Glucose transport and apoptosis after gene therapy with HSV thymidine kinase

The relation between tumour metabolism and induction of apoptosis by gene t herapy was investigated in a rat Morris hepatoma (MH3924A) model expressing the HSV thymidine kinase (HSVtk) gene. In vivo the amount of glucose trans porter (GLUT1 and GLUT3 isoforms) expressing cells was determined in tumour s of untreated and treated animals using immunohistochemistry. In vitro upt ake studies with 2-fluoro-2-deoxy-D-glucose (FDG), 3-O-methylglucose and th ymidine (TdR) and a TUNEL (TdT-mediated dUTP nick end labelling) assay for the assessment of apoptosis were done immediately and 24 h after treatment of the recombinant cells with different doses of ganciclovir (GCV). Immunoh istochemistry revealed a significant increase in GLUT1 in treated tumours w hich showed enhanced transport activity for FDG. In vitro the FDG and 3-O-m ethylglucose uptake increased to 186% when compared with that of the non-tr eated cells immediately after incubation with GCV. However, 24 h later the FDG uptake had declined to its normal level, whereas the accumulation of 3- O-methylglucose remained elevated. The uptake of TdR, which was determined simultaneously, decreased in the acid-insoluble fraction of the cells to 27 % and 11%, respectively, immediately and 24 h after therapy, while in the a cid-soluble fraction it increased to 229% and to 167%. respectively. Employ ing the TUNEL technique, 25% of cells were found to be apoptotic 24 h after the termination of GCV treatment. Inhibition of glucose transport by cytoc halasin B or competition with deoxyglucose resulted in a 78% (cytochalasin B) and 88% (deoxyglucose) decrease in FDG uptake in the recombinant hepatom a cells and in an increase in the apoptotic cell fraction. It is concluded that inhibition of enhanced glucose transport in GCV-treated cells increase d apoptosis. Therefore, enhanced glucose transport seems to represent a str ess reaction of tumour cells dedicated for the prevention of cell death.