Ultraviolet-B-Induced Apoptosis and Cytokine Release in Xeroderma Pigmentosum Keratinocytes
2000; Elsevier BV; Volume: 115; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2000.00093.x
ISSN1523-1747
AutoresEmily Capulas, Jillian E. Lowe, Michael H.L. Green, C.F. Arlett, Corinne Petit-Frére, Peter H. Clingen, Leena Koulu, Reijo J. Marttila, Nicolaas G.J. Jaspers,
Tópico(s)Fungal Infections and Studies
ResumoWe have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-α from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B. We have assessed the ability of xeroderma pigmentosum and normal keratinocytes grown out from skin biopsies to undergo apoptosis after irradiation with ultraviolet B. Keratinocytes have been studied from xeroderma pigmentosum complementation groups A (three biopsies), C (three biopsies), D (one biopsy), xeroderma pigmentosum variant (two biopsies), and Cockayne syndrome (one biopsy). The three xeroderma pigmentosum group A and the xeroderma pigmentosum group D samples were at least six times more sensitive than normal cells to ultraviolet B-induced apoptosis. The xeroderma pigmentosum variant samples showed intermediate susceptibility. Xeroderma pigmentosum group C samples proved heterogeneous: one showed high sensitivity to apoptosis, whereas two showed near normal susceptibility. The Cockayne syndrome sample showed the high susceptibility of xeroderma pigmentosum groups A and D only at a higher fluence. These results suggest that the relationships between repair deficiency, apoptosis, and susceptibility to skin cancer are not straightforward. Ultraviolet B-induced skin cancer is also thought to be due in part to ultraviolet B-induced impairment of immune responses. The release of the inflammatory cytokines interleukin-6 and tumor necrosis factor-α from cultured xeroderma pigmentosum keratinocytes tended to occur at lower fluences than in normals, but was less extensive, and was more readily inhibited at higher fluences of ultraviolet B. Cockayne syndrome ultraviolet B, 280–315 nm (in this instance from Westinghouse FS20 broad spectrum lamps) XPA, XPC, XPD, XPV, xeroderma pigmentosum, XP complementation group A, C, D, variant Skin cancer is the most prevalent type of cancer in light-skinned individuals, and occurs as a direct consequence of exposure to solar ultraviolet (UV) radiation, in particular UVB (280–315 nm) (IARC, 1992IARC Solar and Ultraviolet Radiation. IARC Monograph, Lyon1992Google Scholar;de Gruijl and van der Leun, 1994de Gruijl F.R. van der Leun J.C. Estimate of the wavelength dependency of ultraviolet carcinogenesis in humans and its relevance to risk assessment of stratospheric ozone depletion.Health Phys. 1994; 67: 319-325Crossref PubMed Scopus (197) Google Scholar;Kraemer, 1997Kraemer K.H. Sunlight and skin cancer: another link revealed.Proc Natl Acad Sci USA. 1997; 94: 11-14Crossref PubMed Scopus (329) Google Scholar). Solar UV radiation induces skin cancer initially through its induction of mutagenic DNA damage, but also via its immunosuppressive action that promotes tumors by allowing them to evade immune surveillance (Liddington et al., 1989Liddington M. Richardson A.J. Higgins R.M. Endre Z.H. Venning V.A. Murie J.A. Morris P.J. Skin cancer in renal transplant recipients.Br J Surg. 1989; 76: 1002-1005Crossref PubMed Scopus (85) Google Scholar;Kripke, 1990Kripke M.L. Photoimmunology.Photochem Photobiol. 1990; 52: 919-924Crossref PubMed Scopus (62) Google Scholar;Streilein, 1995Streilein J.W. Photoimmunology of non-melanoma skin cancer.Cancer Surv. 1995; 26: 207-218Google Scholar;Kraemer, 1997Kraemer K.H. Sunlight and skin cancer: another link revealed.Proc Natl Acad Sci USA. 1997; 94: 11-14Crossref PubMed Scopus (329) Google Scholar;Longstreth et al., 1998Longstreth J. de Gruijl F.R. Kripke M.L. et al.Health risks.J Photochem Photobiol B Biol. 1998; 46: 20-39Crossref PubMed Scopus (176) Google Scholar). In addition, apoptosis may play a protective part by eliminating damaged keratinocytes (Brash, 1997Brash D.E. Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (259) Google Scholar). Malignant melanoma, derived from melanocytes, accounts for approximately 10% of skin cancers, but is the leading cause of death (Elwood and Russell, 1989Elwood J.M. Epidemiology of melanoma: its relationship to ultraviolet radiation and ozone depletion.in: Russell Jones R. Wigley T. Ozone Depletion: Health and Environmental Consequences. John Wiley, Chichester1989: 169-190Google Scholar). Commoner forms of skin cancer are basal cell (70% of cases) and squamous cell (20% of cases) carcinomas, which arise from epidermal keratinocytes. While less feared, these cancers place a substantial strain on health resources, cause a considerable frequency of long-term disability, and may be responsible for up to 30% of skin cancer related deaths (Weinstock, 1997Weinstock M.A. Death from skin cancer among the elderly–-Epidemiological patterns.Arch Dermatol. 1997; 133: 1207-1209Crossref PubMed Google Scholar). Xeroderma pigmentosum (XP) is a rare, sun sensitive and cancer prone genetic disorder with a 100–1000-fold increased risk of patients developing all forms of skin cancer in sun-exposed areas (Kraemer et al., 1987Kraemer K.H. Lee M.M. Scotto J. Xeroderma pigmentosum. Cutaneous, ocular and neurologic abnormalities in 830 published cases.Arch Dermatol. 1987; 123: 241-250Crossref PubMed Scopus (918) Google Scholar;Arlett and Lehmann, 1996Arlett C.F. Lehmann A.R. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: Sun sensitivity, DNA repair defects and skin cancer.in: Eeles R. Ponder B. Easton D. Horwich E.A. Genetic Predisposition to Cancer. Chapman & Hall, London1996: 185-206Crossref Google Scholar;Kraemer, 1997Kraemer K.H. Sunlight and skin cancer: another link revealed.Proc Natl Acad Sci USA. 1997; 94: 11-14Crossref PubMed Scopus (329) Google Scholar). Cells from 80% of patients are defective in nucleotide excision repair (Cleaver, 1968Cleaver J.E. Deficiency in repair replication of DNA in xeroderma pigmentosum.Nature. 1968; 218: 652-656Crossref PubMed Scopus (1230) Google Scholar), the pathway responsible for the removal of major UV-induced lesions, cyclobutane pyrimidine dimers, and (6–4) photoproducts. XP cells exhibit increased UV radiation induced mutagenesis (Maher et al., 1979Maher V.M. Dorney D.J. Mendrala A.L. Konze-Thomas B. McCormick J.J. DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation.Mutat Res. 1979; 62: 311-323Crossref PubMed Scopus (190) Google Scholar;Patton et al., 1984Patton J.D. Rowan L.A. Mendrala A.L. Howell J.N. Maher V.M. McCormick J.J. Xeroderma pigmentosum fibroblasts including cells from XP variants are abnormally sensitive to the mutagenic and cytotoxic action of broad-spectrum simulated sunlight.Photochem Photobiol. 1984; 39: 37-42Crossref PubMed Scopus (47) Google Scholar). Eight complementation groups have been described and the proteins encoded by genes A–G are essential for specific steps of nucleotide excision repair (Friedberg et al., 1995Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. ASM Press, Washington1995Google Scholar;Wood, 1997Wood R.D. Nucleotide excision repair in mammalian cells.J Biol Chem. 1997; 272: 23465-23468Crossref PubMed Scopus (365) Google Scholar). The remaining group, XP variant (XPV), is defective in daughter strand break rejoining (Lehmann et al., 1975Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H.M. de Weerd-Kastelein E.A. Bootsma D. Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.Proc Natl Acad Sci USA. 1975; 72: 219-223Crossref PubMed Scopus (514) Google Scholar). The gene responsible for the XPV defect has recently been identified and shown to be a DNA polymerase (Masutani et al., 1999Masutani C. Kusumoto R. Yamada A. et al.The, XPV, (xeroderma pigmentosum variant) gene encodes human, DNA, polymerase eta.Nature. 1999; 399: 700-704Crossref PubMed Scopus (1095) Google Scholar) allowing synthesis bypassing cyclobutane pyrimidine dimers. Recognition and excision of certain types of UV damage, in particular cyclobutane pyrimidine dimers, occurs somewhat differently and more efficiently in transcribed regions of DNA (Hanawalt, 1994Hanawalt P.C. Transcription-coupled repair and human disease.Science. 1994; 266: 1957-1958Crossref PubMed Scopus (439) Google Scholar). Although XPC cells are deficient in overall excision repair and sensitive to UV killing, they are proficient in the repair of transcribed regions of the genome (Venema et al., 1991Venema J. van Hoffen A. Karcagi V. Natarajan A.T. van Zeeland A.A. Mullenders L.H.F. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes.Mol Cell Biol. 1991; 11: 4128-4134Crossref PubMed Scopus (285) Google Scholar). In contrast, in a second UV-sensitive syndrome, Cockayne syndrome (CS), there is a specific defect in the repair of transcribed DNA (Barrett et al., 1991Barrett S.F. Robbins J.H. Tarone R.E. Kraemer K.H. Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells.Mutat Res. 1991; 255: 281-291Crossref PubMed Scopus (39) Google Scholar;Hanawalt, 1994Hanawalt P.C. Transcription-coupled repair and human disease.Science. 1994; 266: 1957-1958Crossref PubMed Scopus (439) Google Scholar), and global repair of damage occurs with reasonable efficiency. There are two complementation groups with specific CS symptoms, and the syndrome is also found in association with some cases of XP (Lehmann, 1982Lehmann A.R. Three complementation groups in Cockayne syndrome.Mutat Res. 1982; 106: 347-356Crossref PubMed Scopus (126) Google Scholar;Broughton et al., 1995Broughton B.C. Thompson A.F. Harcourt S.A. et al.Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D with the clinical features of xeroderma pigmentosum and Cockayne syndrome.Am J Hum Genet. 1995; 56: 167-174PubMed Google Scholar). Patients from the two CS complementation groups without XP are sun-sensitive, their cells are sensitive to UV killing and are hypermutable, but the patients are not cancer prone (Lehmann, 1990Lehmann A.R. Cockayne syndrome.in: Buyse M.L. Birth Defects Encyclopedia. Center for Birth Defects Information Services Inc., 1990: 420-421Google Scholar;Arlett and Lehmann, 1996Arlett C.F. Lehmann A.R. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy: Sun sensitivity, DNA repair defects and skin cancer.in: Eeles R. Ponder B. Easton D. Horwich E.A. Genetic Predisposition to Cancer. Chapman & Hall, London1996: 185-206Crossref Google Scholar). XPV patients are highly cancer prone. Their cells are hypermutable (Maher et al., 1976Maher V.M. Ouellette L.M. Curren R.D. McCormick J.J. Frequency of ultraviolet light-induced mutations is higher in xeroderma pigmentosum variant cells than in normal human cells.Nature. 1976; 261: 593-595Crossref PubMed Scopus (259) Google Scholar), but appear to show normal levels of excision repair and are only slightly hypersensitive to killing. The lack of any simple relationship between susceptibility to cancer and hypermutability, specificity of repair, or susceptibility to killing, suggests that the effects of repair deficiency on immunosuppression or apoptosis need to be investigated. It has been proposed that impaired immune function (Bridges et al., 1982Bridges B.A. Strauss G.H. Hall-Smith P. Price M. Induction of somatic mutations and impairment of immune capacity by PUVA treatment and their relation to skin cancer in man.Psoralens in Cosmetics and Dermatology. pp. Pergamon Press, Paris1982: 287-294Google Scholar;Norris et al., 1990Norris P.G. Limb G.A. Hamblin A.S. et al.Immune function, mutant frequency and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.J Invest Dermatol. 1990; 94: 94-100Abstract Full Text PDF PubMed Google Scholar;Mariani et al., 1992Mariani E. Facchini A. Honorati M.C. et al.Immune defects in families and patients with xeroderma-pigmentosum and trichothiodystrophy.Clin Exp Immunol. 1992; 88: 376-382Crossref PubMed Scopus (45) Google Scholar;Bridges, 1998Bridges B.A. UV-induced mutations and skin cancer: how important is the link?.Mutat Res. 1998; 422: 23-30Crossref PubMed Scopus (10) Google Scholar) is a factor involved in the development of skin cancer in XP. Another potential mechanism for the prevention of cancer is the apoptotic elimination of cells that have been subject to DNA damage (Brash, 1997Brash D.E. Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (259) Google Scholar;Kondo, 1998Kondo S. Apoptotic repair of genotoxic tissue damage and the role of p53 gene.Mutat Res. 1998; 402: 311-319Crossref PubMed Scopus (24) Google Scholar) and it has been suggested that this process may be altered in specific XP complementation groups (Ljungman and Zhang, 1996Ljungman M. Zhang F.F. Blockage of RNA polymerase as a possible trigger for u.v. light-induced apoptosis.Oncogene. 1996; 13: 823-831PubMed Google Scholar;Wang et al., 1996Wang X.W. Vermeulen W. Coursen J.D. et al.The XPB and XPD DNA helicases are components of the p53-mediated apoptosis pathway.Genes Dev. 1996; 10: 1219-1232Crossref PubMed Scopus (306) Google Scholar). The commonest forms of skin cancer arise in keratinocytes, and the formation of apoptotic keratinocytes or sunburn cells is a well-known outcome following the exposure of skin to UV radiation (Young, 1987Young A.R. The sunburn cell.Photodermatology. 1987; 4: 127-134PubMed Google Scholar). p53 knockout mice develop very few sunburn cells in comparison with control animals (Ziegler et al., 1994Ziegler A. Jonason A.S. Leffell D.J. et al.Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1299) Google Scholar) and the incidence of sunburn cells can be partially reduced by the use of a topical cyclobutane pyrimidine dimer-specific repair enzyme (Wolf et al., 1995Wolf P. Cox P. Yarosh D.B. Kripke M.L. Sunscreens and T4n5 liposomes differ in their ability to protect against ultraviolet-induced sunburn cell formation, alterations of dendritic epidermal cells, and local suppression of contact hypersensitivity.J Invest Dermatol. 1995; 104: 287-292Crossref PubMed Scopus (98) Google Scholar). UV-induced apoptosis may occur via a DNA damage dependent pathway through p53 stabilization and p21 induction (Yamaizumi and Sugano, 1994Yamaizumi M. Sugano T. UV-induced nuclear accumulation of P53 is evoked through DNA-damage of actively transcribed genes independent of the cell-cycle.Oncogene. 1994; 9: 2775-2784PubMed Google Scholar) or by a membrane-dependent pathway (Bender et al., 1997Bender K. Blattner C. Knebel A. Iordanov M. Herrlich P. Rahmsdorf H.J. New trends in photobiology (Invited review). UV-induced signal transduction.J Photochem Photobiol B Biol. 1997; 37: 1-17Crossref PubMed Scopus (227) Google Scholar;Gniadecki et al., 1997Gniadecki R. Hansen M. Wulf H.C. Two pathways for induction of apoptosis by ultraviolet radiation in cultured human keratinocytes.J Invest Dermatol. 1997; 109: 163-169Crossref PubMed Scopus (84) Google Scholar;Aragane et al., 1998Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L.J Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (424) Google Scholar;Herrlich et al., 1999Herrlich P. Bender K. Knebel A. et al.Radiation-induced signal transduction. Mechanisms and consequences.Comptes Rendus Serie III–-Life Sci. 1999; 322: 121-125Crossref PubMed Scopus (22) Google Scholar).Kulms et al., 1999Kulms D. Poppelmann B. Yarosh D. Luger T.A. Krutmann J. Schwarz T. Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation.Proc Natl Acad Sci USA. 1999; 96: 7974-7979Crossref PubMed Scopus (174) Google Scholar have recently demonstrated in HeLa cells that the nuclear and cell membrane contribution to apoptosis are independent. A number of studies have demonstrated impaired immune function in XP patients, including deficiencies in contact allergy reactions (Morison et al., 1985Morison W.L. Bucana C. Hashem N. Kripke M.L. Cleaver J.E. German J.L. Impaired immune function in patients with xeroderma pigmentosum.Cancer Res. 1985; 45: 3929-3931PubMed Google Scholar), contact hypersensitivity (Wysenbeek et al., 1986Wysenbeek A.J. Weiss H. Duczyminer-Kahana M. Grunwald M.H. Pick A.I. Immunologic alterations in xeroderma pigmentosum patients.Cancer. 1986; 58: 219-221Crossref PubMed Scopus (46) Google Scholar), increased depletion of epidermal Langerhans cells (Koulu and Jansen, 1983Koulu L.M. Jansen C.T. Langerhans cells in xeroderma pigmentosum.J Invest Dermatol. 1983; 80: 374Google Scholar), and reduced natural killer cell function (Norris et al., 1990Norris P.G. Limb G.A. Hamblin A.S. et al.Immune function, mutant frequency and cancer risk in the DNA repair defective genodermatoses xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.J Invest Dermatol. 1990; 94: 94-100Abstract Full Text PDF PubMed Google Scholar;Anstey et al., 1991Anstey A. Arlett C.F. Cole J. et al.Long term survival and preservation of natural killer cell activity in a xeroderma pigmentosum patient with spontaneous regression and multiple deposits of malignant melanoma.Br J Dermatol. 1991; 125: 272-278Crossref PubMed Scopus (28) Google Scholar;Gaspari et al., 1993Gaspari A.A. Fleisher T.A. Kraemer K.H. Impaired interferon production and natural killer cell activation in patients with the skin cancer prone disorder, xeroderma pigmentosum.J Clin Invest. 1993; 92: 1135-1142Crossref PubMed Scopus (66) Google Scholar) Altered natural killer cell function (Miyauchi-Hashimoto et al., 1999Miyauchi-Hashimoto H. Okamoto H. Tanaka K. Horio T. Ultraviolet radiation-induced suppression of natural killer cell activity is enhanced in xeroderma pigmentosum group A (XPA) model mice.J Invest Dermatol. 1999; 112: 965-970Crossref PubMed Scopus (24) Google Scholar), and local and systemic immunosuppression are enhanced in XPA knockout mice (Miyauchi-Hashimoto et al., 1996Miyauchi-Hashimoto H. Tanaka K. Horio T. Enhanced inflammation and immunosuppression by ultraviolet radiation in xeroderma pigmentosum group A (XPA) model mice.J Invest Dermatol. 1996; 107: 343-348Crossref PubMed Scopus (80) Google Scholar), although the pattern is not identical to that in human XP patients. Langerhans cells are seen as a major target in the modification of antigen presentation following UV irradiation (Kripke, 1990Kripke M.L. Photoimmunology.Photochem Photobiol. 1990; 52: 919-924Crossref PubMed Scopus (62) Google Scholar;Dandie et al., 1998Dandie G.W. Clydesdale G.J. Jacobs I. Muller H.K. Effects of UV on the migration and function of epidermal antigen presenting cells.Mutat Res. 1998; 422: 147-154Crossref PubMed Scopus (36) Google Scholar;Dittmar et al., 1999Dittmar H.C. Weiss J.M. Termeer C.C. et al.In vivo UVA-1 and UVB irradiation differentially perturbs the antigen-presenting function of human epidermal Langerhans cells.J Invest Dermatol. 1999; 112: 322-325Crossref PubMed Scopus (35) Google Scholar). A role for DNA damage in the modulation of the activity of Langerhans cells is seen from the effects of the cyclobutane pyrimidine dimer-specific enzymes photolyase and T4 endonuclease V (Kripke et al., 1992Kripke M.L. Cox P.A. Alas L.G. Yarosh D.B. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.Proc Natl Acad Sci USA. 1992; 89: 7516-7520Crossref PubMed Scopus (441) Google Scholar;Vink et al., 1997Vink A.A. Moodycliffe A.M. Shreedhar V. Ullrich S.E. Roza L. Yarosh D.B. Kripke M. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers.Proc Natl Acad Sci USA. 1997; 94: 5255-5260Crossref PubMed Scopus (126) Google Scholar;Yarosh et al., 1999Yarosh D.B. O'connor A. Alas L. Potten C. Wolf P. Photoprotection by topical DNA repair enzymes: Molecular correlates of clinical studies.Photochem Photobiol. 1999; 69: 136-140PubMed Google Scholar) and in the study of excision repair of UVB-induced lesions (Jimbo et al., 1992Jimbo T. Ichihashi M. Mishima Y. Fujiwara Y. Role of excision repair in UVB-induced depletion and recovery of human epidermal Langerhans cells.Arch Dermatol. 1992; 128: 61-67Crossref PubMed Scopus (19) Google Scholar). Recent attention has focused on the role of cytokines in UV radiation-induced immunosuppression (Boonstra and Savelkoul, 1997Boonstra A. Savelkoul H.F.J. The role of cytokines in ultraviolet-B induced immunosuppression.Eur Cytokine Net. 1997; 8: 117-123PubMed Google Scholar). UVB-induced immune modulation is mediated in part by the release of immunosuppressive and inflammatory cytokines such as interleukin (IL) -10 (Grewe et al., 1995Grewe M. Gyufko K. Krutmann J. Human keratinocytes produce IL-10: modulation by ultraviolet B and ultraviolet A1 radiation.J Invest Dermatol. 1995; 104: 3-6Crossref PubMed Scopus (171) Google Scholar), IL-6 (de Vos et al., 1994de Vos S. Brach M. Budnik A. Grewe M. Herrmann F. Krutmann J. Post-transcriptional regulation of interleukin-6 gene expression in human keratinocytes by ultraviolet B radiation.J Invest Dermatol. 1994; 103: 92-96Crossref PubMed Scopus (51) Google Scholar), and tumor necrosis factor-α (TNF-α) (Köck et al., 1990Köck A. Schwarz T. Kirnbauer R. Urbanski A. Perry P. Ansel J.C. Luger T.A. Human keratinocytes are a source for tumor necrosis factor alpha: Evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.J Exp Med. 1990; 172: 1609-1614Crossref PubMed Scopus (611) Google Scholar) from epidermal keratinocytes. DNA damage is an important intermediate required for the induction of IL-6 by normal human keratinocytes (Petit-Frère et al., 1998Petit-Frère C. Clingen P.H. Grewe M. Krutmann J. Roza L. Arlett C.F. Green M.H.L. Induction of interleukin-6 production by ultraviolet radiation in normal human epidermal keratinocytes and in a human keratinocyte cell line is mediated by DNA damage.J Invest Dermatol. 1998; 111: 354-359Crossref PubMed Scopus (75) Google Scholar) and for cytokine induction in animal models (Yarosh and Kripke, 1996Yarosh D.B. Kripke M.L. DNA repair and cytokines in antimutagenesis and anticarcinogenesis.Mutat Res. 1996; 350: 255-260Crossref PubMed Scopus (25) Google Scholar). Consequently, we hypothesized that XP keratinocytes would exhibit an altered pattern of cytokine induction after UV irradiation. Despite the fact that the most common forms of skin cancer are derived from keratinocytes, the relationships between apoptosis, cancer, and DNA repair in XP have, with few exceptions (e.g.,Otto et al., 1999Otto A.I. Riou L. Marionnet C. Mori T. Sarasin A. Magnaldo T. Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients.Cancer Res. 1999; 59: 1212-1218PubMed Google Scholar), been deduced from studies using primary or transformed human skin fibroblasts. The aim of this study was to investigate whether XP keratinocytes differ from normal keratinocytes in cytokine induction or apoptotic responses following UVB irradiation. Primary human keratinocytes are very much more demanding than fibroblasts to culture, and have a short lifespan. Nevertheless, we have found that it is possible to perform short-term culture of keratinocytes using residual material from small skin biopsies obtained for diagnostic studies. Sufficient material has been obtained to allow us to assess apoptosis using a terminal deoxyribonucleotidyl transferase nick end-labelling (TUNEL) assay (Apoptag), and in a number of cases IL-6 and TNF-α release using enzyme-linked immunosorbent assay. These assays provide a model system for investigating end-points relevant to skin cancer using the relevant damaging agent and the cell type that is subject to photocarcinogenesis. UVB irradiation was performed through the bottom of slides or Petri dishes using a bank of four Westinghouse FS20 sunlamps as described previously (Arlett et al., 1993Arlett C.F. Lowe J.E. Harcourt S.A. et al.Hypersensitivity of human lymphocytes to UV-B and solar irradiation.Cancer Res. 1993; 53: 609-614PubMed Google Scholar). Fluence rates were determined with an International Light radiometer (IL1350) and were typically 5 W per m2 for UVB. Punch biopsies (4 mm) were obtained with informed consent from four normal human donors, nine XP individuals (three XPA, three XPC, one XPD, two XPV), and one CS individual Table 1. A fragment of each XP biopsy was used to isolate a fibroblast culture according to standard procedures (Arlett et al., 1993Arlett C.F. Lowe J.E. Harcourt S.A. et al.Hypersensitivity of human lymphocytes to UV-B and solar irradiation.Cancer Res. 1993; 53: 609-614PubMed Google Scholar). The fibroblasts were cultured to perform tests to confirm the XP phenotype, and complementation analysis (De Weerd-Kastelein et al., 1972De Weerd-Kastelein E.A. Keijzer W. Bootsma D. Genetic heterogeneity of xeroderma pigmentosum demonstrated by somatic cell hybridisation.Nature (New Biol). 1972; 238: 80-83Crossref PubMed Scopus (228) Google Scholar;Vermeulen et al., 1991Vermeulen W. Stefanini M. Giliani S. Hoeijmakers J.H.J. Bootsma D. Xeroderma pigmentosum complementation group H falls into complementation group D.Mutat Res. 1991; 255: 201-208Crossref PubMed Scopus (65) Google Scholar) was performed in Rotterdam. A second fragment of the biopsy was cryopreserved.Table IKeratinocyte cultures used in this investigationCell designationOriginComplementation groupXP1TUFTurku, FinlandXPAXP3TUFTurku, FinlandXPAXP5TUFTurku, FinlandXPAXP2TUFTurku, FinlandXPCXP4TUFTurku, FinlandXPCXP29BRAmersham, U.K.XPCXP31BRBarcelona, SpainXPDXP7BRBasingstoke, U.K.XPVXP11BRLeicester, U.K.XPVCS13BRSheffield, U.K.CS?MCBrighton, U.K.NormalMMBrighton, U.K.NormalTEBrighton, U.K.NormalWWBrighton, U.K.Normal Open table in a new tab The remainder of the biopsy was cut into small fragments which were incubated for 4–5 d in two chamber Permanox slides (Nunc, Roskilde, Denmark) (two fragments per chamber) in Eagle's minimum essential medium (MEM) with 20 U penicillin per ml, 20 μg streptomycin per ml, 2 mM glutamine (Gibco, Paisley, U.K.), and 15% fetal bovine serum (15% MEM). Procedures were similar for normal biopsies, except that fibroblasts were not isolated. When sufficient keratinocytes appeared around the skin fragment, usually at day 4, the medium was aspirated, cells were washed with phosphate-buffered saline and irradiated in 1 ml phosphate-buffered saline. The cells were then incubated in 0.75 ml of 15% MEM for 24 h at 37°C in a humidified atmosphere containing 5% CO2. At the end of the incubation the medium was removed, slides were air dried and kept at -20°C until staining for apoptotic cells. Although apoptosis could be determined directly on the keratinocytes growing out from skin fragments, it was not possible to obtain quantitative data for cytokine release in this way, because of the variation in size between fragments and sector of keratinocytes. Therefore keratinocytes outgrown from the explants were expanded following dispersion with trypsin/ethylenediamine tetraacetic acid (Clonetics, San Diego, CA). The cells were cultured on a feeder layer of 1.5 × 104 per cm2 normal fibroblasts derived from a normal donor (1BR.3) which had received 30 Gy γ-radiation in 1.5 cm dishes (Nunc). The medium was composed of 1 ml Dulbecco's MEM (DMEM)/F12 mix (1:1) with 20 U penicillin per ml, 20 μg streptomycin per ml, 2 mM glutamine (Gibco), 5 μg per ml insulin, 1 ng epidermal growth factor per ml, 0.4 μg hydrocortisone per ml (Clonetics), 0.1 nM cholera toxin (Calbiochem, Nottingham, U.K.), and 10% fetal bovine serum. When the cells reached 70–80% confluency, they were treated first with 0.05% ethylenediamine tetraacetic acid for 5 min to remove the fibroblasts, then with trypsin/ethylenediamine tetraacetic acid for 5–10 min. The keratinocytes were cultured successively in 3.5 cm dishes and 25 cm2 tissue culture flasks (Nunc) with DMEM/F12 mix. The ultimate yield of keratinocytes was limited and varied considerably between biopsies, so that experiments on an individual culture could not be repeated and in most instances not all the desired experiments could
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