Development of Oral Epithelial Cell Line ROE2 with Differentiation Potential from Transgenic Rats Harboring Temperature-Sensitive Simian Virus40 Large T-Antigen Gene

Abstract

We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.

Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

“…We monitored whether NaF affected cell number and cell viability in oral epithelial ROE2 cells [ 25 ]. NaF at concentrations of 2 and 4 mM resulted in a significant decrease in cell number by the trypan blue dye exclusion assay, and the inhibition percentages were 59.3% and 74.4%, respectively ( Figure 1A ).…”

“…Recently, we succeeded in establishing the immortalized oral epithelial cell line ROE2 from fetal transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen [ 25 ]. The cells with stratified epithelial-like morphology proliferated continuously at a permissive temperature of 33 °C and at an intermediate temperature of 37 °C.…”

“…The cells with stratified epithelial-like morphology proliferated continuously at a permissive temperature of 33 °C and at an intermediate temperature of 37 °C. ROE2 cells were maintained in Dulbecco’s modified Eagle medium/Ham F-12 (1:1) (DMEM/F12) medium (Life Technologies Co., Grand Island, NY, USA) supplemented with 2% fetal bovine serum (FBS; Equitech-Bio Inc., Kerrville, TX, USA), 20 μg/L insulin, 20 μg/L transferrin, 1.22 μg/L ethanolamine, 91.4 ng/L sodium selenite and 10 μg/L epidermal growth factor (Sigma-Aldrich Co., St. Louis, MO, USA) in a collagen type I-precoated culture vessel (Asahi Glass Co., Ltd., Tokyo, Japan) at 33 °C [ 25 ]. The human malignant oral epithelial cell line HSC-3 was obtained from the Human Science Research Resources Bank of the Japan Health Sciences Foundation (Tokyo, Japan).…”