Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM1)

Abstract

The long-lived keratinocyte line, NM 1, was isolated from the epidermis of a pool of foreskins obtained from apparently normal neonates at the time of circumcision. Cultures were initiated in Dulbecco's minimal essential medium containing 20% fetal bovine serum, 0.4 micrograms/ml hydrocortisone, 10(-9) M cholera toxin, and 10 ng/ml epidermal growth factor using mitomycin C-treated 3T3 cells as a feeder layer. Unlike normal keratinocytes which survive for only 150 generations these cells have been in culture for more than a year and have been carried for more than 400 doublings. The cells seem to follow a pathway of growth and differentiation that is very similar to normal keratinocytes. Cytokeratin fibrils, intercellular attachments, and cornified envelopes were observed. The keratin polypeptides isolated from the NM 1 cells were similar to those previously described in normal cultured cells; the presence of profilaggrin and involucrin was demonstrated by sodium dodecyl sulfate electrophoresis and immunoblotting with monoclonal antibodies specific to these proteins. The NM 1 cells showed a reduced dependency on 3T3 feeder cells but did not form tumors when placed into athymic nude mice. Screening of the cells for SV40, BK, HPV 16, and HPV 18 viruses was negative. The NM 1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of keratinocytes.