Summary
The long-lived keratinocyte line, NM1, was isolated from the epidermis of a pool of foreskins obtained from apprently, normal neonates at the time of circumcision. Cultures were initiated in Dulbecco’s minimal essential medium containing 20% fetal bovine serum, 0.4 μg/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 NM1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of kerationocytes.
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Arrighi, F. E.; Hsu, T. C. Localization of heterochromatin in human chromosomes Cytogenetics 10:81–86; 1971.
Baden, H. P.; Kubilus, J. The growth and differentiation of cultured newborn rat keratinocytes. J. Invest. Dermatol. 80:124–130; 1983.
Baden, H. P., Kubilus, J. Effect of minoxidil on cultured keratinocytes. J. Invest. Dermatol. 81:558–560; 1983.
Berger, R. The chromosomes in hematology. Cancer Genet. Cytogenet. 4:69–88; 1981.
Boshart, M., Gissman, L., Ikenberg, H., et al. A new type of papillomavirus DNA and its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 3:1151–1157; 1984.
Cohen, J.; Li, F. P., Berg, S., et al. Hereditary renal-cell carcinoma associated with a chromosomal translocation. N. Engl. J. Med. 301;592–595; 1979.
Dalla-Favera, R., Martinotti, S., Gallo, R. C., et al. Human c-myc oncogene locus in human undifferentiated B-cell lymphomas. Science 219:963–967; 1983.
Defendi, V., Naimski, P., Steinberg, M. L. Human cells transformed by SV40 revisited: The epithelial cells. J. Cell. Physiol. [Suppl.] 3:131–140; 1982.
deGrouchy, J.; Turleau, C. Clinical atlas of human chromosomes, 2nd ed. New York: John Wiley & Sons; 1984:126–133.
Dvrst, M., Gissman, L.; Idenberg, H., et al. A papilloma-virus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Natl. Acad. Sci. USA 80:3812–3815; 1983.
Ehrlich, H. P.; Griswold, T. R. Epidermolysis bullosa dystrophica recessive fibroblasts produce increased concentrations of cAMP within a collagen matrix. J. Invest. Dermatol. 83:230–233; 1984.
Eva, A.; Robbins, K. C.; Andersen, P. R., et al. Cellular genes analogous to retroviral one genes are transcribed in human tumour cells. Nature 295:116–119; 1982.
Fleckman, P.; Dale, B. A.; Holbrook, K. A. Profilaggrin, a highmolecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes. J. Invest. Dermatol. 85:507–512; 1985.
Fuchs, E.; Green, H. The expression of keratin genes in epidermis and cultured cells. Cell 15:887–897; 1978.
Green, H.; Kehinde, O.; Thomas, J. Growth of cultured epidermal cells into multiple epithelia, suitable for grafting. Proc. Natl. Acad. Sci. USA 76:5665–5668; 1979.
Green, H.; Fuchs, E.; Watt, F. Differentiated structural components of the keratinocyte. Cold Spring Harbor Symp. Quant. Biol., vol. 46:293–301; 1982.
Hirs, C. H. W. Detection of peptides by chemical methods. In: Hirs, C. H. W., ed., Methods in Enzymology, vol. 11, New York: Academic Press; 1967:325–329.
Kohler, G.; Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–498; 1975.
Kubilus, J.; MacDonald, M. J.; Baden, H. P. Epidermal proteins of cultured human and bovine keratinocytes. Biochim. Biophys. Acta. 578:484–492; 1979.
Kubilus, J.; Baden, H. P. Isolation of two immunologically related transglutaminase substrates from cultured human keratinocytes. In Vitro 18:447–455; 1982.
Kubilus, J.; Scott, I.; Harding, C. R., et al. The occurence, of profilaggrin and its processing in cultured keratinocytes. J. Invest. Dermatol. 85:513–517; 1985.
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685; 1970.
Lee, L. D.; Baden, H. P.; Kubilus, J., et al. Immunology of epidermal fibrous proteins. J. Invest. Dermatol. 67:521–525; 1976.
Mann, R. S.; Carroll, R. B. Cross-reaction of BK virus large T antigen with monoclonal antibodies directed against SV40 large T antigen. Virology 138:379–385; 1984.
Mark, J.; Dahlenfors, R.; Ekedahl, C.; Stenman, G. The mixed salivary gland tumor a usually benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genetics and Cytogenetics 2:231–241; 1980.
Mitelman, F.; Levan, G. Clustering of aberrations to specific chromosomes in human neoplasms. IV. A survey of 1,871 cases. Hereditas 95:79–139; 1981.
Moll, R.; Franke, W. W.; Schiller, D. L.; Geiger, B.; Krepler, R. The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors, and cultured cells. Cell 31:11–24;1982.
Morris. A.; Steinberg, M. L.; Defendi, V. Keratin gene expression in SV40 transformed human keratinocytes. Proc. Natl. Acad. Sci. USA. In Press. 1986.
Naimski, P.; Steinberg, M. L. Analysis of stable and unstable viral forms of SV40-infected human keratinocytes. J. Cell. Biochem. 29:95–103; 1985.
Neel, B. G.; Jhanwar, S. C.; Chagannti, R. S. K.; Hayward, W. S. Two human c-onc genes are located on the long arm of chromosome 8. Proc. Natl. Acad. Sci. USA 79:7842–7846; 1982.
Portolani, M.; Barbati-Brodano, G.; LaPlaca, M. Malignant transformation of hamster kidney cells by BK virus. J. Virol. 15:420–422; 1975.
Reichmann, A.; Martin, P.; Levin, B. Chromosomal banding patterns in human large bowel cancer. Int. J. Cancer. 28:431–440; 1981.
Rheinwald, J. G.; Green, H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6:331–344; 1975.
Rheinwald, J. G.; Green, H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265:421–424; 1977.
Rheinwald, J. G.; Beckett, M. A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinoma. Cancer Res. 41:1657–1663; 1981.
Sandberg, A. A. A chromosomal hypothesis of oncogenesis. Cancer Genet. Cytogenet. 9:277–285; 1983.
Schwarz, E.; Freese, U. K.; Gissmann, L., et al. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314:111–114; 1985.
Shah, K. V.; Daniel, R. W.; Strandberg, J. D. Sarcoma in a rat inoculated with BK virus, a human papovavirus. JNCI 54:945–949; 1975.
Slamon, D. J.; deKernion, J. B.; Verman, I. M., et al. Expression of cellular oncogenes in human malignancies. Science 224:256–262; 1984.
Steinberg, M. L.; Defendi, V. Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40. J. Cell Phys. 123:117–125; 1985.
Stewart, T. A.; Bellve, A. R.; Leder, P. Transcription and promoter usage of the myc gene in normal somatic and spermatogenic cells. Science 226:707–710; 1984.
Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamid gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354; 1979.
White, B. A.; Bancroft, F. C. Cytoplasmic dot hybridization. J. Biol. Chem. 257:8569–8572; 1982.
Wolman, S. R. Karyotypic progression in human tumors. Cancer Metastasis Reviews, 2:257–293; 1983.
Wolman, S. R.; Lanfrancone, L.; Dalla-Favera, R.; Ripley, S.; Henderson, A. S. Oncogene mobility in a human leukemia line HL-60. Cancer Genetics and Cytogenetics 17:133–141; 1985.
Worst, P. K. M.; MacKenzie, I. C.; Fusenig, N. E. Reformation of organized epidermal structure by transplantation of suspensions and cultures of epidermal and dermal cells. Cell Tissue Res. 225:65–77; 1982.
Wuepper, K. D.; Holbrook, K. A.; Karasek, M.; Marcelo, C.; Prunieras, eds. Biology of the Keratinocyte In Vitro. J. Invest. Dermatol. 81: 1s-194s; 1983.
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Baden, H.P., Kubilus, J., Kvedar, J.C. et al. Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM1). In Vitro Cell Dev Biol 23, 205–213 (1987). https://doi.org/10.1007/BF02623581
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DOI: https://doi.org/10.1007/BF02623581