Characterization of human buccal epithelial cells transfected with the simian virus 40 T-antigen gene

Abstract

Serum-free cultures of normal human buccal epithelial cells were transfected with a plasmid containing the SV40 T-antigen (SV40T) gene. Two major lines developed that showed extended lifespans (between 30 and 40 weeks) as compared with the controls (approximately 6 weeks). Continued growth through one or two crises generated several sublines. They expressed the epithelial marker keratin and also exhibited nuclear expression of SV40T. The lines showed abnormal karyotypes with both numerical and structural aberrations and variably responded to agents that normally inhibit growth and/or induce terminal differentiation, i.e. transforming growth factor-beta 1 and fetal bovine serum. One of the lines, termed SVpgC2a, developed into an apparently immortal line, since it had undergone more than 700 population doublings from over 2 years in culture. Further characterization of this line demonstrated its clonal origin, with integration of two copies of SV40T at the same site and the presence of both normal retinoblastoma and wild-type p53 proteins. This line showed high resistance to growth inhibition by transforming growth factor-beta 1 and serum similar to that shown by buccal carcinoma cell line SqCC/Y1. Neither SVpgC2a nor its parental lines were tumorigenic when injected into athymic nude mice, whereas the SqCC/Y1 cells induced tumors. The various lines with extended but finite lifespans, complemented by one immortalized line, which retained non-malignant properties upon extended culture, provide a battery of model systems that will be useful for studying mechanisms of human oral carcinogenesis.

Expression of retinoid-related genes in serum-free cultures of normal, immortalized and malignant human oral keratinocytes

“…Primary keratinocyte lines were derived following incubation of tissue with 0.17% trypsin (Sigma) in phosphate buffered saline at 4°C for 18-24 h, and subsequent seeding of keratinocyte aggregates and single cells at 5x10 3 cells/cm 2 onto fibronectin/ collagen-coated (Sigma) dishes in serum-free EMHA(epithelial medium with high levels of amino acids) (16). The immortal cell line SVpgC2a, derived by transfection and stable integration of the SV40T antigen into buccal keratinocytes, and a serum-free strain of the buccal carcinoma cell line SqCC/Y1 were also cultured in EMHA (16,17). The normal keratinocyte lines were subjected to the transcript analysis in passage 2, the SVpgC2a line in passages 63 and 64, and the SqCC/Y1 line in passages 125 and 128.…”

“…Normal (NOK), SV40 T antigen-immortalized (SVpgC2a) and malignant (SqCC/Y1) human buccal keratinocytes model oral cancer development on the basis that they reflect the eventual acquisition of immortality, loss of p53 tumor suppressor function and gain of a tumorigenic phenotype (16,17). Furthermore, these normal and transformed keratinocyte cell lines demonstrate keratin expression patterns similar to normal tissue, oral dysplasia and well-differentiated oral squamous cell carcinoma, respectively (18).…”

Expression of Alcohol Dehydrogenase 3 in Tissue and Cultured Cells from Human Oral Mucosa

“…29 EMA was reconstituted from MCDB 153 medium and supplemented with 1 mol/L hydrocortisone, 0.77 mol/L insulin, 1.64 nmol/L epidermal growth factor, 100 mol/L each of ethanolamine and phosphoethanolamine, and 50 g/ml Gentamicin (Life Technologies Ltd., Paisly, Scotland). 5 The immortal cell line SVpgC2a, derived by transfection and stable integration of the SV40T antigen into buccal keratinocytes, 30 and the buccal carcinoma cell line SqCC/Y1 29 were cultured in EMA. Primary outgrowths of fibroblasts were obtained from tissue explants maintained in CRML 1066 medium supplemented with 10% fetal bovine serum, 440 nmol/L hydrocortisone, 1.83 nmol/L epidermal growth factor, 0.25 mol/L ethanolamine, 0.25 nmol/L phosphoethanolamine, and 50 g/ml Gentamicin (Life Technologies Ltd.), and the resulting cell lines grown and transferred in a 1:1 mixture of MCDB 153 and M199 media and was supplemented with 1.25% fetal bovine serum, 440 nmol/L hydrocortisone, 0.83 nmol/L epidermal growth factor, 0.25 mol/L ethanolamine, 0.25 nmol/L phosphoethanolamine, 63 nmol/L transferrin, and 50 g/ml Gentamicin.…”

“…28 The serum-free methods developed for oral normal keratinocytes are also applicable to the various transformed oral keratinocyte lines, including SV40 T antigen-immortalized keratinocyte line SVpgC2a and the squamous carcinoma cell line SqCC/Y1. 29,30 These transformed keratinocyte lines model the step-wise development of oral cancer on the basis that they reflect acquisition of immortality (the SvpgC2a cells), loss of p53 tumor suppressor functions, and eventually gain of the tumorigenic phenotype (the SqCC/Y1 cells). 28,31,32 Studies of expression, activity, and regulation of human ADH and ALDH enzymes are not available in normal or transformed oral cell lines.…”

Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen‐immortalized oral keratinocytes

“…18 The latter cell line models an early step in tumor progression on the basis of its acquisition of immortality, lack of tumorigenicity in athymic nude mice, loss of p53 tumor suppressor function and an expression pattern of cytokeratins similar to a severe epithelial dysplasia. [17][18][19][20] A need for genetic characterization of non-malignant, immortalized cell lines is implied from the fact that efforts in cancer research have primarily utilized malignant cell lines. Furthermore, immortalized cell lines are easily and reproducibly grown and potentially useful in pharmaco-toxicological studies.…”