Role for the double-stranded RNA-activated protein kinase PKR in Ad-TNF-α gene therapy in esophageal cancer

Background. Tumor necrosis factor alpha (TNT-alpha) is a cytokine with direct antitumor activity. Clinical trials with TAT-a have been limited because of the severe side effects of systemic administration. Gene therapy with an adenoviral vector allows delivery of high local doses of TNF-alpha. Activation of protein kinase R (PKR) has been implicated as a general transducer of apaptosis in response to a variety of different stimuli including TNT-alpha. We, therefore, evaluated the role of PKR in Ad-TNT alpha-induced apoplosis in esophageal cancer cells. Methods. A tetracycline-responsive adenoviral vector was used, to transfect the TAT-a gene (Ad-TNF-alpha) into human esophageal cancer cell lines Bic1, Seg1 and TT, as well (is in transformed PKR+/+ and PYR-/- early-passage mouse embryo fibroblasts. Ad-luciferase, Ad-Bak, and mock infection with buffered saline solution were used as controls. Gene expression was determined by Western blot phosphate analysis. Apoptosis was detected by propidium iodide staining and fluorescence activated cell sorter analysis. Results. Overexpression of TAT-alpha in the lysate was evident in all cell lines treated with Ad-TNT-alpha. Treatment with Ad-TNT alpha was associated with PKR-upregulation and induction of apoptosis. Inhibition of TNF-alpha expression by tetracycline resulted in downregulation of PKR and decreased apoptosis. Transduction of PKR+/+, and PKR-/- mouse embryo fibroblasts with Ad-TNF-alpha demonstrated that Ad-TNF-alpha-induced opoptosis was mediated in part through, a PKR-dependent process. Conclusions. These results suggest that Ad-TNT-alpha-mediated apoptosis in esophageal cancer cell lines is dependent in part on PKR upregulation. Strategies to enhance PKR upregulation may allow increased Ad-TNF-alpha antitumoral activity in the treatment of esophageal cancer.