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

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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.