of wtp53. Thus, utilization of this novel mutant p53 gene may augment the antitumor effect of p53 gene therapy for cancer and enable tumor-specific promoter to be applicable overcoming the low promoter activity.