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  • Acquired Diseases
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Selection of HSV-1 TK gene-transfected murine mammary carcinoma cells resistant to (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and ganciclovir (GCV)

Gene Therapy volume 7, pages 1543–1552 (2000)Cite this article

Abstract

We evaluated the molecular mechanism of resistance in herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene-transfected murine mammary carcinoma (FM3ATK−/HSV-1 TK+) cells, that were selected for resistance against (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and ganciclovir (GCV) by prolonged exposure of the cell cultures to dose-escalating concentrations of these compounds. Drug-resistant FM3ATK−/HSV-1 TK+ cells showed marked differences in their sensitivity spectrum to a series of antiherpetic nucleoside analogues. BVDU-resistant FM3ATK−/HSV-1 TK+ cells displayed the same sensitivity profile as wild-type FM3A/0 cells. In contrast, GCV-resistant FM3ATK−/HSV-1 TK+ cells were still sensitive to BVDU, (E)-5-(2-iodovinyl)-2′-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2′-deoxycytidine (BVDC), a typical feature of FM3ATK− cells lacking cytosolic TK. Southern blot and PCR analysis revealed that HSV-1 TK genes were not deleted from the genome of the drug-resistant FM3ATK−/HSV-1 TK+cells. However, the TK genes in drug-resistant FM3ATK−/HSV-1 TK+cells were shown to be heavily methylated. Accordingly, RT-PCR demonstrated the complete abrogation of TK mRNA production resulting in a complete loss of TK enzyme activity in drug-resistant FM3ATK−/HSV-1 TK+ cells.

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Acknowledgements

We thank Christiane Callebaut and Inge Aerts for dedicated editorial help and Lizette van Berckelaer for excellent technical help. This work was supported by Project G0140–98 from the Flemish ‘Fonds Voor Wetenschappelijk Onderzoek’, Project 00/12 from the Flemish ‘Geconcerteerde Onderzoeksacties’, and the ‘Belgische Federatie tegen kanker’. Bart Degrève is the recipient of a fellowship from the ‘Belgische Federatie tegen kanker’.

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  1. Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium

    B Degrève, E De Clercq & J Balzarini

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Degrève, B., De Clercq, E. & Balzarini, J. Selection of HSV-1 TK gene-transfected murine mammary carcinoma cells resistant to (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) and ganciclovir (GCV). Gene Ther 7, 1543–1552 (2000). https://doi.org/10.1038/sj.gt.3301278

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