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The SOS response in human skin
The SOS Response in Human Skin Steven Mayer, Mark S. Eller, and Barbara A. Gilchrest, MD
The wavelengths of sunlight transmitted to the earth’s surface include ultraviolet radiation that is harmful to living organisms. The damage is primarily genetic: shorter wavelengths (UV-B, 290 to 320 nm) catalyze formation of covalent bonds between adjacent nucleotides in DNA molecules, and longer wavelengths (UV-A, 320 to 400 nm) oxidize membrane lipids and other cellular constituents to generate free radicals that then modify individual nucleotides. All terrestrial organisms appear to have evolved both constitutive and inducible photoprotective strategies that provide shielding from damaging ultraviolet wavelengths or enhance repair of damaged DNA. Constitutive DNA repair was first elucidated in bacteria and is now also well understood in mammals. It comprises two strategies for recognizing and replacing damaged nucleotides, as described below. By far the best characterized inducible or photoadaptive behavior is the SOS response, also first described in bacteria, in the 1970s by Radman.1 This response, now understood in detail at the molecular level, appears designed evolutionarily to protect these single-celled organisms from frequently encountered DNA-damaging agents. Human skin also responds to ultraviolet radiation in a manner that decreases damage from subsequent exposures. To a degree determined by as yet unknown genetic factors, after a lag period of several days, skin increases its synthesis and intraepidermal dispersion of melanin, a pigment that directly absorbs ultraviolet photons and free radicals to protect tissues from their destructive effects on DNA.2,3 This well-recognized process is termed tanning. Lack of melanin protection increases skin cancer risk, as evidenced by cancerprone fair-skinned people4,5 or patients with albinism.4,6 Very recently, however, human skin cells have also been shown to induce DNA repair enzymes and other photoprotective gene products in response to ultraviolet irradiation,7 suggesting the existence of a photoadaptive response far broader than increased melanin alone.
Curr Probl Dermatol, May/June 2001
Repair of Ultraviolet-Induced DNA Damage Terrestrial sunlight damage to DNA is principally caused by the UV-B wavelengths. More than 90% of the lesions are cyclobutyl pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts.8-10 These lesions are largely repaired by a process called nucleotide excision repair (NER) in which incisions are made in the DNA on both sides of the lesion. The damaged strand is then displaced, the gap (20 to 25 nucleotides in bacteria and 29 to 32 in human beings) is filled in by DNA polymerase, and the new strand is religated to repair the lesion.11-13 Oxidized nucleotide bases resulting from either UV-B or UV-A–induced free radical damage are replaced individually by base excision repair,14 a constitutive process essential for cell viability. NER is not essential for cell viability but is evolutionarily conserved from bacteria to mammals.14 In human beings, defects in genes encoding any of at least 7 NER pathway proteins cause xeroderma pigmentosum (XP), a recessively inherited disease that manifests ultraviolet hypersensitivity with various photoaging-like changes such as dryness and irregular pigmentation for which the disease is named.14 In addition, patients with XP show a greater than 1000-fold increased risk for skin cancer compared with the general population.15 The XP phenotype in human beings confirms the major photoprotective function of NER.
The SOS Response in Bacteria The SOS response to DNA damage was first described in Escherichia coli as a system promoting DNA repair, cell cycle control, and recombination.16 In bacteria, single-stranded DNA generated during the course of DNA damage and repair interacts with and activates a protease, leading ultimately to derepression of at least 20 genes involved in DNA repair, replication, and cell survival16 (Fig 1). This is termed the SOS response and serves to enhance survival after the inciting irradiation and to increase bacterial resistance to subsequent ultraviolet-induced DNA damage. Thus if bacteria are
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FIG 1. Single-stranded DNA induces expression of SOS genes in bacteria. The LexA protein normally represses genes that are induced as part of the SOS response. After DNA damage, single-stranded DNA is generated by the excision repair process and at stalled replication forks. This single-stranded DNA interacts with and activates the RecA protease (designated as RecA*) which then cleaves the LexA protein, de-repressing and inducing the transcription of the SOS response genes.
exposed to a sublethal dose of ultraviolet irradiation, and those that survive are subsequently exposed to the same ultraviolet dose, the organisms more efficiently process the DNA damage and manifest enhanced survival. Another feature of the SOS response is bypassing the block on DNA replication induced by DNA damage. Induction of two genes, UmuC and UmuD, allows DNA polymerase to replicate a damaged template by relaxing normal Watson-Crick base-pairing requirements.17 This repair is error-prone but immediately beneficial for cell survival, even though it increases the risk of mutations that may affect cellular function in subsequent generations. Indeed, it has been postulated that error-prone repair of the ultraviolet-irradiated bacterial genome is evolutionarily beneficial because it encourages the appearance of mutations, some of which are likely to improve cell function or confer better survival in the presence of the environmental DNA-damaging agent.
The SOS Response in Human Skin Because ultraviolet-induced tanning is the major recognized defense of human skin against subsequent ultraviolet damage,2,18 we postulated that tanning might be part of a mammalian SOS response and, by analogy to bacteria, that melanogenesis might be stimulated by ultraviolet damage–induced single-stranded DNA fragments. The postulated link between tanning and DNA
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damage in human skin was supported by the observation that treatment of cultured pigment cells with a liposomeencapsulated prokaryotic DNA repair protein, T4 endonuclease V, known to catalyze the rate-limiting step of excision repair of thymine dimers in bacteria, enhances melanogenesis after ultraviolet irradiation.19 Thymine dimers are the most common ultraviolet photoproducts,20 and therefore thymidine dinucleotides (abbreviated pTpT to indicate their phosphate linkage in the DNA backbone) would be the most common sequence-specific element in ultravioletdamaged DNA. Indeed, pTpT, like ultraviolet radiation exposure, was found to markedly increase cellular melanin within 5 days in cultured mouse and human pigment cells and in intact guinea pig skin.21,22 As a control, the adenine dinucleotide pApA, which does not commonly form dimers, did not elicit a tanning response. These studies also found that the tanning response to pTpT mimicked ultraviolet-induced melanogenesis mechanistically, in that both ultraviolet radiation and pTpT induced tyrosinase expression and alpha-melanocyte–stimulating hormone binding to the cell surface and had a similar time course, requiring several days for maximal response.23
DNA Fragments Up-regulate DNA Repair Capacity In bacteria, the ultraviolet radiation–induced SOS response consists of transcriptional up-regulation of genes involved in DNA repair and cell survival.16 Although a comparable response was not recognized in human beings, there were isolated reports of DNA repair enzyme induction after DNA damage in mammalian cells.24-26 Up-regulation of DNA repair capacity in human skin would be expected to have a clinically meaningful protective effect, in that impaired DNA repair capacity is known to be associated with the development of skin cancer. The most dramatic example of this relationship is XP, described above, in which repair capacity is reduced by at least 50% and in some patients by more than 95%, depending on the specific mutation and the repair assay used. However, skin cancer occurrence in otherwise normal individuals has also been linked to more subtle reductions in DNA repair capacity,27 and in large populations, decreased DNA repair capacity is inversely related to mutation frequency,28 with mutations in key regulatory genes playing a major role in carcinogenesis. We therefore investigated whether
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pTpT might also stimulate DNA repair capacity in human skin–derived cells. The effect of pTpT on DNA repair was determined by comparing the ability of pTpT-treated cells versus controls to repair and then transcribe a reporter plasmid containing the bacterial chloramphenicol acetyl transferase gene in a host cell reactivation assay. In this test system, human keratinocytes and fibroblasts pretreated with pTpT for 3 days and then transfected with the reporter plasmid were twice as effective as control cells in repair and subsequent expression of this ultraviolet-damaged chloramphenicol acetyl transferase gene.23 Similar results were obtained in a second assay in which pTpTtreated cells or controls were ultraviolet irradiated, and the rate of removal of DNA photoproducts was determined directly in multiple DNA samples harvested over a 24-hour period. The pTpT-treated cultures removed photoproducts at a statistically more rapid rate. In ultraviolet-irradiated guinea pig skin, pTpT-treated sites also showed more rapid removal of photoproducts, compared with control vehicle–treated sites, as indicated by loss of antidimer antibody binding to nuclei in skin biopsy cross-sections.7 These data demonstrate that dinucleotide treatment is effective, both in vivo and in vitro, in preparing cells to combat ultraviolet-induced DNA damage. The mechanism by which pTpT increases DNA repair capacity proved to be a 2- to 3-fold up-regulation of mRNA and protein levels of several DNA repair enzymes and cell cycle regulation proteins,23,29 although it remains unknown whether all are similarly rate-limiting in normal cells under basal conditions. Because SOS repair in bacteria is error-prone, as noted above, and because mutations in human cells may lead to dysfunction or malignant transformation, experiments were performed to determine whether the enhanced DNA repair induced by pTpT is also error-prone. A transgenic mouse model was used in which all cells carry multiple copies of a bacterial Lac-Z reporter plasmid stably integrated into the genome that can be isolated to determine quantitatively the number of mutations caused by a treatment of interest.29,30 Mouse skin in vivo or cultured cells derived from the mice were treated with pTpT or vehicle alone and then ultraviolet irradiated, after which the Lac-Z plasmid was harvested and analyzed for mutations. As expected, ultraviolet irradiation caused mutations in a dose-dependent manner. Of critical importance, however, pTpT pretreatment decreased the mutation rate by approximately 50%.33 These results
Curr Probl Dermatol, May/June 2001
TABLE 1. Photoprotective responses inducible by oligonucleotides
Response
p53 Mediated?
Induced gene products identified to date
Tanning Cell cycle arrest Enhanced DNA repair
Yes Yes Yes
Immunosuppression Enhanced repair of oxidative damage Apoptosis
Unknown Unknown
Tyrosinase p21 XPA, PCNA, ERCC3, RPA, GADD45 TNFα, IL-10 SOD 1
Yes
p53, E2F1
TNFα, Tumor necrosis factor–α; IL-10, interleukin 10; SOD 1, superoxide dismutase.
are consistent with the lack of known human homologs for the umuC and umuD genes responsible for errorprone DNA repair in bacteria and with the concept that reduced levels of specific DNA repair proteins in human beings may be causally related to increased ultravioletinduced mutation frequency and skin cancer.28
Molecular Mechanism of the Human SOS-Like Response The 53-kD transcription factor and tumor suppressor protein, p53, is termed the guardian of the genome because of its central role in DNA repair. To determine whether p53 mediates the effects of pTpT, dinucleotide treatments were compared in an established cell line lacking the p53 gene and in the same cell line transfected with wild-type (normal) p53. The pTpT effects were observed only in cells containing p53.23 Furthermore, pTpT treatment was found to activate p53 protein in a number of established assay systems23,32 and to increase mRNA and protein expression of genes known to be p53 regulated, including p21, PCNA, and GADD45, known to be involved in DNA repair.23 As predicted by the SOS hypothesis, ultraviolet irradiation similarly up-regulates these genes.33 Recently, even tyrosinase, the rate-limiting enzyme in melanogenesis, was found to be transcriptionally regulated by p53,34,35 explaining its up-regulation by pTpT and presumably also ultraviolet irradiation, which is well documented to increase p53 levels and activity. Early experiments focused on pTpT, with the rationale that adjacent thymidines frequently participated in ultraviolet-induced DNA photoproduct formation. However, it was subsequently found that several other oligonucleotides, arbitrarily chosen and up to 9 bases in length, but not all such oligomers, were equally or more effective in
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FIG 2. Consequences of SOS response in human skin. In skin, under basal conditions of relatively low DNA repair capacity and melanin content, an initial ultraviolet exposure (UV-1) results in substantial immediate DNA damage (solid line). Repair, as measured by removal of DNA photoproducts after irradiation of cultured newborn fibroblasts, has been shown to occur exponentially, and the time for 50% of total repair (T1) has been determined to be approximately 24 hours for thymine dimers and 6 hours for (6-4) photoproducts. Concomitantly, over approximately 3 to 5 days after the ultraviolet exposure, tyrosinase gene expression and melanin content of skin progressively increase, leading to a visible tan, and rate-limiting DNA repair proteins are progressively induced 2- to 3-fold (dashed line), as determined experimentally. Thereafter, a second equal ultraviolet exposure (UV-2) results in less initial damage (primarily because of greater absorption of ultraviolet photons by melanin) and the 50% repair time (T2) is approximately half, as determined experimentally in cultured human-derived cells. After each ultraviolet exposure, there is a dose-dependent p53-mediated cell cycle arrest, on the order of 1 to 2 days, during which DNA repair occurs and mutations (consequences of DNA replication past unrepaired photoproducts) do not form. The length of this arrest (L1 and L2, shaded areas) depends on the extent of p53 activation and resulting level and duration of induction of p21 and other cell cycle inhibitors. Hence, it is probably greater after the second ultraviolet exposure when p53 induction is more pronounced, but it has not been determined experimentally. In combination, these phenomena result in a period of time after a first ultraviolet exposure when considerable DNA damage persists, cells have resumed DNA synthesis, DNA repair capacity is submaximal, and therefore there is a relatively high risk of mutation (striped area below curve). In contrast, after a second ultraviolet exposure that occurs during the period of induced photoprotection, growth arrest persists for essentially the entire period required for DNA repair, greatly reducing the risk of ultraviolet-induced mutation. Eventually, in the absence of further ultraviolet exposures, the induced melanin content and repair proficiency return to baseline; and any subsequent exposure is handled like UV-1. However, if exposures are repeated during the period encompassed by the SOS response (assumed to be 1 to 2 weeks, depending on inciting ultraviolet exposure), increased melanin content and repair proficiency are retained or further induced and each exposure is handled like UV-2. For these reasons, the pattern of ultraviolet exposures, in addition to the total cumulative ultraviolet dose, has major long-term impacts on skin.
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stimulating melanogenesis and other photoadaptive responses,36 although effective sequences did not necessarily contain adjacent thymidines. These findings suggest that the base sequence of DNA fragments is critical to signal the presence of at least some types of DNA damage within cells and that effective sequences include but are not limited to pTpT. Thus, as with the bacterial SOS response, the adaptive human response to DNA injury appears to involve transcriptional up-regulation of multiple gene products critical for DNA repair and subsequent survival. Moreover, it is mimicked by short single-stranded DNA fragments (Table 1), suggesting that this is the physiological signal after ultraviolet irradiation.
DNA Fragments Mimic Other Ultraviolet-Induced Photoprotective Responses Ultraviolet irradiation has numerous recognized effects on human skin that are used therapeutically and can be interpreted as photoprotective. These include transient arrest of the cell cycle (or epidermal turnover), a response that allows time for DNA repair before cell division; apoptosis or programmed cell suicide of severely DNA-damaged cells, a response that reduces the risk of subsequent mutation and malignant transformation; and transient immunosuppression, a response postulated to avoid eliciting immunity to self-antigens such as DNA that has been altered by introduction of DNA photoproducts. Reversible cell cycle arrest and slowed growth of treated cell cultures, in the absence of cellular toxicity, had been noted routinely in pTpT experiments.22,23 Elucidation of the role of p53 led to recognition that this growth arrest was mediated at least in part by the p53-regulated gene p21.23 The effect could also be observed in vivo as a reduced epidermal labeling index.7 Under the experimental conditions initially used, apoptosis was not observed in keratinocytes, fibroblasts, or melanocytes after pTpT treatment. However, later experiments with apoptosis-prone human T cells and treatment with pTpT or, more strikingly, other oligonucleotides induced classic apoptosis over a period of several days.36 Whether the mechanism is the same as, for example, that underlying the therapeutic response of cutaneous T-cell lymphoma to psoralen–UV-A therapy or photopheresis remains to be determined.
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To determine whether, like ultraviolet irradiation, pTpT is immunosuppressive, dinucleotide treatment was compared with ultraviolet in a standard mouse model. Animals are allergically sensitized to dinitrofluorobenzene (DNFB) by topical application to abdominal skin and the intensity of sensitization determined 2 weeks later by application of a challenge dose of DNFB to the ear.37 As expected, control animals sensitized to DNFB showed marked ear swelling after the challenge dose, and animals whose abdominal skin was ultraviolet irradiated before application of DNFB failed to become sensitized and showed no ear swelling after application of the challenge dose. In this assay system, pretreatment of the abdominal skin with topical pTpT was as effective as ultraviolet irradiation in blocking allergic sensitization.37 Further testing in a transgenic mouse model revealed that, again as with ultraviolet irradiation, pTpT transcriptionally up-regulated tumor necrosis factor–α,37 a cytokine known to mediate immunosuppression under these conditions. Most recently, studies were performed to determine whether pTpT also elicits responses protective against the oxidative damage known to result from generation of free radical species within irradiated cells. The literature regarding effects of ultraviolet irradiation in this context is controversial, with most studies showing a transient decrease in antioxidant cellular defenses after ultraviolet irradiation and some suggesting a subsequent upregulation. In these experiments, cultured cells were exposed to hydrogen peroxide (H2O2), a source of pure oxidative damage, after pretreatment with pTpT or diluent alone. As expected, H2O2 caused dose-dependent cell killing, but cells pretreated with pTpT had approximately twice the survival rate of controls. Preliminary experiments indicate that this enhanced survival can be attributed at least in part to up-regulation of manganesedependent superoxide dismutase 1, with consequent increase in cellular antioxidant capacity.38 Overall, these data suggest that the SOS-like response in mammalian skin that follows ultraviolet-induced DNA damage is very broad. In addition to tanning, it appears to consist of enhanced DNA repair, transient cell cycle arrest, apoptosis in severely damaged or genetically predisposed cells, transient immunosuppression, and enhanced antioxidant capacity. The ability of pTpT and other oligonucleotides to mimic these responses suggests that all are stimulated by generation of singlestranded DNA that in turn activates p53, leading to
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induction of p53-regulated genes and ultimately to the photoprotective cellular changes.
Summary and Conclusions Increasing evidence suggests that human cells respond to DNA damage by activating p53 and inducing p53-regulated gene products that then enhance the cell’s ability to survive subsequent damage of the same type (Fig 2). Identical responses can be induced in the absence of DNA damage by providing cells single-stranded DNA fragments such as thymidine dinucleotides that apparently mimic the DNA damage signal within cells. This response thus appears highly analogous to the wellstudied SOS response in bacteria that is also initiated by single-stranded DNA and mediated by transcriptional up-regulation of key genes. In human skin, the SOS-like response includes not only up-regulation of DNA repair capacity (as in bacteria), but also increased melanogenesis, transient cell cycle arrest, transient immunosuppression, and up-regulation of antioxidant defenses. These observations suggest that topical treatment with pTpT or other effective DNA fragments might permit stimulation of these photoprotective and often therapeutically beneficial responses in human skin, in the absence of the DNA damage that inevitably accompanies natural sun exposure and phototherapy.
REFERENCES 1. Radman M. phenomenology of an inducible mutageneic DNA repair pathway in Escherichia coli: SOS repair hypothesis. In: Prakash L, Sherman R, Miller M, Lawrence C, Tabor HW, editors. Molecular and environmental aspects of mutagenesis. Springfield (Ill): Charles C. Thomas; 1974. p. 128-42. 2. Nordlund J, Boissy RE, Hearing VJ, King RA, Ortonne J-P. The pigmentary system. New York: Oxford University Press, 1998. 3. Pathak MA. Functions of melanin and protection by melanin. In: Zeise L, Chedekel MR, Thomas B, editors. Melanin: its role in human photoprotection. Overland Park: Valdenmar Publishing; 1995. p. 125-34. 4. Kripke ML. Carcinogenesis: ultraviolet. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austin KF, editors. Dermatology in general medicine, vol. I. New York: McGraw-Hill; 1993. p. 797-804. 5. Mark R, Sober JA. Skin cancer. In: Lim HW, Soter NA, editors. Clinical photomedicine. New York: Marcel Dekker; 1993. p. 113-35. 6. Pathak MA, Fitzpatrick TB. The role of natural photoprotective agents in human skin. In: Pathak MA, Harber LC, Seiji M, Kukita A, Fitzpatrick TB, editors. Sunlight and man. Tokyo: University of Tokyo Press, 1974. p. 725-50.
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7. Gilchrest BA, Eller MS. DNA Photodamage stimulates melanogenesis and other photoprotective responses. J Invest Dermatol Symposium Proc 1999;4:35-40. 8. Freeman SE, Hacham H, Gange RW, Maytum DJ, Sutherland JC, Sutherland BM. Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light. Proc Natl Acad Sci USA 1989;86:5605. 9. Mitchell DL, Nairn RS. The biology of the (6-4) photoproduct. Photochem Photobiol 1989;49:805-19. 10. Mitchell DL, Jen J, Cleaver JE. Relative induction of cyclobutane dimers and cytosine photohydrates in DNA irradiated in vitro and in vivo with ultraviolet-C and ultraviolet-B light. Photochem Photobiol 1991;54:741-6. 11. Eller MS. Repair of DNA photodamage in human skin. In: Gilchrest BA, editor. Photodamage. New York: Blackwell Science; 1995. p. 26-50. 12. Wood RD. DNA repair in eukaryotes. Annu Rev Biochem 1996;65:135-67. 13. de Laat WL, Jaspers NG, et al. Molecular mechanism of nucleotide excision repair. Genes Dev 1999;13:768-85. 14. Freidberg EC, Walker GC, Siede W. DNA repair and mutagenesis. Washington (DC): ASM Press; 1995. 15. Kraemer KH, Lee MM, Scotto J. Xeroderma pigmentosum: cutaneous ocular and neurologic abnormalities in 830 published cases. Arch Dermatol 1987;123:241-50. 16. Walker GC. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 1984;48:60-93. 17. Reuven, NB, Tomer G, Livneh Z. The mutagenesis proteins UmuD’ and UmuC prevent lethal frameshifts while increasing base substitution mutations. Mol Cell 1998;2:191-9. 18. Jimbow K, Quevado WC, Fitzpatrick TB, Szabo G. Biology of melanocytes. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF, editors. Dermatology in general medicine. 4th ed. Vol I. New York: McGraw-Hill; 1993. p. 261-89. 19. Gilchrest BA, Zhai S, Eller MS, Yarosh DB, Yaar M. Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation. J Invest Dermatol 1993;101:666-72. 20. Setlow R, Carrier WL. Pyrimidine dimers in ultraviolet-irradiated DNAs. J Mol Biol 1966;17:237-54. 21. Eller MS, Yaar M, Gilchrest BA. DNA damage and melanogenesis. Nature 1994;372:413-4. 22. Pedeux R, Al-Irani N, Marteau C, et al. Thymidine dinucleotides induce S phase cell cycle arrest in addition to increased melanogenesis in human melanocytes. J Invest Dermatol 1998;3:472-7. 23. Eller MS, Maeda T, Magnoni C, Atwal D, Gilchrest BA. Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response. Proc Natl Acad Sci USA 1997;94:12627-32. 24. Protic M, Roilides E, Levin AS, Dixon K. Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment. Somat Cell MolGenet 1988;14:351-7.
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25. McKay BC, Rainbow AJ. Heat-shock enhanced reactivation of a UV-damaged reporter gene in human cells involves the transcription coupled DNA repair pathway. Mutat Res 1996;363: 125-35. 26. McKay BC, Francis MA, Rainbow AJ. Wild type Q53 is required for heat shock and ultraviolet light enhanced repair of UV-damaged reporter gene. Carcinogenesis 1997;18:245-9. 27. Wei Q, Matanoski GM, Farmer ER, Hedayati MA, Grossman L. DNA repair and aging in basal cell carcinoma: a molecular epidemiology study. Proc Natl Acad Sci USA 1993;90:1614-8. 28. Moriwaki S-I, Ray S, Tarone RE, Kraemer KH, Grossman L. The effect of donor age on the processing of UV-damaged DNA by cultured human cells: reduced DNA repair capacity and increased DNA mutability. Mutat Res 1996;364:117-23. 29. Dolle MET, Martus H-J, Gossen JA, Boerrigter METI, Vijg J. Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations. Mutagenesis 1996;11:111-8. 30. Boerrigter METI, Dolle MET, Martus H-J, Gossen JA, Vijg J. Plasmid-based transgeneic mouse model for studying in vivo mutations. Nature 1995;377:657-9. 31. Hadshiew IM, Khlagatian M, Giese H, Eller MS, Vijg, Gilchrest BA. Reduktion de UV-induzierten Mutationsrate durch Behandlung mit dem Thymidin Dinucleotid (pTpT). In: Plettenbert A, Meigel WN, Moll I, editors. Dermatologie an der Schwelle zum neuen Jahrtausent – Aktueller Stand von Klinik und Forschung. Berlin: Springer Verlag, 1999. p. 652-4. 32. Maeda T, Eller MS, Hodayati M, Grossman L, Gilchrest BA. Enhanced repair of benzo(a)pyrene-induced DNA damage in human cells treated with thymidine dinucleotides. Mut Res 1999;433:137-45. 33. Goukassian D, Eller MS, Gilchrest BA. Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins [abstract]. J Invest Dermatol 1998; 110:474. 34. Nylander K, Bourdon JC, Bray SE, Gibbs NK, Kay R, Hart I, Hall PA. Transcriptional activation of tyrosinase and TRP-1 by p53 links UV irradiation to the protective tanning response. J Pathol 2000;190:39-46. 35. Khlgatian M, Asawanonda P, Eller M, Yaar M, Fujita M, Norris DA, et al. Tyrosinase expression is regulated by p53. J Invest Dermatol In press. 36. Eller MS, Hadshiew IM, Puri N, Curiel-Lewandrowski C, Venna S, Gilchrest BA. The single-stranded telomeric DNA induces DNA damage responses [abstract]. J Invest Dermatol 2000;114:756. 37. Cruz PD, Leverkus M, Dougherty I, Gleason MJ, Eller MS, Yaar M, et al. Thymidine dinucleotides inhibit contact hypersensitivity and activate the gene for tumor necrosis factor. J Invest Dermatol 2000;114:253-8. 38. Lee MS, Li GS, Yaar M, Ruenger T, Gilchrest BA. Thymidine dinucleotide (pTpT) protects cells against oxidative damage [abstract]. J Invest Dermatol 2000;114:882. 54/1/111435 doi:10.1067/mdm.2001.111435
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The wavelengths of sunlight transmitted to the earth’s surface include ultraviolet radiation that is harmful to living organisms. The damage is primarily genetic: shorter wavelengths (UV-B, 290 to 320 nm) catalyze formation of covalent bonds between adjacent nucleotides in DNA molecules, and longer wavelengths (UV-A, 320 to 400 nm) oxidize membrane lipids and other cellular constituents to generate free radicals that then modify individual nucleotides. All terrestrial organisms appear to have evolved both constitutive and inducible photoprotective strategies that provide shielding from damaging ultraviolet wavelengths or enhance repair of damaged DNA. Constitutive DNA repair was first elucidated in bacteria and is now also well understood in mammals. It comprises two strategies for recognizing and replacing damaged nucleotides, as described below. By far the best characterized inducible or photoadaptive behavior is the SOS response, also first described in bacteria, in the 1970s by Radman.1 This response, now understood in detail at the molecular level, appears designed evolutionarily to protect these single-celled organisms from frequently encountered DNA-damaging agents. Human skin also responds to ultraviolet radiation in a manner that decreases damage from subsequent exposures. To a degree determined by as yet unknown genetic factors, after a lag period of several days, skin increases its synthesis and intraepidermal dispersion of melanin, a pigment that directly absorbs ultraviolet photons and free radicals to protect tissues from their destructive effects on DNA.2,3 This well-recognized process is termed tanning. Lack of melanin protection increases skin cancer risk, as evidenced by cancerprone fair-skinned people4,5 or patients with albinism.4,6 Very recently, however, human skin cells have also been shown to induce DNA repair enzymes and other photoprotective gene products in response to ultraviolet irradiation,7 suggesting the existence of a photoadaptive response far broader than increased melanin alone.
Curr Probl Dermatol, May/June 2001
Repair of Ultraviolet-Induced DNA Damage Terrestrial sunlight damage to DNA is principally caused by the UV-B wavelengths. More than 90% of the lesions are cyclobutyl pyrimidine dimers or pyrimidine (6-4) pyrimidone photoproducts.8-10 These lesions are largely repaired by a process called nucleotide excision repair (NER) in which incisions are made in the DNA on both sides of the lesion. The damaged strand is then displaced, the gap (20 to 25 nucleotides in bacteria and 29 to 32 in human beings) is filled in by DNA polymerase, and the new strand is religated to repair the lesion.11-13 Oxidized nucleotide bases resulting from either UV-B or UV-A–induced free radical damage are replaced individually by base excision repair,14 a constitutive process essential for cell viability. NER is not essential for cell viability but is evolutionarily conserved from bacteria to mammals.14 In human beings, defects in genes encoding any of at least 7 NER pathway proteins cause xeroderma pigmentosum (XP), a recessively inherited disease that manifests ultraviolet hypersensitivity with various photoaging-like changes such as dryness and irregular pigmentation for which the disease is named.14 In addition, patients with XP show a greater than 1000-fold increased risk for skin cancer compared with the general population.15 The XP phenotype in human beings confirms the major photoprotective function of NER.
The SOS Response in Bacteria The SOS response to DNA damage was first described in Escherichia coli as a system promoting DNA repair, cell cycle control, and recombination.16 In bacteria, single-stranded DNA generated during the course of DNA damage and repair interacts with and activates a protease, leading ultimately to derepression of at least 20 genes involved in DNA repair, replication, and cell survival16 (Fig 1). This is termed the SOS response and serves to enhance survival after the inciting irradiation and to increase bacterial resistance to subsequent ultraviolet-induced DNA damage. Thus if bacteria are
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FIG 1. Single-stranded DNA induces expression of SOS genes in bacteria. The LexA protein normally represses genes that are induced as part of the SOS response. After DNA damage, single-stranded DNA is generated by the excision repair process and at stalled replication forks. This single-stranded DNA interacts with and activates the RecA protease (designated as RecA*) which then cleaves the LexA protein, de-repressing and inducing the transcription of the SOS response genes.
exposed to a sublethal dose of ultraviolet irradiation, and those that survive are subsequently exposed to the same ultraviolet dose, the organisms more efficiently process the DNA damage and manifest enhanced survival. Another feature of the SOS response is bypassing the block on DNA replication induced by DNA damage. Induction of two genes, UmuC and UmuD, allows DNA polymerase to replicate a damaged template by relaxing normal Watson-Crick base-pairing requirements.17 This repair is error-prone but immediately beneficial for cell survival, even though it increases the risk of mutations that may affect cellular function in subsequent generations. Indeed, it has been postulated that error-prone repair of the ultraviolet-irradiated bacterial genome is evolutionarily beneficial because it encourages the appearance of mutations, some of which are likely to improve cell function or confer better survival in the presence of the environmental DNA-damaging agent.
The SOS Response in Human Skin Because ultraviolet-induced tanning is the major recognized defense of human skin against subsequent ultraviolet damage,2,18 we postulated that tanning might be part of a mammalian SOS response and, by analogy to bacteria, that melanogenesis might be stimulated by ultraviolet damage–induced single-stranded DNA fragments. The postulated link between tanning and DNA
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damage in human skin was supported by the observation that treatment of cultured pigment cells with a liposomeencapsulated prokaryotic DNA repair protein, T4 endonuclease V, known to catalyze the rate-limiting step of excision repair of thymine dimers in bacteria, enhances melanogenesis after ultraviolet irradiation.19 Thymine dimers are the most common ultraviolet photoproducts,20 and therefore thymidine dinucleotides (abbreviated pTpT to indicate their phosphate linkage in the DNA backbone) would be the most common sequence-specific element in ultravioletdamaged DNA. Indeed, pTpT, like ultraviolet radiation exposure, was found to markedly increase cellular melanin within 5 days in cultured mouse and human pigment cells and in intact guinea pig skin.21,22 As a control, the adenine dinucleotide pApA, which does not commonly form dimers, did not elicit a tanning response. These studies also found that the tanning response to pTpT mimicked ultraviolet-induced melanogenesis mechanistically, in that both ultraviolet radiation and pTpT induced tyrosinase expression and alpha-melanocyte–stimulating hormone binding to the cell surface and had a similar time course, requiring several days for maximal response.23
DNA Fragments Up-regulate DNA Repair Capacity In bacteria, the ultraviolet radiation–induced SOS response consists of transcriptional up-regulation of genes involved in DNA repair and cell survival.16 Although a comparable response was not recognized in human beings, there were isolated reports of DNA repair enzyme induction after DNA damage in mammalian cells.24-26 Up-regulation of DNA repair capacity in human skin would be expected to have a clinically meaningful protective effect, in that impaired DNA repair capacity is known to be associated with the development of skin cancer. The most dramatic example of this relationship is XP, described above, in which repair capacity is reduced by at least 50% and in some patients by more than 95%, depending on the specific mutation and the repair assay used. However, skin cancer occurrence in otherwise normal individuals has also been linked to more subtle reductions in DNA repair capacity,27 and in large populations, decreased DNA repair capacity is inversely related to mutation frequency,28 with mutations in key regulatory genes playing a major role in carcinogenesis. We therefore investigated whether
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pTpT might also stimulate DNA repair capacity in human skin–derived cells. The effect of pTpT on DNA repair was determined by comparing the ability of pTpT-treated cells versus controls to repair and then transcribe a reporter plasmid containing the bacterial chloramphenicol acetyl transferase gene in a host cell reactivation assay. In this test system, human keratinocytes and fibroblasts pretreated with pTpT for 3 days and then transfected with the reporter plasmid were twice as effective as control cells in repair and subsequent expression of this ultraviolet-damaged chloramphenicol acetyl transferase gene.23 Similar results were obtained in a second assay in which pTpTtreated cells or controls were ultraviolet irradiated, and the rate of removal of DNA photoproducts was determined directly in multiple DNA samples harvested over a 24-hour period. The pTpT-treated cultures removed photoproducts at a statistically more rapid rate. In ultraviolet-irradiated guinea pig skin, pTpT-treated sites also showed more rapid removal of photoproducts, compared with control vehicle–treated sites, as indicated by loss of antidimer antibody binding to nuclei in skin biopsy cross-sections.7 These data demonstrate that dinucleotide treatment is effective, both in vivo and in vitro, in preparing cells to combat ultraviolet-induced DNA damage. The mechanism by which pTpT increases DNA repair capacity proved to be a 2- to 3-fold up-regulation of mRNA and protein levels of several DNA repair enzymes and cell cycle regulation proteins,23,29 although it remains unknown whether all are similarly rate-limiting in normal cells under basal conditions. Because SOS repair in bacteria is error-prone, as noted above, and because mutations in human cells may lead to dysfunction or malignant transformation, experiments were performed to determine whether the enhanced DNA repair induced by pTpT is also error-prone. A transgenic mouse model was used in which all cells carry multiple copies of a bacterial Lac-Z reporter plasmid stably integrated into the genome that can be isolated to determine quantitatively the number of mutations caused by a treatment of interest.29,30 Mouse skin in vivo or cultured cells derived from the mice were treated with pTpT or vehicle alone and then ultraviolet irradiated, after which the Lac-Z plasmid was harvested and analyzed for mutations. As expected, ultraviolet irradiation caused mutations in a dose-dependent manner. Of critical importance, however, pTpT pretreatment decreased the mutation rate by approximately 50%.33 These results
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TABLE 1. Photoprotective responses inducible by oligonucleotides
Response
p53 Mediated?
Induced gene products identified to date
Tanning Cell cycle arrest Enhanced DNA repair
Yes Yes Yes
Immunosuppression Enhanced repair of oxidative damage Apoptosis
Unknown Unknown
Tyrosinase p21 XPA, PCNA, ERCC3, RPA, GADD45 TNFα, IL-10 SOD 1
Yes
p53, E2F1
TNFα, Tumor necrosis factor–α; IL-10, interleukin 10; SOD 1, superoxide dismutase.
are consistent with the lack of known human homologs for the umuC and umuD genes responsible for errorprone DNA repair in bacteria and with the concept that reduced levels of specific DNA repair proteins in human beings may be causally related to increased ultravioletinduced mutation frequency and skin cancer.28
Molecular Mechanism of the Human SOS-Like Response The 53-kD transcription factor and tumor suppressor protein, p53, is termed the guardian of the genome because of its central role in DNA repair. To determine whether p53 mediates the effects of pTpT, dinucleotide treatments were compared in an established cell line lacking the p53 gene and in the same cell line transfected with wild-type (normal) p53. The pTpT effects were observed only in cells containing p53.23 Furthermore, pTpT treatment was found to activate p53 protein in a number of established assay systems23,32 and to increase mRNA and protein expression of genes known to be p53 regulated, including p21, PCNA, and GADD45, known to be involved in DNA repair.23 As predicted by the SOS hypothesis, ultraviolet irradiation similarly up-regulates these genes.33 Recently, even tyrosinase, the rate-limiting enzyme in melanogenesis, was found to be transcriptionally regulated by p53,34,35 explaining its up-regulation by pTpT and presumably also ultraviolet irradiation, which is well documented to increase p53 levels and activity. Early experiments focused on pTpT, with the rationale that adjacent thymidines frequently participated in ultraviolet-induced DNA photoproduct formation. However, it was subsequently found that several other oligonucleotides, arbitrarily chosen and up to 9 bases in length, but not all such oligomers, were equally or more effective in
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FIG 2. Consequences of SOS response in human skin. In skin, under basal conditions of relatively low DNA repair capacity and melanin content, an initial ultraviolet exposure (UV-1) results in substantial immediate DNA damage (solid line). Repair, as measured by removal of DNA photoproducts after irradiation of cultured newborn fibroblasts, has been shown to occur exponentially, and the time for 50% of total repair (T1) has been determined to be approximately 24 hours for thymine dimers and 6 hours for (6-4) photoproducts. Concomitantly, over approximately 3 to 5 days after the ultraviolet exposure, tyrosinase gene expression and melanin content of skin progressively increase, leading to a visible tan, and rate-limiting DNA repair proteins are progressively induced 2- to 3-fold (dashed line), as determined experimentally. Thereafter, a second equal ultraviolet exposure (UV-2) results in less initial damage (primarily because of greater absorption of ultraviolet photons by melanin) and the 50% repair time (T2) is approximately half, as determined experimentally in cultured human-derived cells. After each ultraviolet exposure, there is a dose-dependent p53-mediated cell cycle arrest, on the order of 1 to 2 days, during which DNA repair occurs and mutations (consequences of DNA replication past unrepaired photoproducts) do not form. The length of this arrest (L1 and L2, shaded areas) depends on the extent of p53 activation and resulting level and duration of induction of p21 and other cell cycle inhibitors. Hence, it is probably greater after the second ultraviolet exposure when p53 induction is more pronounced, but it has not been determined experimentally. In combination, these phenomena result in a period of time after a first ultraviolet exposure when considerable DNA damage persists, cells have resumed DNA synthesis, DNA repair capacity is submaximal, and therefore there is a relatively high risk of mutation (striped area below curve). In contrast, after a second ultraviolet exposure that occurs during the period of induced photoprotection, growth arrest persists for essentially the entire period required for DNA repair, greatly reducing the risk of ultraviolet-induced mutation. Eventually, in the absence of further ultraviolet exposures, the induced melanin content and repair proficiency return to baseline; and any subsequent exposure is handled like UV-1. However, if exposures are repeated during the period encompassed by the SOS response (assumed to be 1 to 2 weeks, depending on inciting ultraviolet exposure), increased melanin content and repair proficiency are retained or further induced and each exposure is handled like UV-2. For these reasons, the pattern of ultraviolet exposures, in addition to the total cumulative ultraviolet dose, has major long-term impacts on skin.
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stimulating melanogenesis and other photoadaptive responses,36 although effective sequences did not necessarily contain adjacent thymidines. These findings suggest that the base sequence of DNA fragments is critical to signal the presence of at least some types of DNA damage within cells and that effective sequences include but are not limited to pTpT. Thus, as with the bacterial SOS response, the adaptive human response to DNA injury appears to involve transcriptional up-regulation of multiple gene products critical for DNA repair and subsequent survival. Moreover, it is mimicked by short single-stranded DNA fragments (Table 1), suggesting that this is the physiological signal after ultraviolet irradiation.
DNA Fragments Mimic Other Ultraviolet-Induced Photoprotective Responses Ultraviolet irradiation has numerous recognized effects on human skin that are used therapeutically and can be interpreted as photoprotective. These include transient arrest of the cell cycle (or epidermal turnover), a response that allows time for DNA repair before cell division; apoptosis or programmed cell suicide of severely DNA-damaged cells, a response that reduces the risk of subsequent mutation and malignant transformation; and transient immunosuppression, a response postulated to avoid eliciting immunity to self-antigens such as DNA that has been altered by introduction of DNA photoproducts. Reversible cell cycle arrest and slowed growth of treated cell cultures, in the absence of cellular toxicity, had been noted routinely in pTpT experiments.22,23 Elucidation of the role of p53 led to recognition that this growth arrest was mediated at least in part by the p53-regulated gene p21.23 The effect could also be observed in vivo as a reduced epidermal labeling index.7 Under the experimental conditions initially used, apoptosis was not observed in keratinocytes, fibroblasts, or melanocytes after pTpT treatment. However, later experiments with apoptosis-prone human T cells and treatment with pTpT or, more strikingly, other oligonucleotides induced classic apoptosis over a period of several days.36 Whether the mechanism is the same as, for example, that underlying the therapeutic response of cutaneous T-cell lymphoma to psoralen–UV-A therapy or photopheresis remains to be determined.
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To determine whether, like ultraviolet irradiation, pTpT is immunosuppressive, dinucleotide treatment was compared with ultraviolet in a standard mouse model. Animals are allergically sensitized to dinitrofluorobenzene (DNFB) by topical application to abdominal skin and the intensity of sensitization determined 2 weeks later by application of a challenge dose of DNFB to the ear.37 As expected, control animals sensitized to DNFB showed marked ear swelling after the challenge dose, and animals whose abdominal skin was ultraviolet irradiated before application of DNFB failed to become sensitized and showed no ear swelling after application of the challenge dose. In this assay system, pretreatment of the abdominal skin with topical pTpT was as effective as ultraviolet irradiation in blocking allergic sensitization.37 Further testing in a transgenic mouse model revealed that, again as with ultraviolet irradiation, pTpT transcriptionally up-regulated tumor necrosis factor–α,37 a cytokine known to mediate immunosuppression under these conditions. Most recently, studies were performed to determine whether pTpT also elicits responses protective against the oxidative damage known to result from generation of free radical species within irradiated cells. The literature regarding effects of ultraviolet irradiation in this context is controversial, with most studies showing a transient decrease in antioxidant cellular defenses after ultraviolet irradiation and some suggesting a subsequent upregulation. In these experiments, cultured cells were exposed to hydrogen peroxide (H2O2), a source of pure oxidative damage, after pretreatment with pTpT or diluent alone. As expected, H2O2 caused dose-dependent cell killing, but cells pretreated with pTpT had approximately twice the survival rate of controls. Preliminary experiments indicate that this enhanced survival can be attributed at least in part to up-regulation of manganesedependent superoxide dismutase 1, with consequent increase in cellular antioxidant capacity.38 Overall, these data suggest that the SOS-like response in mammalian skin that follows ultraviolet-induced DNA damage is very broad. In addition to tanning, it appears to consist of enhanced DNA repair, transient cell cycle arrest, apoptosis in severely damaged or genetically predisposed cells, transient immunosuppression, and enhanced antioxidant capacity. The ability of pTpT and other oligonucleotides to mimic these responses suggests that all are stimulated by generation of singlestranded DNA that in turn activates p53, leading to
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induction of p53-regulated genes and ultimately to the photoprotective cellular changes.
Summary and Conclusions Increasing evidence suggests that human cells respond to DNA damage by activating p53 and inducing p53-regulated gene products that then enhance the cell’s ability to survive subsequent damage of the same type (Fig 2). Identical responses can be induced in the absence of DNA damage by providing cells single-stranded DNA fragments such as thymidine dinucleotides that apparently mimic the DNA damage signal within cells. This response thus appears highly analogous to the wellstudied SOS response in bacteria that is also initiated by single-stranded DNA and mediated by transcriptional up-regulation of key genes. In human skin, the SOS-like response includes not only up-regulation of DNA repair capacity (as in bacteria), but also increased melanogenesis, transient cell cycle arrest, transient immunosuppression, and up-regulation of antioxidant defenses. These observations suggest that topical treatment with pTpT or other effective DNA fragments might permit stimulation of these photoprotective and often therapeutically beneficial responses in human skin, in the absence of the DNA damage that inevitably accompanies natural sun exposure and phototherapy.
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