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

ABSTRACT

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is a cytokine with direct antitumor activity. Clinical trials with TNF-alpha 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 apoptosis in response to a variety of different stimuli including TNF-alpha. We, therefore, evaluated the role of PKR in Ad-TNF-alpha-induced apoptosis in esophageal cancer cells. METHODS: A tetracycline-responsive adenoviral vector was used to transfect the TNF-alpha gene (Ad-TNF-alpha) into human esophageal cancer cell lines Bic1, Seg1 and TT, as well as in transformed PKR(+/+) and PKR(-/-) early-passage mouse embryo fibroblasts. Ad-luciferase, Ad-Bak, and mock infection with phosphate buffered saline solution were used as controls. Gene expression was determined by Western blot analysis. Apoptosis was detected by propidium iodide staining and fluorescence-activated cell sorter analysis. RESULTS: Overexpression of TNF-alpha in the lysate was evident in all cell lines treated with Ad-TNF-alpha. Treatment with Ad-TNF-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 apoptosis was mediated in part through a PKR-dependent process. CONCLUSIONS: These results suggest that Ad-TNF-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.