Ranajoy Chattopadhyay

Oxidative Stress: An Essential Factor in the Pathogenesis of Gastrointestinal Mucosal Diseases

Reactive oxygen species (ROS) are generated as by-products of normal cellular metabolic activities. Superoxide dismutase, glutathione peroxidase, and catalase are the enzymes involved in protecting cells from the damaging effects of ROS. ROS are produced in response to ultraviolet radiation, cigarette smoking, alcohol, nonsteroidal anti-inflammatory drugs, ischemia-reperfusion injury, chronic infections, and inflammatory disorders. Disruption of normal cellular homeostasis by redox signaling may result in cardiovascular, neurodegenerative diseases and cancer. ROS are produced within the gastrointestinal (GI) tract, but their roles in pathophysiology and disease pathogenesis have not been well studied. Despite the protective barrier provided by the mucosa, ingested materials and microbial pathogens can induce oxidative injury and GI inflammatory responses involving the epithelium and immune/inflammatory cells. The pathogenesis of various GI diseases including peptic ulcers, gastrointestinal cancers, and inflammatory bowel disease is in part due to oxidative stress. Unraveling the signaling events initiated at the cellular level by oxidative free radicals as well as the physiological responses to such stress is important to better understand disease pathogenesis and to develop new therapies to manage a variety of conditions for which current therapies are not always sufficient.

Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating 35S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.

Regulatory Role of Human AP-Endonuclease (APE1/Ref-1) in YB-1-Mediated Activation of the Multidrug Resistance Gene MDR1†

ABSTRACT Human AP-endonuclease (APE1/Ref-1), a central enzyme involved in the repair of oxidative base damage and DNA strand breaks, has a second activity as a transcriptional regulator that binds to several trans-acting factors. APE1 overexpression is often observed in tumor cells and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to such agents. Because the involvement of APE1 in repairing the DNA damage induced by many of these drugs is unlikely, drug resistance may be linked to APE1s transcriptional regulatory function. Here, we show that APE1, preferably in the acetylated form, stably interacts with Y-box-binding protein 1 (YB-1) and enhances its binding to the Y-box element, leading to the activation of the multidrug resistance gene MDR1. The enhanced MDR1 level due to the ectopic expression of wild-type APE1 but not of its nonacetylable mutant underscores the importance of APE1s acetylation in its coactivator function. APE1 downregulation sensitizes MDR1-overexpressing tumor cells to cisplatin or doxorubicin, showing APE1s critical role in YB-1-mediated gene expression and, thus, drug resistance in tumor cells. A systematic increase in both APE1 and MDR1 expression was observed in non-small-cell lung cancer tissue samples. Thus, our study has established the novel role of the acetylation-mediated transcriptional regulatory function of APE1, making it a potential target for the drug sensitization of tumor cells.

Stimulation of NEIL2-mediated Oxidized Base Excision Repair via YB-1 Interaction during Oxidative Stress

The recently characterized enzyme NEIL2 (Nei-like-2), one of the four oxidized base-specific DNA glycosylases (OGG1, NTH1, NEIL1, and NEIL2) in mammalian cells, has poor base excision activity from duplex DNA. To test the possibility that one or more proteins modulate its activity in vivo, we performed mass spectrometric analysis of the NEIL2 immunocomplex and identified Y box-binding (YB-1) protein as a stably interacting partner of NEIL2. We show here that YB-1 not only interacts physically with NEIL2, but it also cooperates functionally by stimulating its base excision activity by 7-fold. Moreover, YB-1 interacts with the other NEIL2-associated BER proteins, namely, DNA ligase IIIα and DNA polymerase β and thus could form a large multiprotein complex. YB-1, normally present in the cytoplasm, translocates to the nucleus during UVA-induced oxidative stress, concomitant with its increased association with and activation of NEIL2. NEIL2-initiated base excision activity is significantly reduced in YB-1-depleted cells. YB-1 thus appears to have a novel regulatory role in NEIL2-mediated repair under oxidative stress.

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1s nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1s N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1s nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.

Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis. METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays. RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE. CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

Cellular and molecular mechanisms of cigarette smoke-induced lung damage and prevention by vitamin C

BackgroundCigarette smoke-induced cellular and molecular mechanisms of lung injury are not clear. Cigarette smoke is a complex mixture containing long-lived radicals, including p-benzosemiquinone that causes oxidative damage. Earlier we had reported that oxidative protein damage is an initial event in smoke-induced lung injury. Considering that p-benzosemiquinone may be a causative factor of lung injury, we have isolated p-benzosemiquinone and compared its pathophysiological effects with cigarette smoke. Since vitamin C is a strong antioxidant, we have also determined the modulatory effect of vitamin C for preventing the pathophysiological events.MethodsVitamin C-restricted guinea pigs were exposed to cigarette smoke (5 cigarettes/day; 2 puffs/cigarette) for 21 days with and without supplementation of 15 mg vitamin C/guinea pig/day. Oxidative damage, apoptosis and lung injury were assessed in vitro, ex vivo in A549 cells as well as in vivo in guinea pigs. Inflammation was measured by neutrophilia in BALF. p-Benzosemiquinone was isolated from freshly prepared aqueous extract of cigarette smoke and characterized by various physico-chemical methods, including mass, NMR and ESR spectroscopy. p-Benzosemiquinone-induced lung damage was examined by intratracheal instillation in guinea pigs. Lung damage was measured by increased air spaces, as evidenced by histology and morphometric analysis. Oxidative protein damage, MMPs, VEGF and VEGFR2 were measured by western blot analysis, and formation of Michael adducts using MALDI-TOF-MS. Apoptosis was evidenced by TUNEL assay, activation of caspase 3, degradation of PARP and increased Bax/Bcl-2 ratio using immunoblot analysis and confocal microscopy.ResultsExposure of guinea pigs to cigarette smoke resulted in progressive protein damage, inflammation, apoptosis and lung injury up to 21 days of the experimental period. Administration of 15 mg of vitamin C/guinea pig/day prevented all these pathophysiological effects. p-Benzosemiquinone mimicked cigarette smoke in causing protein modification and apoptosis in vitro and in A549 cells ex vivo as well as apoptosis and lung damage in vivo. All these pathophysiological events were also prevented by vitamin C.Conclusionp-Benzosemiquinone appears to be a major causative factor of cigarette smoke-induced oxidative protein damage that leads to apoptosis and lung injury. The pathophysiological events are prevented by a moderately large dose of vitamin C.

Dual Regulation by Apurinic/Apyrimidinic Endonuclease-1 Inhibits Gastric Epithelial Cell Apoptosis during Helicobacter pylori Infection

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.

Regulation of Noxa-mediated apoptosis in Helicobacter pylori–infected gastric epithelial cells

Helicobacter pylori induces the antiapoptotic protein myeloid cell leukemia 1 (Mcl1) in human gastric epithelial cells (GECs). Apoptosis of oncogenic protein Mcl1‐expressing cells is mainly regulated by Noxa‐mediated degradation of Mcl1. We wanted to elucidate the status of Noxa in H. pylori‐infected GECs. For this, various GECs such as AGS, MKN45, and KATO III were either infected with H. pylori or left uninfected. The effect of infection was examined by immunoblotting, immunoprecipitation, chromatin immunoprecipitation assay, in vitro binding assay, flow cytometry, and confocal microscopy. Infected GECs, surgical samples collected from patients with gastric adenocarcinoma as well as biopsy samples from patients infected with H. pylori showed significant up‐regulation of both Mcl1 and Noxa compared with noninfected samples. Coexistence of Mcl1 and Noxa was indicative of an impaired Mcl‐Noxa interaction. We proved that Noxa was phosphorylated at Ser13 residue by JNK in infected GECs, which caused cytoplasmic retention of Noxa. JNK inhibition enhanced Mcl1‐Noxa interaction in the mitochondrial fraction of infected cells, whereas overexpression of nonphosphorylatable Noxa resulted in enhanced mitochondria‐mediated apoptosis in the infected epithelium. Because phosphorylation‐dephosphorylation can regulate the apoptotic function of Noxa, this could be a potential target molecule for future treatment approaches for H. pylori‐induced gastric cancer.—Rath, S., Das, L., Kokate, S. B., Pratheek, B. M., Chattopadhyay, S., Goswami, C., Chattopadhyay, R., Crowe, S. E., Bhattacharyya, A., Regulation of Noxa‐mediated apoptosis in Helicobacter pylori–infected gastric epithelial cells. FASEB J. 29, 796–806 (2015). www.fasebj.org