The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro.
To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.
Raslova
H
H
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Matis
J
J
Rezuchova
I
I
Macakova
K
K
Berebbi
M
M
Kudelova
M
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D005467:Floxuridine / Q000494:pharmacology / Q000633:toxicity; D018142:Gammaherpesvirinae / Q000201:enzymology* / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology* / Q000633:toxicity; D018259:Herpesvirus 1, Human / Q000201:enzymology; D051379:Mice; D009705:Nucleosides / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D015215:Zidovudine / Q000494:pharmacology / Q000633:toxicity
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D009705:Nucleosides; D011355:Prodrugs; D005467:Floxuridine; C020235:brivudine; D015215:Zidovudine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D015774:Ganciclovir