Viral HSV1-TK gene, radiolabeled FIAU, and ganciclovir : combined gene targeted radiotherapy and suicide gene therapy for prostate cancer

The strategy of suicide gene therapy in cancer is based on the idea of enabling tumour cells, by gene transfer, to convert a non-toxic pro-drug into a toxic product. Previous work has shown that the combination of herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) transfer with the pro-drug ganciclovir (GCV) to be a promising suicide gene therapy in cancer. Unlike several other gene therapy systems, early-phase clinical trials of this strategy have shown encouraging results. Therefore, methods to improve its therapeutic efficacy are urgently sought. The thymidine analogue 5-iodo-2’-fluoro-2’-deoxy-1-s-D-arabino-furonosyluracil (FIAU) is an alternative substrate of the HSV1-TK enzyme. The iodine atom of FIAU can be substituted with radioactive iodine, for example; [123I]-iodine, and thereby utilised for the delivery of ionising radiation into tumour cells expressing the viral tk gene. The aim of this study was primarily to investigate the therapeutic potential of combining HSV1-tk gene transfer and [123I]FIAU for the targeted radiation cytotherapy of prostate cancer cells alone or in combination with GCV. The HSV1-tk gene was cloned into the plasmid vector pcDNA3.1. This plasmid, driven by the ubiquitous promoter of CMV, was then used to transfect the prostate cancer cell line DU145 and the glioma cell line UVW. A viral TK positive, commercially available cell line derived from osteosarcoma (143B-TK) along with its TK-negative clone were also used for comparison. The viral tk gene transfection efficiency was assessed by three independent methods. Firstly, the uptake of [123I]FIAU normalised to the uptake of tritiated thymidine ([methyl-3H]TdR); secondly, GCV sensitivity, assessed by the MTT assay; and thirdly, by the detection of HSV1-tk gene by RT-PCR. The highest specific activity of [123I]FIAU was obtained by the use of a no-carrier added method of synthesis. The cytotoxicity of [123I]FIAU was assessed by clonogenic assay after incubating monolayers of parental and TK-positive clones of the cell lines with a range of doses of [123I]FIAU for the periods of 4 h, 8 h and a period equal to their doubling times. The effect of this treatment on cell cycle progression was assessed by FACS analysis after staining the cellular DNA with propodium iodide. Combination therapy using GCV and [123I]FIAU for the treatment of TK-positive clones of the prostate cancer cell line DU-145 and the osteosarcoma cell line 143B was assessed by the method of median effect analysis and combination index. Monolayers were treated with a constant ratio of various doses of [123I]FIAU for 4h or GCV for 72h. The combination therapy followed three different timing schedules of GCV-before-[123I]FIAU, [123I]FIAU-before-GCV, or simultaneous therapy. The expression of HSV1-tk gene by the three cell lines was confirmed by the three methods described above. For instance, the TK positive clone of the cell line DU145 exhibited 4.25 ± 0.15 times higher [123I]FIAU/ [methyl-3H]TdR uptake ratio and 43 times higher sensitivity to GCV compared with the parental cell line. The three cell lines demonstrated sensitivity to radiolabelled FIAU, which was significantly enhanced by HSV1-tk gene expression. This sensitivity was time-, dose-, and proliferation-dependent. Maximum cell kill was achieved when the monolayers were exposed to [123I]FIAU for a period equavelant to the cellular doubling time. For example, the sensitivity enhancement factor by tk gene expression of the cell line DU145 increased from 5.2 to 7.6 when the treatment period was prolonged from 4 h to 26 h (doubling time of DU145). Following the treatment with [123I]FIAU for a period equal to the doubling time, cells were arrested at G2/M phase of the cell cycle. For instance, 49% of DU145-TK cells treated with 1 MBq/ml for 26 h were at G2/M phase compared with 21.9% of the untreated cells. In contrast, incubation of DU145-TK or 143B-TK cell lines with lethal doses of [123I]FIAU for 4 h and GCV for 72 h had no significant effect on cell cycle progression. Comparison of the effectiveness of [123I]FIAU in the monolayer and spheroid cultures indicated that clonogenic cell kill resulting from Auger electron bombardment was restricted to targeted rather than bystander cells. The combination therapy of [123I]FIAU and GCV of the cell line DU145-TK resulted in antagonistic effect throughout the examined dose range of the schedules of FIAU-before-GCV and simultaneous therapy and the low toxicity concentration range (lower surviving fractions) of the GCV-before-FIAU schedule. The high toxicity concentration range of the latter schedule has shown evidence of additive effect. For the osteosarcoma cell line 143B-TK, synergistic effect was observed at the high toxicity concentration range of the three combination schedules and antagonism at the low toxicity concentration range of the combinations. We concluded from this in vitro study that the combination of HSV1-tk gene transfer and the delivery of radiolabelled FIAU is a promising strategy for targeted radiation cytotherapy of prostate cancer. This proliferation-dependent therapy has caused significant cell cycle arrest that warrants further investigation. Furthermore, the combination of GCV and radiolabelled FIAU for the treatment of tumour cells expressing the gene of viral TK resulted in a dose- and schedule-dependent synergism. We believe that these encouraging results should be substantiated by in vivo experiments in the near future.