VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death

Background The inefficiency of herpes simplex virus thymidine kinase (TK) g ene transfer and toxicity of ganciclovir (GCV) at high concentrations in vi vo limits the use of this suicide gene therapy approach for the treatment o f cancers in clinical settings, To overcome the problem, we have sought evi dence of amplification of cytotoxicity by co-transfer of the TK gene fused with the gene encoding HSV-1 structural protein VP22 which has a remarkable ability fbr intercellular trafficking. Methods The expression of the fusion proteins from the chimeric VP22-TK or VP22-EGFP genes was shown by Western blot and VP22 promoted TK or EGFP inte rcellular trafficking by an indirect immunofluorescent assay. The cytotoxic ity was demonstrated by a colorimetric cell proliferation assay followed by an assessment of the bystander effect on admixtures of transfected with no n-transfected naive cells. Results Our results show the expression of the VP22 fusion proteins and the ir spread to varying numbers of bystander cells (up to 30, observed in viab le cells with VP22-EGFP as well as after methanol fixation), confirming tha t VP22 assisted intercellular trafficking of the fusion proteins. This VP22 promoted TK spreading resulted in killing by 2.5 mug/ml GCV of virtually a ll cells in cultures that had been transfected at an efficiency of only 27. 5%. In contrast, fewer than 80% of cells were killed when transfected with 'tk alone' at the same efficiency. The cell killing effect was exponentiall y dependent on GCV concentration in cells transfected with 'tk alone' at GC V concentrations between 0.25 and 0.5 mug/ml, but not those transfected wit h VP22-TK, probably due to the continuously variable, high sensitivity of a bout 50% of cells. Even at low concentration of GCV (0.2 mug/ml), the enhan cement of cell killing by VP22 was four-fold higher in cells transfected wi th VP22-TK than in cells tranfected with 'tk alone'. Conclusions VP22 enhanced intercellular trafficking of TK and amplified the TK/GCV killing effect, especially in the lower range of GCV concentrations . This offers a new strategy to enhance the effectiveness of suicide gene t herapy for the treatment of cancers. Copyright (C) 2001 John Wiley & Sons, Ltd.