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Tumor necrosis factor α (TNFα) shows a strong direct anti-tumor activity. However, severe side effects of systemic administration of TNFα have limited clinical trials with this cytokine. High doses of TNFα can be delivered by an adenoviral vector without the severe systemic toxicity but the mechanism of apoptosis induction is not well defined. PKR and NF-kB activation have been described following TNF-α protein administration. We, therefore, evaluated the role of PKR and NF-kB following Ad-TNFα transduction in esophageal cancer cells. A tetracycline responsive adenoviral vector was used to transfect the TNFα gene (Ad-TNFα) into human esophageal cancer cell lines Bic1, Seg1 and TT, as well as in transformed PKR +/+ and PKR -/- early passage mouse embryo fibroblasts (MEF). The proapoptotic gene Bak (Ad-Bak), Ad-Luciferase (Ad-Luc) and mock infection with PBS were used as controls. The serine kinase inhibitor 2-aminopurine was used to chemically inhibit PKR activation. Poly dI:dC (dsRNA) was used to induce PKR. Gene expression was determined by Western Blot analysis. Apoptosis was detected by propidium iodide staining, FACS analysis and PARP cleavage. NF-kB activation was determined by electrophoretic mobility shift assay. TNFα was overexpressed in the lysate of all esophageal cancer cell lines treated with Ad-TNFα but not in those infected with control vectors. Treatment of esophageal cancer cells with Ad-TNFα was associated with PKR upregulation, increased expression of the pro-apoptotic gene Bax, and induction of apoptosis in the Bic1 and TT cell lines but not the Seg1 cell line. PKR +/+ MEFs showed increased sensitivity to Ad-TNFα as opposed to Ad-Bak induced apoptosis compared to PKR -/- MEFs. Western Blot analysis showed a dose dependent downregulation of PKR and its downstream target, phosphorylated eIF2α, as well as downregulation of the pro-apoptotic gene Bax when cells were treated with 2-aminopurine. An electrophoretic mobility shift assay (EMSA) was performed to investigate the role of NF-kB in Ad-TNFα mediated apoptosis, and failed to show NF-kB activation 24, 48 and 72 h following Ad-TNFα infection. These data suggest that Ad-TNFα-mediated apoptosis in esophageal cancer cell lines may be dependent on PKR activation. However, the role of NF-kB activation in Ad-TNFα gene therapy remains unclear. Strategies to enhance PKR upregulation may allow increased Ad-TNFα anti-tumoral activity in the treatment of esophageal cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]