title: Macrophages, nitric oxide and microRNAs are associated with DNA damage response pathway and senescence in inflammatory bowel disease.
abstract: BACKGROUND
  Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation.
  
  
  RESULTS
  Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn's disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression.
  
  
  CONCLUSIONS
  Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.
authors.forename: Jane J
authors.initials: JJ
authors.lastname: Sohn
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Schetter
authors.forename: Harris G
authors.initials: HG
authors.lastname: Yfantis
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Ridnour
authors.forename: Izumi
authors.initials: I
authors.lastname: Horikawa
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Khan
authors.forename: Ana I
authors.initials: AI
authors.lastname: Robles
authors.forename: S Perwez
authors.initials: SP
authors.lastname: Hussain
authors.forename: Akiteru
authors.initials: A
authors.lastname: Goto
authors.forename: Elise D
authors.initials: ED
authors.lastname: Bowman
authors.forename: Lorne J
authors.initials: LJ
authors.lastname: Hofseth
authors.forename: Jirina
authors.initials: J
authors.lastname: Bartkova
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Gerald N
authors.initials: GN
authors.lastname: Wogan
authors.forename: David A
authors.initials: DA
authors.lastname: Wink
authors.forename: Curtis C
authors.initials: CC
authors.lastname: Harris
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics*; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003093:Colitis, Ulcerative / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D003106:Colon / Q000201:enzymology / Q000473:pathology; D003424:Crohn Disease / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006657:Histones / Q000378:metabolism; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000201:enzymology / Q000235:genetics* / Q000473:pathology*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006657:Histones; D035683:MicroRNAs; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D017346:Protein Serine-Threonine Kinases; C518294:CBX3 protein, human; C067980:CD68 antigen, human; D000097382:CD68 Molecule; C468783:H2AX protein, human; C496319:NOS2 protein, human; C578634:CHEK2 protein, human; C578633:Chek2 protein, mouse