title: New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression.
abstract: OBJECTIVE
  To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection.
  
  
  METHODS
  Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence.
  
  
  RESULTS
  Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Le(x)), Le(y) and Le(a) and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Le(x) and Le(a) glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.
  
  
  CONCLUSIONS
  These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Kathy
authors.initials: K
authors.lastname: Saraiva-Pava
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Nazanin
authors.initials: N
authors.lastname: Navabi
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Emma C
authors.initials: EC
authors.lastname: Skoog
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Sara K
authors.initials: SK
authors.lastname: Lindén
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Mónica
authors.initials: M
authors.lastname: Oleastro
authors.affiliation: Kathy Saraiva-Pava, Faculdade de Engenharia, Universidade Católica Portuguesa, Campus de Sintra, 2635-631 Rio de Mouro, Portugal.
authors.forename: Mónica
authors.initials: M
authors.lastname: Roxo-Rosa
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D020134:Catalytic Domain; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D004553:Electric Conductivity; D004790:Enzyme Induction; D004847:Epithelial Cells / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology; D005753:Gastric Mucosa / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology; D016480:Helicobacter pylori / Q000472:pathogenicity; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D010641:Phenotype; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014774:Virulence
chemical_list: D015415:Biomarkers; C508606:CXCL8 protein, human; D016209:Interleukin-8; C509186:TERT protein, human; D019098:Telomerase
keywords: Cellular model; Helicobacter pylori infection; Human gastric epithelium; NCI-N87 cells; Pathogenesis