New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

Kathy D. Saraiva-Pava, Nazanin Navabi, Emma C Skoog, Sara K. Linden, Mónica Oleastro, Mónica Roxo-Rosa

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Abstract

AIM: 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. CONCLUSION: 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.
Original languageEnglish
Pages (from-to)6526-6542
Number of pages18
JournalWorld Journal of Gastroenterology
Volume21
Issue number21
DOIs
Publication statusPublished - 7 Jun 2015

Fingerprint

Telomerase
Stomach
Clone Cells
Helicobacter pylori
Periodic Acid
Mucins
Enzyme Precursors
Coculture Techniques
Phenotype
Cell Line
Polysaccharides
Virulence
Mucin 5AC
ABO Blood-Group System
Staining and Labeling
Pepsinogen A
Antigens
Alcian Blue
Intercellular Junctions
human TERT protein

Keywords

  • Helicobacter pylori infection
  • Pathogenesis
  • Human gastric epithelium
  • Cellular model
  • NCI-N87 cells

Cite this

Saraiva-Pava, Kathy D. ; Navabi, Nazanin ; Skoog, Emma C ; Linden, Sara K. ; Oleastro, Mónica ; Roxo-Rosa, Mónica . / New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression. In: World Journal of Gastroenterology. 2015 ; Vol. 21, No. 21. pp. 6526-6542.
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New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression. / Saraiva-Pava, Kathy D.; Navabi, Nazanin; Skoog, Emma C; Linden, Sara K.; Oleastro, Mónica; Roxo-Rosa, Mónica .

In: World Journal of Gastroenterology, Vol. 21, No. 21, 07.06.2015, p. 6526-6542.

Research output: Contribution to journalArticle

TY - JOUR

T1 - New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

AU - Saraiva-Pava, Kathy D.

AU - Navabi, Nazanin

AU - Skoog, Emma C

AU - Linden, Sara K.

AU - Oleastro, Mónica

AU - Roxo-Rosa, Mónica

N1 - info:eu-repo/grantAgreement/FCT/3599-PPCDT/82565/PT# info:eu-repo/grantAgreement/FCT/3599-PPCDT/125876/PT# Supported by Grants from the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal), No. PPCDT/SAL-IMI/57297/2004 and No. PTDC/BIM-MEC/1051/2012; The Swedish Cancer foundation; The Swedish Research Council, No. K2010-79X-21372-01-3; Forska utan djurforsok, Animal Free Research; and by Research fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia (Portugal).

PY - 2015/6/7

Y1 - 2015/6/7

N2 - AIM: 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. CONCLUSION: 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.

AB - AIM: 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. CONCLUSION: 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.

KW - Pathogenesis

KW - BARRIER

KW - ESTABLISHMENT

KW - HELICOBACTER-PYLORI INFECTION

KW - IN-VITRO MODEL

KW - DISEASE

KW - E-CADHERIN

KW - PATHOGENESIS

KW - Helicobacter pylori infection

KW - CANCER CELL-LINES

KW - Human gastric epithelium

KW - NCI-N87 cells

KW - IMMORTALIZATION

KW - Cellular model

KW - MUCIN

KW - Helicobacter pylori infection

KW - Pathogenesis

KW - Human gastric epithelium

KW - Cellular model

KW - NCI-N87 cells

U2 - 10.3748/wjg.v21.i21.6526

DO - 10.3748/wjg.v21.i21.6526

M3 - Article

VL - 21

SP - 6526

EP - 6542

JO - World Journal of Gastroenterology

JF - World Journal of Gastroenterology

SN - 1007-9327

IS - 21

ER -