Hexokinase type II: a novel tumor-specific promoter for gene-targeted therapy differentially expressed and regulated in human cancer cells.

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. We 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%, respectively, relative to the activation observed with a constitutive promoter, while it was only 0.9% in different preparations of primary normal human bronchial epithelial cells (NHBECs). 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 NHBECs is suppressed by glucagon. H460 and H661 cells infected with a recombinant adenovirus carrying an HK II/LacZ expression cassette, AdHexLacZ, demonstrated beta-galactosidase activity that correlated with the level of HK II promoter activation in these cells. Under similar conditions, no enzyme activity was observed in NHBECs. Cells were then infected with AdHexTk and treated with GCV. Our results demonstrate selectivity in toxicity, with a 10- to 100-fold increase in IC50 between lung cancer cell lines H661 and H460, respectively, and NHBECs. There was also a 100-fold increase in IC50 in NHMECs relative to breast carcinoma cell line MCF-7. In HepG2 cells, an IC50 of 1 microg/ml was observed, comparable to that of other tumor cell lines. This represents a novel use of the hexokinase type II as a selective promoter in cancer gene therapy.

Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.

The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.