A previous study has shown that uracil DNA glycosylase SMUG1 efficiently removes 5-FU from DNA and in cells to generate AP sites that are repaired by APE1-mediated BER (80,105)
A previous study has shown that uracil DNA glycosylase SMUG1 efficiently removes 5-FU from DNA and in cells to generate AP sites that are repaired by APE1-mediated BER (80,105). the most aggressive and lethal solid malignancies, PDAC is usually highly resistant to chemotherapy and radiation and remains a major clinical challenge (2,3). The proto-oncogene, impacts the viability of pancreatic malignancy cells. Thus, the absolute importance of mutant expression in PDAC has driven multiple investigations to target it both at the transcriptional and translational levels (5,15,16). Several studies have exhibited that this promoter region of many cancer-driving oncogenes, including and expression (17,30C33). Furthermore, oxidative stress that induced guanine (G) oxidation in the regulatory G4 motif in was shown to upregulate expression (34). Despite these well-established functions of G4 in transcriptional regulation, little is known concerning the molecular machinery that regulates the formation and stability of these G4 structures in cells. Recently, we exhibited a genome-wide correlation between the occupancy of apurinic/apyrimidinic endonuclease 1 (APE1) and G4 structures in the cells (35). Originally FGFR1/DDR2 inhibitor 1 discovered as a DNA repair enzyme, human APE1 plays a central role in the repair of endogenous oxidative and alkylating DNA damage through the well-conserved base excision repair (BER) pathway (36,37). Later, APE1 was independently identified as a multifunctional protein involved not only in RHCE DNA damage repair but also in regulating gene expression by its redox activity and is hence referred to as reductionCoxidation factor 1 (Ref-1) (38). APE1 reduces redox-sensitive cysteine residues in many oxidized TFs, including AP-1 (39), nuclear factor kappa B?(40) and p53 (41), and increases their DNA binding by maintaining them in a reduced active state. Studies by us and others have also exhibited that APE1 can act as a direct transcriptional co-activator or co-repressor of several different genes involved in cell growth, proliferation and chemotherapeutic drug resistance (42C46). APE1 is usually overexpressed in a variety of cancers, including pancreatic (47), prostate (48), cervical (49), gliomas (50), ovarian (51,52), lung (53) and colon (54). This increased expression has been associated with increased tumor growth, cell migration and drug resistance, as well as patients poor prognosis (50,51,55,56). In this study, we demonstrate that APE1 is a G4-binding protein that plays a critical role in the formation of stable G4 structures in the genome and regulates expression in PDAC cells. Our study shows that recombinant APE1 binds to promoter G4 with high affinity expression in PDAC cell lines. Knockdown (KD)?of APE1 sensitizes PDAC cells to chemotherapy both and Red Starter Kit Mouse/Rabbit (Sigma-Aldrich, # DUO92101). Main antibodies used in western blot studies include mouse monoclonal anti-APE1 (1:5000; Novus Biologicals, Cat # NB100-116) and FGFR1/DDR2 inhibitor 1 mouse monoclonal anti-HSC70 (1:10?000; Santa Cruz Biotechnology, Cat # sc-7298). Main antibodies used for the chromatin immunoprecipitation (ChIP) assay include mouse monoclonal anti-APE1 (Novus Biologicals, Cat # NB100-116), mouse monoclonal anti-G4 clone 1H6 (Millipore Sigma, Cat # MABE1126), mouse monoclonal IgG2a anti-MAZ (133.7) (Santa Cruz Biotechnology, Cat # sc-130915) and mouse monoclonal IgG2a anti-PARP1 (F-2) (Santa Cruz Biotechnology, Cat # sc-8007). Reagents used in the study include RNase A from bovine pancreas (Sigma, Cat # R4875-100MG), pyridostatin hydrochloride (PDS; Sigma, Cat # SML2690), doxycycline (Dox;?Sigma, Cat # D9891), TMPyP4 (Millipore Sigma, Kitty # 613560), gemcitabine hydrochloride (Millipore Sigma, Kitty # G6423), oxaliplatin (Supelco, Kitty # PHR1528)?and 5-fluorouracil (5-FU;?Sigma, Kitty # F6627). All recombinant protein used in the analysis had been purified as referred to previously (58). All man made single-stranded DNA oligonucleotides, either unlabeled or 5-6-carboxyfluorescein (6-FAM) tagged, had been HPLC-purified quality and bought from Integrated DNA Technology in lyophilized type. These were dissolved in TE buffer to produce a stock focus of 100 M and kept at ?20C. Oligonucleotide brands and sequences are the following:?G4-forming KRAS oligo (5-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3) and non-G4-forming KRAS oligo (5-AGTTCGGTGTGTTAAGAGTTAAGAGTTGGAGG-3). Biological assets The individual embryonic kidney HEK-293T (ATCC, Kitty # CRL-3216), mutant KRAS expressing pancreatic ductal adenocarcinoma PANC-1 (ATCC, Kitty # CRL-3216) and MIA PaCa-2 (ATCC, Kitty # CRM-CRL-1420) cell lines had been taken care of in high-glucose Dulbeccos customized Eagles moderate (DMEM; Thermo FGFR1/DDR2 inhibitor 1 Fisher Scientific, Kitty # 11965084) supplemented with 10% fetal bovine serum (FBS; Sigma, Kitty # F2442) and an antibiotic combination of 100 U/ml penicillin and 100 g/ml streptomycin (Gibco-BRL). The wild-type (WT) KRAS expressing BxPC-3 (ATCC, Kitty # CRL-1687) PDAC cell range was taken care of in RPMI 1640 moderate (1) (Gibco, Kitty # A10491-01) supplemented with 10% FBS and an antibiotic combination of 100 U/ml penicillin and 100 g/ml streptomycin. All cell lines had been authenticated by STR DNA profiling by Genetica DNA Laboratories, Burlington, NC. For APE1 KD research, PANC-1 and MIA PaCa-2 cell lines stably expressing APE1shRNA or non-targetable control (NTC) shRNA had been taken care of in 10% FBS supplemented DMEM with 1 g/ml puromycin. To create.