Fusion of herpes simplex virus thymidine kinase to VP22 does not result in intercellular trafficking of the protein.
Suicide gene therapy is a promising approach for the treatment of cancer. Current protocols, however, suffer from low efficiency. We tried to alleviate this problem by developing a transgene that will spread from the initially transduced cell to the surrounding cells (transmission). We used herpes simplex virus (HSV) VP22 as a signal for cellular uptake of HSV-1 thymidine kinase (TK). By co-culturing naive cells with cells producing a TK-VP22 fusion protein, we detected intercellular trafficking of this protein. We used a variety of techniques, including two-color flow cytometry and cytotoxicity assays to detect the presence of TK in the non-producing cells. We confirmed intercellular migration of VP22. We did not detect any intercellular trafficking of the TK-VP22 fusion protein, by various fixation methods or flow cytometry. In ganciclovir sensitivity assays, we found no difference between the efficiency of TK (IC50=3.15+/-0.76 microg/ml) and TK-VP22 (IC50=2.27+/-0.59 microg/ml). Using a cell-free enzyme activity assay we showed that fusion of TK to VP22 did not change the enzyme activity. In conclusion, we described novel and robust methods to detect intercellular trafficking. From our data we concluded that protein transmission of TK by VP22 for gene therapy is not likely to be successful. In addition, we described a useful and quantifiable assay to measure the enzymatic activity of TK and TK fusion proteins, and described some common properties of VP22 fusion proteins that may explain the different results that have been obtained by others.
Beerens
A M J
AM
Department of Therapeutic Gene Modulation, University Centre for Pharmacy, University of Groningen, 9700 AD Groningen, The Netherlands.
Rots
M G
MG
de Vries
E F J
EF
Haisma
H J
HJ
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology; D041722:Genes, Transgenic, Suicide; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D007150:Immunohistochemistry; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D019076:Transgenes; D015678:Viral Structural Proteins / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22; D013937:Thymidine Kinase; D015774:Ganciclovir