Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
권호 표지
DOI QR코드

DOI QR Code

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction

  • Choi, Moonju (College of Pharmacy, Dongguk University) ;
  • Park, Minkyung (College of Pharmacy, Dongguk University) ;
  • Lee, Suhyon (R&D Institute, Biosolution Co., Ltd.) ;
  • Lee, Jeong Woo (Department of Urology, Dongguk University Ilsan Hospital, Dongguk University College of Medicine) ;
  • Cho, Min Chul (Department of Urology, Seoul Metropolitan Government-Seoul National University (SMG-SNU) Boramae Medical Center) ;
  • Noh, Minsoo (College of Pharmacy, Seoul National University) ;
  • Lee, Choongho (College of Pharmacy, Dongguk University)
  • Received : 2017.02.28
  • Accepted : 2017.03.08
  • Published : 2017.05.01
Download PDF
⟨ Previous Next ⟩

Abstract

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced block-age of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

Keywords

References

  1. Allen-Hoffmann, B. L., Schlosser, S. J., Ivarie, C. A., Sattler, C. A., Meisner, L. F. and O'Connor, S. L. (2000) Normal growth and differentiation in a spontaneously immortalized near-diploid human keratinocyte cell line, NIKS. J. Invest. Dermatol. 114, 444-455. https://doi.org/10.1046/j.1523-1747.2000.00869.x
  2. Baden, H. P., Kubilus, J., Kvedar, J. C., Steinberg, M. L. and Wolman, S. R. (1987) Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM 1). In Vitro Cell. Dev. Biol. 23, 205-213. https://doi.org/10.1007/BF02623581
  3. Ben-Porath, I. and Weinberg, R. A. (2004) When cells get stressed: an integrative view of cellular senescence. J. Clin. Invest. 113, 8-13. https://doi.org/10.1172/JCI200420663
  4. Ben-Porath, I. and Weinberg, R. A. (2005) The signals and pathways activating cellular senescence. Int. J. Biochem. Cell Biol. 37, 961-976. https://doi.org/10.1016/j.biocel.2004.10.013
  5. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A. and Fusenig, N. E. (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761-771. https://doi.org/10.1083/jcb.106.3.761
  6. Cheong, K. A., Kim, H. J., Kim, J. Y., Kim, C. H., Lim, W. S., Noh, M. and Lee, A. Y. (2014) Retinoic acid and hydroquinone induce inverse expression patterns on cornified envelope-associated proteins: implication in skin irritation. J. Dermatol. Sci. 76, 112-119. https://doi.org/10.1016/j.jdermsci.2014.08.003
  7. Choi, M. and Lee, C. (2015) Immortalization of primary keratinocytes and its application to skin research. Biomol. Ther. (Seoul) 23, 391-399. https://doi.org/10.4062/biomolther.2015.038
  8. Choi, M., Lee, S., Choi, T. and Lee, C. (2014) Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes. Virus Genes. 48, 224-232. https://doi.org/10.1007/s11262-013-1017-9
  9. Durst, M., Dzarlieva-Petrusevska, R. T., Boukamp, P., Fusenig, N. E. and Gissmann, L. (1987) Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1, 251-256.
  10. Flores, E. R., Allen-Hoffmann, B. L., Lee, D., Sattler, C. A. and Lambert, P. F. (1999) Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line. Virology 262, 344-354. https://doi.org/10.1006/viro.1999.9868
  11. Fridman, A. L. and Tainsky, M. A. (2008) Critical pathways in cellular senescence and immortalization revealed by gene expression profiling. Oncogene 27, 5975-5987. https://doi.org/10.1038/onc.2008.213
  12. Halbert, C. L., Demers, G. W. and Galloway, D. A. (1991) The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65, 473-478.
  13. Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R. and Schiller, J. T. (1989) HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 3905-3910.
  14. Hayflick, L. and Moorhead, P. S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585-621. https://doi.org/10.1016/0014-4827(61)90192-6
  15. Kiyono, T., Foster, S. A., Koop, J. I., McDougall, J. K., Galloway, D. A. and Klingelhutz, A. J. (1998) Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396, 84-88. https://doi.org/10.1038/23962
  16. Lee, C. and Laimins, L. A. (2004) Role of the PDZ domain-binding motif of the oncoprotein E6 in the pathogenesis of human papillomavirus type 31. J. Virol. 78, 12366-12377. https://doi.org/10.1128/JVI.78.22.12366-12377.2004
  17. Lee, C., Wooldridge, T. R. and Laimins, L. A. (2007) Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle. Virology 358, 201-210. https://doi.org/10.1016/j.virol.2006.08.028
  18. Lee, E., Kim, H. J., Lee, M., Jin, S. H., Hong, S. H., Ahn, S., Kim, S. O., Shin, D. W., Lee, S. T. and Noh, M. (2016) Cystathionine metabolic enzymes play a role in the inflammation resolution of human keratinocytes in response to sub-cytotoxic formaldehyde exposure. Toxicol. Appl. Pharmacol. 310, 185-194. https://doi.org/10.1016/j.taap.2016.09.017
  19. Lehman, T. A., Modali, R., Boukamp, P., Stanek, J., Bennett, W. P., Welsh, J. A., Metcalf, R. A., Stampfer, M. R., Fusenig, N., Rogan, E. M. and Harris, C. C. (1993) p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14, 833-839. https://doi.org/10.1093/carcin/14.5.833
  20. McGhee, E. M., Cotter, P. D., Weier, J. F., Berline, J. W., Turner, M. A., Gormley, M. and Palefsky, J. M. (2006) Molecular cytogenetic characterization of human papillomavirus16-transformed foreskin keratinocyte cell line 16-MT. Cancer Genet. Cytogenet. 168, 36-43. https://doi.org/10.1016/j.cancergencyto.2005.12.015
  21. Narita, M., Nunez, S., Heard, E., Lin, A. W., Hearn, S. A., Spector, D. L., Hannon, G. J. and Lowe, S. W. (2003) Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113, 703-716. https://doi.org/10.1016/S0092-8674(03)00401-X
  22. Regan, J. A. and Laimins, L. A. (2013) Viral transformation of epithelial cells. Methods Mol. Biol. 945, 449-465.
  23. Serrano, M. and Blasco, M. A. (2001) Putting the stress on senescence. Curr. Opin. Cell Biol. 13, 748-753. https://doi.org/10.1016/S0955-0674(00)00278-7
  24. Shelton, D. N., Chang, E., Whittier, P. S., Choi, D. and Funk, W. D. (1999) Microarray analysis of replicative senescence. Curr. Biol. 9, 939-945. https://doi.org/10.1016/S0960-9822(99)80420-5
  25. Stewart, S. A. and Weinberg, R. A. (2002) Senescence: does it all happen at the ends? Oncogene 21, 627-630. https://doi.org/10.1038/sj.onc.1205062
  26. Stoppler, H., Hartmann, D. P., Sherman, L. and Schlegel, R. (1997) The human papillomavirus type 16 E6 and E7 oncoproteins dissociate cellular telomerase activity from the maintenance of telomere length. J. Biol. Chem. 272, 13332-13337. https://doi.org/10.1074/jbc.272.20.13332

Cited by

  1. In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom vol.12, pp.4, 2018, https://doi.org/10.1371/journal.pntd.0006427
  2. Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation vol.14, pp.1, 2019, https://doi.org/10.1371/journal.pone.0210504
  3. A long-wave UVA filter avobenzone induces obesogenic phenotypes in normal human epidermal keratinocytes and mesenchymal stem cells vol.93, pp.7, 2017, https://doi.org/10.1007/s00204-019-02462-1
  4. Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes vol.140, pp.5, 2017, https://doi.org/10.1016/j.jid.2019.09.022
  5. Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer vol.10, pp.1, 2017, https://doi.org/10.1038/s41598-020-72027-1
  6. 3D skin models in domestic animals vol.52, pp.1, 2021, https://doi.org/10.1186/s13567-020-00888-5