Abstract
To monitor therapeutic transgene expression, we developed fusion genes of enhanced green fluorescent protein (EGFP) with two different prodrug-activating enzyme genes: herpes simplex virus type 1 thymidine kinase (HSV-tk) and rabbit cytochrome P450 4B1 (cyp4b1). Expression of the resulting fusion proteins, TK-EGFP and 4B1-EGFP, rendered transduced human and rodent glioma cells sensitive to cytotoxic treatment with the corresponding prodrugs ganciclovir and 4-ipomeanol. Ganciclovir and 4-ipomeanol sensitivity was comparable with that achieved with the native HSV-TK and CYP4B1 proteins. As shown by fluorescence microscopy, TK-EGFP was expressed predominantly intranuclearly, whereas 4B1-EGFP was detectable in the cytoplasm, thereby displaying the orthotopic subcellular distribution of the corresponding native enzymes. The fluorescence intensity correlated well with the corresponding prodrug sensitivity, as shown by fluorescence-activated cell sorter analysis. EGFP expression was also used for the selection of stably HSV-tk-transduced cells by flow cytometric cell sorting. Resulting cell populations showed a homogeneity of fluorescence intensity similar to single-cell clones after antibiotic selection. In conclusion, tk-egfp and 4b1-egfp fusion genes are valuable tools for monitoring prodrug-activating gene therapy in living cells. EGFP fusion genes/proteins provide a simple and reproducible means for the detection, selection, and characterization of cells expressing enzyme genes for prodrug activation.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Steffens, S., Frank, S., Fischer, U. et al. Enhanced green fluorescent protein fusion proteins of herpes simplex virus type 1 thymidine kinase and cytochrome P450 4B1: Applications for prodrug-activating gene therapy. Cancer Gene Ther 7, 806–812 (2000). https://doi.org/10.1038/sj.cgt.7700173
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.cgt.7700173
Keywords
This article is cited by
-
Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells
Nuclear Medicine and Molecular Imaging (2010)
-
Suicide gene therapy of sarcoma cell lines using recombinant adeno-associated virus 2 vectors
Cancer Gene Therapy (2004)
-
Expression of a mutant hTERT in human bladder carcinoma cell line T24 and its clinical significance
Chinese Journal of Cancer Research (2004)
-
Differential cytotoxicity and bystander effect of the rabbit cytochrome P450 4B1 enzyme gene by two different prodrugs: Implications for pharmacogene therapy
Cancer Gene Therapy (2002)