HIGH-EFFICIENCY GENE-TRANSFER AND HIGH-LEVEL EXPRESSION OF WILD-TYPE P53 IN HUMAN LUNG-CANCER CELLS MEDIATED BY RECOMBINANT ADENOVIRUS

A replication-defective and helper-independent recombinant p53 adenovi rus was generated. The virus, Ad5CMV-p53, carries an expression casset te that contains human cytomegalovirus E1 promoter, human wild-type p5 3 cDNA, and SV40 early polyadenylation signal. Four human non-small-ce ll lung cancer cell lines representing differences in p53 configuratio n were used to evaluate the Ad5CMV-p53 virus. In the H358 cell line, w hich has a homozygous deletion of p53, the p53 gene was transferred wi th 97% to 100% efficiency, as detected by immunohistochemical analysis , when the cells were infected with Ad5CMV-p53 at a multiplicity of in fection of 30 to 50 plaque-forming units/cell. Western blots showed th at the p53 protein was expressed at a high level. The protein expressi on peaked at day 3 after infection and lasted for at least 15 days. Gr owth of the Ad5CMV-p53 virus-infected H358 cells was inhibited 79%, wh ereas that of noninfected cells or the cells infected with the control virus was not inhibited. Growth of cell line H322, which has a point mutation in p53, was inhibited 72% by Ad5CMV-p53, while that of cell l ine H460 containing wild-type p53 was less affected (28% inhibition). Tests in nude mice demonstrated that tumorigenicity of the Ad5CMV-p53- treated H358 cells was greatly inhibited. In a mouse model of orthotop ic human lung cancer, the tumorigenic H226Br cells, with a point mutat ion in p53, were inoculated intratracheally 3 days before the virus tr eatment. Intratracheal instillation of Ad5CMV-p53 prevented tumor form ation. These results suggest that adenovirus is an efficient vector fo r mediating transfer and expression of tumor suppressor genes in human cancer cells and that the Ad5CMV-p53 virus may be further developed i nto a therapeutic agent for use in cancer gene therapy.