Optimizing prostate cancer suicide gene therapy using herpes simplex virus thymidine kinase active site variants.
The herpes simplex virus (HSV) thymidine kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV thymidine kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.
Pantuck
Allan J
AJ
Department of Urology, School of Medicine, University of California-Los Angeles, Los Angeles, CA 90095, USA.
Matherly
Jamie
J
Zisman
Amnon
A
Nguyen
David
D
Berger
Frank
F
Gambhir
Sanjiv S
SS
Black
Margaret E
ME
Belldegrun
Arie
A
Wu
Lily
L
D000212:Acyclovir / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D017209:Apoptosis; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D018450:Disease Progression; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D006151:Guanosine / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009154:Mutation; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D011355:Prodrugs; D011419:Propidium / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000628:therapy*; D018139:Simplexvirus / Q000201:enzymology; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics*; D014055:Tomography, Emission-Computed; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D008164:Luminescent Proteins; D011355:Prodrugs; D013778:Tetrazolium Salts; D013844:Thiazoles; D006151:Guanosine; D049452:Green Fluorescent Proteins; D011419:Propidium; D013937:Thymidine Kinase; C022616:thiazolyl blue; D015774:Ganciclovir; D000212:Acyclovir