RESUMEN
PURPOSE: We developed a prostate cancer tumor model capable of being noninvasively imaged using positron emission tomography (PET) based on expression of the herpes simplex virus thymidine kinase (HSV1-tk) reporter gene. MATERIALS AND METHODS: The androgen independent, metastatic prostate cancer cell lines CL1 and CL1-GFP were stably transfected with the mutant HSV1-tk gene pcDNA3.1/pCMV-sr39tk, which has increased ability to phosphorylate penciclovir. The presence of the sr39tk gene product was analyzed by Western blot analysis and relative thymidine kinase enzyme activity was assessed by a functional thymidine kinase enzyme activity assay. Subcutaneous and orthotopic CL1 and CL1-SR39 tumor xenografts were established in SCID mice. The ability to image CL1-SR39 was assessed using fluorodeoxyglucose and F-penciclovir ( F-FHBG) micro-PET (a rodent PET scanner). To investigate the systemic distribution of intratumoral sr39tk injections established CL1 tumors were transiently injected with first generation adenoviral vectors carrying the sr39tk gene under control of the strong cytomegalovirus promoter Ad-CMV-HSV1-sr39tk and imaged using micro-PET. RESULTS: Transfection of sr39tk into CL1 cells was successful. CL1-SR39 thymidine kinase enzyme activity was greater than twice the activity of the glioma cell line C6-SR39 control and above the threshold necessary for micro-PET detection. Fluorodeoxyglucose micro-PET in SCID mice was positive for CL1 and CL1-SR39 tumors. Selective micro-PET of subcutaneous CL1-SR39 tumors was done using F-FHBG. Micro-PET imaging after systemic and intratumoral injection of Ad-CMV-HSV1-sr39tk revealed significant systemic transgene leakage with significant hepatic expression of sr39TK protein. CONCLUSIONS: Molecular based imaging of sr39tk transfected prostate cancer tumors and adenoviral delivered HSV1-tk suicide gene therapy based on the selective conversion and intracellular trapping of F-FHBG by sr39tk is feasible. Potential applications include noninvasive monitoring of the location, duration and intensity of gene constructs, which may contribute to the safety of clinical gene therapy protocols, and noninvasive imaging of the prostate cancer xenograft response to experimental therapy.
Asunto(s)
Aciclovir/análogos & derivados , Terapia Genética , Neoplasias de la Próstata/terapia , Tomografía Computarizada de Emisión , Aciclovir/metabolismo , Animales , Western Blotting , Fluorodesoxiglucosa F18 , Genes Reporteros , Vectores Genéticos , Guanina , Herpesvirus Humano 1/genética , Humanos , Masculino , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/enzimología , Radiofármacos , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
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.