Skip to main content
SpringerLink
Log in

Induction of apoptosis in human HaCaT keratinocytes

  • Original Paper
  • Published:
Archives of Dermatological Research Aims and scope Submit manuscript

Abstract

Although cell death by apoptosis has been recognized as an important control mechanism in the maintenance of tissue homeostasis and in the elimination of cells with damaged DNA, information on the induction and characteristics of apoptosis in keratinocytes is rather scarce. Apoptotic mechanisms may play an important role in normal and disturbed homeostasis of the skin. In the present study, we therefore investigated the effects of several potential inducers of apoptosis in the human keratinocyte cell line HaCaT. Apoptosis was assessed with respect to morphological changes by light and electron microscopic examinations and to DNA integrity by a specific ELISA. UVB irradiation induced the morphology and internucleosomal DNA fragmentation characteristic of apoptosis in a dose- and time-dependent manner. Interferon-γ caused DNA cleavage at the linker regions without producing morphological features consistent with apoptotic cell death. In contrast, treatment with dithranol and NP-40 resulted in necrotic alterations in the keratinocytes. Treatment with the calcium ionophore A23187 caused morphological changes which were similar to the characteristics of ‘nonapoptotic programmed cell death’. Dexamethasone, tumor necrosis factor-α, transforming growth factor-β, TPA, retinoic acid, the podophyllin derivative etoposide, the thromboxane A2 analogue U46619, cycloheximide, and the nitric oxide donors sodium nitroprusside and S-nitroso-glutathione, which are all known to induce apoptosis in other cell types, did not affect HaCaT keratinocytes. These results demonstrate that apoptosis can be induced in keratinocytes in vitro but the apoptosis differs from that in other cell types, such as haematopoietic cells, with regard to the type of inducer and/or the sensitivity of the target cells. Since keratinocytes are affected by numerous external and internal stimuli, they might posses several protective mechanisms to prevent apoptosis and to ensure the structural integrity of the outermost barrier of the body.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Kerr JFR, Wyllie AH, Currie AR (1972), Apoptosis. A basic phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26: 239–257

    PubMed  CAS  Google Scholar 

  2. Duvall E, Wyllie AH (1986) Death and the cell. Immunol Today 7: 115–118

    Article  CAS  Google Scholar 

  3. Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endonuclease activation. Nature 284: 555–556

    Article  PubMed  CAS  Google Scholar 

  4. Arends MJ, Morris MG, Wyllie AH (1990) Apoptosis. The role of the endonuclease. Am J Pathol 136: 593–608

    PubMed  CAS  Google Scholar 

  5. Lockshin RA, Zakeri Z (1991) Programmed cell death and apoptosis. In: Tomei D, Cope F (eds) Apoptosis: the molecular basis of cell death. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 47–60

    Google Scholar 

  6. Sellins K, Cohen JJ (1987) Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J Immunol 139: 3199–3206

    PubMed  CAS  Google Scholar 

  7. Martin SJ, Cotter TG (1991) Ultraviolet B irradiation of human leukemia HL-60 cells in vitro induces apoptosis. Int J Radiat Biol 59: 1001–1016

    PubMed  CAS  Google Scholar 

  8. Kizaki H, Tadakuma T, Odaka C, Muramatsu J, Ishimura U (1989) Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters. J Immunol 143: 1790–1794

    PubMed  CAS  Google Scholar 

  9. Fairbairn LJ, Cowling GJ, Reipert BM, Dexter TM (1993) Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 74: 823–832

    Article  PubMed  CAS  Google Scholar 

  10. Martin DP, Schmidt RE, Di Stefano PS, Lowry OH, Carter JG, Johnson EM (1988) Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J Cell Biol 106: 829–844

    Article  PubMed  CAS  Google Scholar 

  11. Larrick JW, Wright SC (1990) Cytotoxic mechanism of TNF-alpha. FASEB J 4: 3215–3223

    PubMed  CAS  Google Scholar 

  12. Novelli F, Di Pierro F, Francia di Celle P, Bertini S, Affaticati P, Garotta G, Forni G (1994) Environmental signals influencing expression of the IFN-γ receptor on human T cells control whether IFN-γ promotes proliferation or apoptosis. J Immunol 152: 496–504

    PubMed  CAS  Google Scholar 

  13. Sarih M, Souvannavong V, Adam A (1993) Nitric oxide synthase induces macrophage death by apoptosis. Biochem Biophys Res Commun 191: 503–508

    Article  PubMed  CAS  Google Scholar 

  14. Trauth BC, Klas C, Peters AMJ, Katzku S, Möller P, Falk W, Debatin KM, Krammer PH (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245: 301–305

    Article  PubMed  CAS  Google Scholar 

  15. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima SI, Sameshima M, Hase A, Seto Y, Nagata S (1991) The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66: 233–243

    Article  PubMed  CAS  Google Scholar 

  16. Kerr JFR, Harmon BV (1991) Definition and incidence of apoptosis: an historical perspective. In: Tomei D, Cope F (eds) Apoptosis: the molecular basis of cell death. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, pp 5–29

    Google Scholar 

  17. Budtz PE, Spies I (1989) Epidermal tissue homeostasis: apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis of the toad, Bufo bufo. Cell Tissue Res 256: 475–486

    Article  PubMed  CAS  Google Scholar 

  18. Haake AR, Polakowska RR (1993) Cell death by apoptosis in epidermal biology. J Invest Dermatol 101: 107–112

    Article  PubMed  CAS  Google Scholar 

  19. Woodcock A, Magnus IA (1976) The sunburn cell in mouse skin: preliminary quantitative studies on its production. Br J Dermatol 95: 459–468

    Article  PubMed  CAS  Google Scholar 

  20. Linse R, Richard G (1990) Zur Histologie UV-induzierter Epidermisreaktionen. 1. Mitteilung: Ein Beitrag zur Differenzierung von sunburn-Zellen. Dermatol Monatsschr 176: 345–348

    PubMed  CAS  Google Scholar 

  21. McCall CA, Cohen JJ (1991) Programmed cell death in terminally differentiating keratinocytes: role for endogenous endonuclease. J Invest Dermatol 9: 111–114

    Article  Google Scholar 

  22. Paus R, Rosenbach T, Haas N, Czarnetzki BM (1993) Patterns of cell death: the significance of apoptosis for dermatology. Exp Dermatol 2: 3–11

    Article  PubMed  CAS  Google Scholar 

  23. Boukamp P, Petrussevska RT, Breitkreuz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106: 761–771

    Article  PubMed  CAS  Google Scholar 

  24. Hart TW (1985) Some observations concerning the S-nitroso and S-phenylsylphonyl derivatives of L-cysteine and glutathione. Tetrahedron Lett 26: 2013–2016

    Article  CAS  Google Scholar 

  25. Kolde G, Knop J (1986) Ultrastructural morphometry of epidermal Langerhans cells: introduction of a simple method for a comprehensive, quantitative analysis of the cells. Arch Dermatol Res 278: 298–301

    Article  PubMed  CAS  Google Scholar 

  26. Godar DE, Miller SA, Thomas DP (1994) Immediate and delayed apoptotic cell death mechanisms: UVA versus UVB and UVC radiation. Cell Death Differ 1: 59–66

    CAS  PubMed  Google Scholar 

  27. Casciola-Rosen LA, Anhalt G, Rosen A (1994) Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 179: 1317–1330

    Article  PubMed  CAS  Google Scholar 

  28. Schwarz A, Bhardwaj R, Aragane Y, Mahnke K, Riemann H, Metze D, Luger TA, Schwarz T (1995) Ultraviolet-B-induced apoptosis of keratinocytes: evidence for partial involvement of tumor necrosis factor-alpha in the formation of sunburn cells. J Invest Dermatol 104: 922–927

    Article  PubMed  CAS  Google Scholar 

  29. Harmon BV, Corder AM, Collins RJ, Gobi GC, Allen J, Allan DJ, Kerr JFR (1990) Cell death induced in a murine mastocytoma by 42–47°C heating in vitro: evidence that the form of death changes from apoptosis to necrosis above a critical heat load. Int J Radiat Biol 58: 845–858

    PubMed  CAS  Google Scholar 

  30. Saunders NA, Jetten AM (1994) Control of growth regulatory and differentiation-specific genes in human epidermal keratinocytes by interferon-γ. Antagonism by retinoic acid and transforming growth factor-β. J Biol Chem 269: 2016–2022

    PubMed  CAS  Google Scholar 

  31. Oyaizu N, McCloskay TW, Than S, Hu R, Kalyanaraman VS, Pahwa S (1994) Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferon-gamma and tumor necrosis factor-alpha secretion. Blood 84: 2622–2631

    PubMed  CAS  Google Scholar 

  32. Sayama K, Yonehara S, Watanabe Y, Miki Y (1994) Expression of Fas antigen on keratinocytes in vivo and induction of apoptosis in cultured keratinocytes. J Invest Dermatol 103: 330–334

    Article  PubMed  CAS  Google Scholar 

  33. Reichert U, Jacques V, Grangeret M, Schmidt R (1985) Antirespiratory and antiproliferative activity of anthralin in cultured human keratinocytes. J Invest Dermatol 84: 130–134

    Article  PubMed  CAS  Google Scholar 

  34. Kanerva L (1990) Electron microscopic observations of dyskeratosis, apoptosis, colloid bodies and fibrillar degeneration after skin irritation with dithranol. J Cutan Pathol 17: 37–44

    Article  PubMed  CAS  Google Scholar 

  35. Sun DY, Jiang S, Zhung LM, Ojcius DM, Young JDE (1994) Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation. J Exp Med 179: 559–568

    Article  PubMed  CAS  Google Scholar 

  36. Schwartz LM, Osborne BA (1993) Programmed cell death, apoptosis and killer genes. Immunol Today 14: 582–590

    Article  PubMed  CAS  Google Scholar 

  37. Alam R, Forsythe P, Stafford S, Fukuda Y (1994) Transforming growth factor β abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J Exp Med 179: 1041–1054

    Article  PubMed  CAS  Google Scholar 

  38. Yoshida A, Ueda T, Takauji R, Liu YP, Fukushima T, Inuzuka M, Nakamura T (1993) Role of calcium ion in induction of apoptosis by etoposide in human leukemia HL-60 cells. Biochem Biophys Res Commun 196: 927–934

    Article  PubMed  CAS  Google Scholar 

  39. Ushikubi F, Aiba YI, Nakamura KI, Namba T, Hirate M, Mazda O, Katsura Y, Narumya S (1993) Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis. J Exp Med 178: 1825–1830

    Article  PubMed  CAS  Google Scholar 

  40. Martin SJ, Bradley JG, Cotter TG (1990) HL-60 cells induced to differentiate towards neutrophils subsequently dia via apoptosis. Clin Exp Immunol 79: 448–453

    Article  PubMed  CAS  Google Scholar 

  41. Martin SJ, Lennon SV, Bonham AM, Cotter TG (1990) Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis. J Immunol 145: 1859–1867

    PubMed  CAS  Google Scholar 

  42. Game SM, Huelsen A, Patel V, Donnel YM, Yeudall WA, Stone A, Fusenig NE, Prime SS (1992) Progressive abrogation of TGF-beta 1 and EGF growth control is associated with tumour progression in ras-transfected human keratinocytes. Int J Cancer 52: 461–470

    PubMed  CAS  Google Scholar 

  43. Winiski AG, Foster CA (1992) ICAM-1 expression in a spontaneously transformed human keratinocyte cell line: characterization by simple cell-ELISA assay. J Invest Dermatol 99: 48–52

    Article  PubMed  CAS  Google Scholar 

  44. Bertrand R, Sarang M, Jenkin J, Kerrigan D, Pommier Y (1991) Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression. Cancer Res 51: 6280–6285

    PubMed  CAS  Google Scholar 

  45. Touchettes N, Fogle S (1991) Apoptosis: it chimers with mitosis. J NIH Res 3: 75–78

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Dermatology, Virchow Clinic, Humboldt University, Augustenburgerplatz 1, D-13344, Berlin, Germany

    Ute Henseleit, Thomas Rosenbach & Gerhard Kolde

Authors
  1. Ute Henseleit
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas Rosenbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gerhard Kolde
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Henseleit, U., Rosenbach, T. & Kolde, G. Induction of apoptosis in human HaCaT keratinocytes. Arch Dermatol Res 288, 676–683 (1996). https://doi.org/10.1007/BF02505277

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02505277

Key words

Search

Navigation