Immortalization and characterization of normal oral epithelial cells without using HPV and SV40 genes

Abstract

a b s t r a c tBackground: As oral neoplasm often originates from epithelium, an immortalized epithelial cell line could be useful for the research of oral carcinogenesis. Although several oral epithelial cell lines were reported, they were either derived from cancer or immortalized by human papilloma virus or simian virus 40 genes, which have the potential to induce carcinogenesis. Materials and methods: We established two immortalized cell lines from human oral epithelium by transducing mutant cyclin dependent kinase 4, cyclin D 1 , and human telomerase reverse transcriptase with or without dominant-negative p53 into primary-cultured normal oral gingival epithelial cells using recombinant lentivirus vectors and named them MOE (mouth-ordinary-epithelium) 1a and MOE1b, respectively. Results: MOE1 cells could be passaged for nine months or more, and the morphology of the cells did not change in comparison with that of fresh primary-cultured epithelial cells. MOE1 cells did not show epithelial-mesenchymal transition. MOE1b cells retain functional p53 and were considered to have less risk of genomic instabilities. Anchorage-independent growth was not observed in MOE1 cells. The expressions of cancer-associated genes including keratin-17 were not elevated in MOE1 cells, whereas oral cancer-derived HSC-2 cells showed overexpression of them. Furthermore, interleukin (IL)-1␤, IL-6, IL-8, tumor necrosis factor-␣, matrix metalloproteinase (MMP)-2, and MMP-9 were induced in response to lipopolysaccharide or heat-killed bacterium in MOE1 cells. Discussion: MOE1 cells kept the characteristics of normal epithelial cells without acquiring typical features of cancer cells and they could be useful not only for the study of oral neoplasm but also for other oral diseases.

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis

“…Primary culture of ameloblastoma was obtained as described previously. 7 In brief, diced ameloblastoma tissue was washed with Dulbecco's modified Eagle's medium (DMEM) 3 times and treated with collagenase solution (20 mg/mL) at 37 C for 2 h with shaking. Ameloblastoma cells were washed with K-SFM and seeded onto a tissue-culture flask for subsequent gene transduction using recombinant lentiviral vectors as described previously.…”

“…21 After viral transduction, cells were maintained with K-SFM in an atmosphere of 5% CO 2 at 37 C. MOE1b human normal oral epithelium-derived and HFF2 human skinderived cell lines were established as described previously. 7,22 MOE1b cells were maintained with K-SFM. 7 HFF2 cells were maintained with DMEM containing 8% fetal bovine serum (FBS).…”

“…7 Microscopic images were obtained using ECLIPSE Ti-E (Nikon Corp., Tokyo, Japan) and PowerShot A640 (Canon Inc., Tokyo, Japan) as described previously. 7 Confocal microscopy was performed using several combinations of antibodies and a confocal laser-scanning microscope (LSM700, Carl-Zeiss, Jena, Germany), as described previously. 7,24 Western blot analysis Cells were scraped with a rubber policeman, washed, and analyzed using antibodies as described previously.…”

“…7 Confocal microscopy was performed using several combinations of antibodies and a confocal laser-scanning microscope (LSM700, Carl-Zeiss, Jena, Germany), as described previously. 7,24 Western blot analysis Cells were scraped with a rubber policeman, washed, and analyzed using antibodies as described previously. 7,24,25 Accumulation of soluble b-catenin was analyzed using sonicated cells as described previously.…”

“…In a previous study, we established immortalized plexiform-type ameloblastoma cells, AM-1, 6 and normal oral epithelial cells, MOE1b. 7 Ameloblastoma has been proposed to stem from odontogenic epithelium 1 and signaling molecules including Wnt, sonic hedgehog, and bone morphogenetic proteins, which are related to the odontogenic epithelialmesenchymal interaction in tooth morphogenesis. 8,9 Therefore, we propose that epithelial-mesenchymal signal molecules might be responsible for the characteristics of ameloblastoma.…”