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

为了研究新型突变型p53基因（p53/S376A）的可行性，我们构建了在巨细胞病毒立即早期启动子/增强子（CMVp）控制下表达新型突变型p53和野生型p53（wtp53）的腺病毒载体（AdV），分别得到AdCMVp/p53/S376A和AdCMV/wtp53。首先，我们研究了p53转导到Hep3B细胞后的易位，其中p53状态为null/null，感染AdCMVp/p53/S376A或AdCMV/wtp53。免疫荧光和Western blotting分析表明，p53/S376A在感染后6h即发生核转位，明显早于野生型p53。接着，在体外检查突变型p53对肿瘤生长的抑制作用。与野生型p53相比，腺病毒转染的突变型p53能显著抑制肿瘤生长。在此基础上，我们进一步探讨了突变型p53对皮下移植瘤小鼠的抗肿瘤作用。与wtp53相比，肿瘤内施用表达突变型p53的AdV显示出显著的肿瘤消退和生存期延长。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效果，并使肿瘤特异性启动子能够克服低启动子活性的缺点。

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.