Sheila E. Crowe

Hypoxia-inducible factor 1 (HIF1) consists of a hypoxia-inducible α subunit and a constitutively expressed β subunit. Reactive oxygen species (ROS) induced by Helicobacter pylori stabilize HIF1α in the human gastric epithelium in normoxia. HIF1α plays crucial role in carcinogenesis and has been associated with malignant progression of gastric cancer. Several genes contain functional hypoxia-response elements (HREs) in their promoters including Bcl2 family member, Mcl1. Cellular ratios of antiapoptotic oncogenic protein, Mcl1, and tumor suppressor proapoptotic protein, Noxa, determine cell fate by regulating normal cellular growth, cell death and oncogenic processes. The aim of the present study was to examine the mechanism of HIF1α induction in the H. pylori -infected gastric epithelium to better understand disease pathogenesis by H. pylori relevant to gastric carcinogenesis. Our data showed that the dose-dependent increase in HIF1α in H. pylori -infected gastric epithelia is mediated by induction of a ROS-inducible protein, apurinic/apyrimidinic endonuclease 1 (APE1), and an enhanced interaction of APE1 with the transcriptional coactivator p300. Surprisingly, with accumulation of HIF1α, further transcriptional activation of mcl1 was not observed. We identified a HIF-binding site (HBS) in the hif1α promoter and showed that increased HIF1α expression, whether H. pylori -induced or hypoxia-mimetic agent, CoCl₂-induced, resulted in enhanced HIF1α binding to its own promoter. This resulted in a transcriptionally inactive hif1α promoter since hif1α HBS lacks HIF ancillary sequence (HAS) required for HIF1 transcriptional activity. We conclude that enhanced binding of “nonfunctional” HIF1α to hif1α promoter and limiting availability of p300 in the cell serves as checkpoints for uncontrolled HIF1α activity.

Human apurinic/apyrimidinic endonuclease-1 (APE-1), a key enzyme involved in repair of oxidative DNA base damage, is an important transcriptional coregulator. We previously reported that Helicobacter pylori infection induces apoptosis and increases APE-1 expression in human gastric epithelial cells (GEC). Although both the DNA repair activity and the acetylation-mediated transcriptional regulation of APE-1 are required to prevent cell death, the mechanisms of APE-1–mediated inhibition of infection-induced apoptosis are unclear. Here, we show that short hairpin RNA–mediated stable suppression of APE-1 results in increased apoptosis in GEC after H. pylori infection. We show that programmed cell death involves both the caspase-9–mediated mitochondrial pathway and the caspase-8–dependent extrinsic pathway by measuring different markers for both the pathways. Overexpression of wild-type APE-1 in APE-1–suppressed GEC reduced apoptosis after infection; however, overexpression of the DNA repair mutant or the nonacetylable mutant of APE-1 alone was unable to reduce apoptosis, suggesting that both DNA repair and acetylation functions of APE-1 modulate programmed cell death. We show for the first time that the DNA repair activity of APE-1 inhibits the mitochondrial pathway, whereas the acetylation function inhibits the extrinsic pathway during H. pylori infection. Thus, our findings establish that the two different functions of APE-1 differentially regulate the intrinsic and the extrinsic pathway of H. pylori–mediated GEC apoptosis. As proapoptotic and antiapoptotic mechanisms determine the development and progression of gastritis, gastric ulceration, and gastric cancer, this dual regulatory role of APE-1 represents one of the important molecular strategies by H. pylori to sustain chronic infection. Cancer Res; 70(7); 2799–808