Published online Jun 7, 2015. doi: 10.3748/wjg.v21.i21.6526
Peer-review started: October 15, 2014
First decision: January 8, 2015
Revised: February 7, 2015
Accepted: March 31, 2015
Article in press: March 31, 2015
Published online: June 7, 2015
Processing time: 239 Days and 14 Hours
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 (Lex), Ley and Lea 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 Lex and Lea 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.
Core tip: In this study, we aimed to establish and characterize novel human gastric epithelial cell lines derived from NCI-N87 cells after over-expression of human telomerase catalytic activity. The two most promising NCI-N87-derived clones were shown to be composed of cells with homogenous phenotype, to produce gastric zymogens and to produce and secrete neutral mucins. In addition, these clones showed very good in vitro growth properties, resistance to microaerobic conditions and good responsiveness to Helicobacter pylori. Due to their improved properties, compared to the heterogeneous parental line, these NCI-N87-derived clones are promising models of the human gastric epithelium.