| The use of tissue or tumor selective promoters in targeted gene therapy for cancer depends on strong and selective activity. Hexokinase type II (HK II) catalyzes the first committed step of glycolysis and is overexpressed in tumors, where it is no longer responsive to normal physiological inhibitors, e.g. glucagon. I show in a reporter gene assay activation of HK II in non-small cell lung carcinomas NCI-H661 and NCI-H460 at 61% and 40% of the activation observed with a constitutive promoter respectively, while it is only 0.9% in a number of different primary normal human bronchial epithelial cell lines (NHBEC). Similar results were observed in a variety of normal and tumor cells. Moreover, treatment of the transfectants with glucagon did not inhibit promoter/activation in the transformed H661 cells, while endogenous HK II in NHBEC is suppressed by glucagon, H460 and H661 cells infected with a recombinant adenovirus carrying a HK II/LacZ expression cassette, Ad HexLacZ, demonstrated beta-galactosidase activity, which correlated with the level of HK II promoter activation in these cells. Under similar conditions, no enzyme activity was observed in NHBEC. Cells were then infected with AdHexTk and treated with GCV. Our results demonstrate selectivity in toxicity with a 10--100 fold increase in IC 50 between lung cancer cells and NHBEC. There was also a 100-fold increase in IC50 in normal human mammary epithelial cells (NHMEC) relative to breast carcinoma cells MCF-7. This represents a novel use of the hexokinase type II as a selective promoter in cancer gene therapy. Other factors important in suicide gene therapy were explored. Pharmacological modulation of the bystander effect using 8-bromo-cAMP, observed in HSVTk/GCV suicide killing, was demonstrated to enhance killing efficacy by 50% when a small proportion of a target population was gene modified. The phenomenon of cellular resistance to HSVTk/GCV was examined and in a novel finding, we demonstrated dramatic interference from adenoviral proteins in cytotoxicity of this combination. Finally, based on indirect evidence suggesting ifosfamide activation by CYP3A4, I evaluated this combination as a potential new prodrug/suicide gene system. It was found to be inefficient at prodrug activation. Furthermore, co-expression of a reductase is not required, as has been suggested for CYP2B1, since reductase expression is induced by CYP3A4. |
