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) gene transfer and toxicity of ganciclovir (GCV) at high concentrations in vivo limits the use of this suicide gene therapy approach for the treatment of cancers in clinical settings. To overcome the problem, we have sought evidence 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 for 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 intercellular trafficking by an indirect immunofluorescent assay. The cytotoxicity was demonstrated by a colorimetric cell proliferation assay followed by an assessment of the bystander effect on admixtures of transfected with non-transfected naive cells.


RESULTS
Our results show the expression of the VP22 fusion proteins and their spread to varying numbers of bystander cells (up to 30, observed in viable cells with VP22-EGFP as well as after methanol fixation), confirming that VP22 assisted intercellular trafficking of the fusion proteins. This VP22 promoted TK spreading resulted in killing by 2.5 microg/ml GCV of virtually all 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 exponentially dependent on GCV concentration in cells transfected with 'tk alone' at GCV concentrations between 0.25 and 0.5 microg/ml, but not those transfected with VP22-TK, probably due to the continuously variable, high sensitivity of about 50% of cells. Even at low concentration of GCV (0.2 microg/ml), the enhancement of cell killing by VP22 was four-fold higher in cells transfected with VP22-TK than in cells transfected 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 therapy for the treatment of cancers.
Liu
C S
CS
Gene Therapy Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Australia.
Kong
B
B
Xia
H H
HH
Ellem
K A
KA
Wei
M Q
MQ
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D015774:Ganciclovir / Q000494:pharmacology*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000502:physiology*
D000998:Antiviral Agents; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D013937:Thymidine Kinase; D015774:Ganciclovir