Replicating adenoviral vector-mediated transfer of a heat-inducible double suicide gene for gene therapy.
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 41 degrees C 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 41 degrees C 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).
Lee
Y J
YJ
Department of Pharmacology and Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. leeyj@msx.upmc.edu
Galoforo
S S
SS
Battle
P
P
Lee
H
H
Corry
P M
PM
Jessup
J M
JM
D000256:Adenoviridae / Q000235:genetics*; D015153:Blotting, Western; D002470:Cell Survival; D015179:Colorectal Neoplasms / Q000378:metabolism; D043525:Cytosine Deaminase; D004926:Escherichia coli / Q000201:enzymology; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D008297:Male; D009700:Nucleoside Deaminases / Q000378:metabolism; D010641:Phenotype; D011355:Prodrugs / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000378:metabolism; D013696:Temperature; D013937:Thymidine Kinase / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D018840:HSP70 Heat-Shock Proteins; D011355:Prodrugs; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase