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 kinas e (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vect or 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 suici de gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovi r (GCV) (IC50=9.7 mu M), cells modified by MHV-72-TK did not show sensitivi ty to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-b romovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expres sing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZ T (0-375 mu M) and for BVDU (0-50 mu M). In contrast, 5'-fluoro-2'-deoxyuri dine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expr essing cells (IC50 value 2.1 mu M). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander e ffect between two heterologous cell types could be mediated by the MHV-72-T K/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 m u M) was added. The cell mixtures contained various ratios of cells express ing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing ce lls were needed to enhance mouse tumour cell killing and to decrease the su rvival 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 para llel, the same concentration of 5-FUdR dose only marginally inhibited tumou r 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-F UdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.