IRF-1 transcriptionally up-regulates PUMA which mediates the mitochondrial apoptotic pathway in IRF-1 induced apoptosis in cancer cells

Interferon Regulatory Factor-1 (IRF-1) is a transcription factor which acts as a tumor suppressor and causes apoptosis in cancer cells. We evaluated IRF-1 induced apoptosis in gastric cancer cell lines. We established stable clones in AGS cells that have a tetracycline inducible IRF-1 expression system. We used these clones and recombinant adenovirus expressing IRF-1 to explore the mechanism of IRF-1 induced apoptosis in gastric cancer. Expression of IRF-1 causes apoptosis in gastric cancer cell lines as demonstrated by phosphatidylserine exposure and cleavage of caspase-8, caspase-3, and Bid with mitochondrial release of cytochrome c. However, inhibition of caspase-8 and Bid did not inhibit apoptosis and did not decrease cleaved caspase-9 or mitochondrial release of cytochrome c. We then demonstrate that IRF-1 up-regulates PUMA (p53 up-regulated modulator of apoptosis), that is known to activate apoptosis by the intrinsic pathway; this can be p53 independent. IRF-1 binds to distinct sites in the promoter of PUMA and activates PUMA transcription. Moreover, molecular markers of mitochondrial apoptosis are eliminated in PUMA knockout and knockdown cells and phospatidylserine exposure is decreased dramatically. Finally, we demonstrate that IFN-γ induces IRF-1 mediated up-regulation of PUMA in cancer cells. We conclude that IRF-1 can induce apoptosis by the intrinsic pathway independent of the extrinsic pathway by up-regulation of PUMA.