Construction of Recombinant p53 Adenovirus ( Ad5CMVp53 ) for Cervical Cancer Gene Therapy

An, W S; Han, Y J; Cho, S D; Kim, J W; Lee, J M; Namkoong, S E; Kim, S J; Kim, J K; Park, Y S; Yang, J M; Park, S H

Abstract

Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit reninoblastoma protein (RB). The p53 protein was first identified in the late 1970s as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein was capable of suppressing the tumorigenic phenotype and regulating cell cycle. Human adenoviruses (Ad) are double-stranded DNA viruses with genome sizes of approximately 36 kb. This group of viruses is easy to grow and manupulate, and they exhibit a broad host range in vivo and in vitro. Adenovirus vectors for gene transfer have distinct advantages when compared with vectors derived from other DNA viruses or from retroviruses. In order to introduce wild type p53 into a cervical cancer for gene therapy, We had constructed p53 recombinant adenovirus (Ad5CMVp53). Ad5CMVp53 was created by inserting the cytomegalovirus (CMV) promoter, human wild-type p53 cDNA, and bovine growth hormone (BGH) polyadenylation signal in a minigene cassette into the E1 deleted region of the modified adenovirus type 5. A method for generating p53 recombinant adenovirus was carried out by using lipofectin-mediated cotransfection and by directly observing for the cytopathic effect (CPE) caused by recombinant adenovirus in transfected 293 cells. A direct analysis by polymeraschain reaction (PCR) on samples from the supernatant of cell cultures with CPE was also carried out, which made identification of newly generated recombinant virus rapid and specific. Finally, we had succeeded for generating recombinant adenovirus by using lipofectin transfection and then monitoring for CPE and confirmed recombianant p53 adenovirus by PCR.