Tumor cells that express a fusion gene of Escherichia coli cytosine deaminase ( CD ) and herpes simplex virus type 1 thymidine kinase ( TK ) sequences activate and are subsequently killed by the nontoxic prodrugs 5 -fluorocytosine and ganciclovir. We have previously developed a recombinant adenovirus containing the CD -TK fusion gene controlled by the human inducible heat shock protein 70 promoter so that heat at 418C for 1 hour induces therapeutic gene expression. This adenovirus effectively transduces heat -inducible expression of the CD -TK gene into human prostate carcinoma cells. However, because a limited number of cells in a tumor can actually be infected, we created a replicating adenoviral vector to increase CD -TK gene expression. This vector is a replication -competent, E1B -attenuated adenoviral vector containing the hsp70 promoter ± driven CD -TK gene ( Ad.E1A( + )HS -CDTK ). When human prostate adenocarcinoma DU -145 cells ( mutant p53 ) were infected with the virus at a multiplicity of infection ( MOI ) of 1 or 10, the viral replication was detected within 2 days at both MOIs. Similar results were observed in human colorectal carcinoma CX -1 cells. When DU -145 cells were infected with the virus at an MOI of 10, incubated for 24 hours, heated at 418C for 4 hours, and then harvested 20 hours later, Western blot analysis demonstrated that this virus successfully produced viral E1A proteins and heat shock stimulated the CD -TK gene expression by 12.3 -fold. In addition, Ad.E1A( + )HS -CDTK effectively suppressed cell proliferation by viral cytopathic effect ). Unlike with a replication -incompetent virus ( Ad.HS -CDTK ), the cytopathic effect of the virus and cytotoxicity in the presence of the prodrugs were still observed even at low MOI ( MOI = 1.0 ). Cancer Gene Therapy ( 2001 ) 8, 397 ± 404

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