School Of Basic And Applied Sciences
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Item Elevated level of acetylation of APE1 in tumor cells modulates DNA damage repair(Impact Journals LLC, 2016) Sengupta, S.; Mantha, Anil K.; Song, H.; Roychoudhury, S.; Nath, S.; Ray, S.; Bhakat, K.K.Apurinic/apyrimidinic (AP) sites are frequently generated in the genome by spontaneous depurination/depyrimidination or after removal of oxidized/modified bases by DNA glycosylases during the base excision repair (BER) pathway. Unrepaired AP sites are mutagenic and block DNA replication and transcription. The primary enzyme to repair AP sites in mammalian cells is AP endonuclease (APE1), which plays a key role in this repair pathway. Although overexpression of APE1 in diverse cancer types and its association with chemotherapeutic resistance are well documented, alteration of posttranslational modification of APE1 and modulation of its functions during tumorigenesis are largely unknown. Here, we show that both classical histone deacetylase HDAC1 and NAD+-dependent deacetylase SIRT1 regulate acetylation level of APE1 and acetylation of APE1 enhances its AP-endonuclease activity both in vitro and in cells. Modulation of APE1 acetylation level in cells alters AP site repair capacity of the cell extracts in vitro. Primary tumor tissues of diverse cancer types have higher level of acetylated APE1 (AcAPE1) compared to adjacent non-tumor tissue and exhibit enhanced AP site repair capacity. Importantly, in the absence of APE1 acetylation, cells accumulate AP sites in the genome and show increased sensitivity to DNA damaging agents. Together, our study demonstrates that elevation of acetylation level of APE1 in tumor could be a novel mechanism by which cells handle the elevated levels of DNA damages in response to genotoxic stress and maintain sustained proliferation.Item Human apurinic/apyrimidinic endonuclease (APE1) is acetylated at DNA damage sites in chromatin, and acetylation modulates its DNA repair activity(American Society for Microbiology, 2016) Roychoudhury, S.; Nath, S.; Song, H.; Hegde, M.L.; Bellot, L.J.; Mantha, Anil K.; Sengupta, S.; Ray, S.; Natarajan, A.; Bhakat, K.K.Apurinic/apyrimidinic (AP) sites, the most frequently formed DNA lesions in the genome, inhibit transcription and block replication. The primary enzyme that repairs AP sites in mammalian cells is the AP endonuclease (APE1), which functions through the base excision repair (BER) pathway. Although the mechanism by which APE1 repairs AP sites in vitro has been extensively investigated, it is largely unknown how APE1 repairs AP sites in cells. Here, we show that APE1 is acetylated (AcAPE1) after binding to the AP sites in chromatin and that AcAPE1 is exclusively present on chromatin throughout the cell cycle. Positive charges of acetylable lysine residues in the N-terminal domain of APE1 are essential for chromatin association. Acetylation-mediated neutralization of the positive charges of the lysine residues in the N-terminal domain of APE1 induces a conformational change; this in turn enhances the AP endonuclease activity of APE1. In the absence of APE1 acetylation, cells accumulated AP sites in the genome and showed higher sensitivity to DNA-damaging agents. Thus, mammalian cells, unlike Saccharomyces cerevisiae or Escherichia coli cells, require acetylation of APE1 for the efficient repair of AP sites and base damage in the genome. Our study reveals that APE1 acetylation is an integral part of the BER pathway for maintaining genomic integrity. ? 2017 Roychoudhury et al.Item An in vitro study ascertaining the role of H2O2 and glucose oxidase in modulation of antioxidant potential and cancer cell survival mechanisms in glioblastoma U-87 MG cells(Springer New York LLC, 2017) Cholia, Ravi P.; Kumari, Sanju; Kumar, Saurabh; Kaur, Manpreet; Kaur, Manbir; Kumar, Raj; Dhiman, Monisha; Mantha, Anil K.Glial cells protect themselves from the elevated reactive oxygen species (ROS) via developing unusual mechanisms to maintain the genomic stability, and reprogramming of the cellular antioxidant system to cope with the adverse effects. In the present study non-cytotoxic dose of oxidants, H2O2 (100??M) and GO (10??U/ml) was used to induce moderate oxidative stress via generating ROS in human glioblastoma cell line U-87 MG cells, which showed a marked increase in the antioxidant capacity as studied by measuring the modulation in expression levels and activities of superoxide dismutase (SOD1 and SOD2) and catalase (CAT) enzymes, and the GSH content. However, pretreatment (3?h) of Curcumin and Quercetin (10??M) followed by the treatment of oxidants enhanced the cell survival, and the levels/activities of the antioxidants studied. Oxidative stress also resulted in an increase in the nitrite levels in the culture supernatants, and further analysis by immunocytochemistry showed an increase in iNOS expression. In addition, phytochemical pretreatment decreased the nitrite level in the culture supernatants of oxidatively stressed U-87 MG cells. Elevated ROS also increased the expression of COX-2 and APE1 enzymes and pretreatment of Curcumin and Quercetin decreased COX-2 expression and increased APE1 expression in the oxidatively stressed U-87 MG cells. The immunocytochemistry also indicates for APE1 enhanced stress-dependent subcellular localization to the nuclear compartment, which advocates for enhanced DNA repair and redox functions of APE1 towards survival of U-87 MG cells. It can be concluded that intracellular oxidants activate the key enzymes involved in antioxidant mechanisms, NO-dependent survival mechanisms, and also in the DNA repair pathways for glial cell survival in oxidative-stress micro-environment. ? 2017, Springer Science+Business Media, LLC.Item Understanding the multifaceted role of ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) and its altered behaviour in human diseases(Bentham Science Publishers B.V., 2015) Cholia, R.P.; Nayyar, H.; Kumar, R.; Mantha, Anil K.Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) also known as Autotaxin, is a secreted lysophospholipase D, which hydrolyzes lysophosphatidylcholine (LPC) into Lysophosphatidic acid (LPA). LPA is the bioactive product of ENPP2 enzyme, which induces diverse signalling pathways via six LPA-G-protein coupled receptors (GPCRs). ENPP2 is an essential protein for normal development and its altered expression is associated with various human diseases. Cellular ENPP2 silencing results in lethality at the embryonic stage in mice. Initially, it is identified as an autocrine factor in melanoma cells. Different research groups are currently exploring to understand the multifaceted role of ENPP2 in various processes such as embryonic and neural development, migration, invasion, differentiation, proliferation, angiogenesis, and survival. Altered expression of ENPP2 is also associated with various diseases like inflammation, cancer, fibrosis, rheumatoid arthritis and neural defects. In this article, we have summarized structural aspects of ENPP2 and biochemical functions associated with its diverse cellular roles in various human diseases including cancer and Alzheimer's disease (AD). In addition, keeping in view and advocating findings, a note on various phytochemicals and synthetic inhibitors, which are currently explored as therapeutic agents targeting functions of ENPP2 for the treatment of various human diseases is also presented. ? 2015 Bentham Science Publishers.