Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (Ͼ10 M) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK (75)) isolated for its ϳ20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1-3 M) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2-4 Gy) of 60 Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches. Cancer Gene Therapy (2000) 7, 879 -884
Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2Ј-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2Ј-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4 -2.3) compared with Adgal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose-(10 -100 M) and time-dependent (24 -48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5-to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100 -150 L) of virus delivered at rates of Յ1 L/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 Ͻ P Ͻ .009) tumor regression 6 days after radiation (30 Gy delivered as 2 ϫ 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas. Cancer Gene Therapy (2000) 7, 778 -788 Key words: Cancer gene therapy; drug delivery; halogenated pyrimidines; osmotic pump; radiation.T he halogenated pyrimidines (HPs) 5-bromo-2Ј-deoxyuridine (BrdU) and 5-iodo-2Ј-deoxyuridine (IdU) have been studied extensively as radiosensitizers of various mammalian cells in culture. [1][2][3] Preclinical and clinical studies have revealed that both BrdU and IdU are relatively efficient radiosensitizers if incorporated at sufficient levels into DNA. 4,5 However, although some clinical success has been achieved with head and neck tumors, attempts to radiosensitize gliomas with HPs have been disappointing, perhaps due to poor incorporation of the halogenated nucleosides into the DNA of these types of tumor cells. 6 Therefore, improved or alternative approaches are needed to treat this disease. The thymidine kinase (tk) gene of herpes simplex virus (HSV) has been used extensively to sensitize cancer cells to the HSV-TK-specific drug ganciclovir (GCV), and quite promising results with glioma have been obtained using various animal tumor mode...
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