The DNA Damage Signal for Mdm2 Regulation, Trp53 Induction, and Sunburn Cell Formation In Vivo Originates from Actively Transcribed Genes
2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês
10.1046/j.0022-202x.2001.01554.x
ISSN1523-1747
AutoresDouglas E. Brash, Norbert M. Wikonkal, Éva Remenyik, Gijsbertus T. J. van der Horst, Errol C. Friedberg, David L. Cheo, Harry van Steeg, Anja Westerman, Henk J. van Kranen,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoThe stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes (“sunburn cells”). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa−/− mice were 7–10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86–90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb−/− mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa−/−, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc−/− mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90–96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in ≈ 89% of the Mdm2 response. The stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes (“sunburn cells”). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa−/− mice were 7–10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86–90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb−/− mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa−/−, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc−/− mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90–96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in ≈ 89% of the Mdm2 response. Cockayne syndrome complementation group B xeroderma pigmentosum complementation group A xeroderma pigmentosum complementation group C human p53 tumor suppressor gene murine p53 gene human p53 protein murine p53 protein Skin irradiated with ultraviolet (UV) B generates characteristic “sunburn cells”—keratinocytes with pyknotic nuclei and intense eosinophilic staining. Sunburn cells arise in the epidermis of many mammalian species, including humans (Danno and Horio, 1987Danno K. Horio T. Sunburn cell: factors involved in its formation.Photochem Photobiol. 1987; 45: 683-690Crossref PubMed Scopus (88) Google Scholar;Young, 1987Young A.R. The sunburn cell.Photodermatology. 1987; 4: 127-134PubMed Google Scholar). They are produced weakly after UVA irradiation (Rosario et al., 1979Rosario R. Mark G.J. Parrish J.A. Mihm M.C. Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens.Br J Dermatol. 1979; 101: 299-308Crossref PubMed Scopus (88) Google Scholar;Lavker and Kaidbey, 1997Lavker R. Kaidbey K. The spectral dependence for UVA-induced cumulative damage in human skin.J Invest Dermatol. 1997; 108: 17-21Crossref PubMed Scopus (101) Google Scholar), whereas UVC is ineffective due to absorption by keratin (Freeman et al., 1989Freeman S.E. Hacham H. Gange R.W. Maytum D.J. Sutherland J.C. Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light.Proc Natl Acad Sci USA. 1989; 86: 5605-5609Crossref PubMed Scopus (252) Google Scholar). These curiosities became of potential importance for human skin cancer after the demonstration that individual sunburn cells contain the DNA double-strand breaks typical of cells undergoing apoptosis (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 (1302) Google Scholar;Brash et al., 1996Brash D.E. Ziegler A. Jonason A. Simon J.A. Kunala S. Leffell D.J. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.J Invest Dermatol Symp Proc. 1996; 1: 136-142PubMed Google Scholar) and that Trp53 knockout mice exhibit an approximately 7-fold deficiency in sunburn cell production (Ziegler et al., 1994Ziegler A. Jonason A.S. 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The involvement of Trp53 in sunburn cell formation provides a connection to cancer, as TP53 mutations are present in most human nonmelanoma skin tumors or precancers (actinic keratoses) and Trp53 mutations are present in most murine skin squamous cell carcinomas induced by UVB (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1644) Google Scholar,Brash et al., 1996Brash D.E. Ziegler A. Jonason A. Simon J.A. Kunala S. Leffell D.J. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.J Invest Dermatol Symp Proc. 1996; 1: 136-142PubMed Google Scholar;Nataraj et al., 1995Nataraj A.J. Trent J.C. 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In skin, apoptosis of keratinocytes after UVB has the important function of preventing the appearance of mutant cells. A 3.6-fold reduction in apoptosis, due to a defective FasL gene, results in a much larger increase in the frequency of UV-induced mutations in the epidermis (Hill et al., 1999Hill L.L. Ouhtit A. Loughlin S.M. Kripke M.L. Ananthaswamy H.N. Owen-Schaub L.B. Fas ligand: a sensor for DNA damage critical in skin cancer etiology.Science. 1999; 285: 898-900Crossref PubMed Scopus (220) Google Scholar). Other gene products now known to participate in UV-induced apoptosis include the apoptosis agonists and antagonists p53IAP1, BCL2, and BCL-xL (Pena et al., 1997Pena J.C. Fuchs E. Thompson C.B. Bcl-x expression influences keratinocyte cell survival but not terminal differentiation.Cell Growth Differ. 1997; 8: 619-629PubMed Google Scholar;Rodriguez-Villanueva et al., 1998Rodriguez-Villanueva J. Greenhalgh D. 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Evaluation of apoptotic cells induced by ultraviolet light B radiation in epidermal sheets stained by the TUNEL technique.J Invest Dermatol. 1999; 113: 802-807https://doi.org/10.1046/j.1523-1747.1999.00757.xCrossref PubMed Scopus (36) Google Scholar). A clue to the localization of this DNA-based apoptosis signal came from experiments with UVC-irradiated immortalized fibroblasts derived from patients with a point mutation in the CSB gene (Cockayne's syndrome complementation group B). These cells are defective in excision repair of the transcribed strand of active genes, a small fraction of the genome. In these fibroblasts, TP53 protein and apoptosis were induced at one-third the dose required for normal cells, although the dose was still 3-fold higher than that required in XPA cells (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;Ljungman and Zhang, 1996Ljungman M. Zhang F. Blockage of RNA polymerase as a possible trigger for u.v. light-induced apoptosis.Oncogene. 1996; 13: 823-831PubMed Google Scholar). The response may be mediated via MDM2, which targets TP53 for degradation, as MDM2 was induced in control but not XPA or CSB patient fibroblasts (Conforti et al., 2000Conforti G. Nardo T. D'Incalci M. Stefanini M. Proneness to UV-induced apoptosis in human fibroblasts defective in transcription coupled repair is associated with the lack of Mdm2 transactivation.Oncogene. 2000; 19: 2714-2720Crossref PubMed Scopus (47) Google Scholar). These results suggest that a major portion of the signal for TP53 induction and apoptosis requires a signal from photoproducts in active genes. This signal appears to originate when RNA polymerase II is stalled at a DNA photoproduct (Ljungman, 2000Ljungman M. Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress.Neoplasia. 2000; 2: 208-225Abstract Full Text PDF PubMed Scopus (179) Google Scholar). Similar experiments in hairless mice indicate the presence of such apoptosis signals in vivo; these may prevent mutations after spontaneous DNA lesions (van Oosten et al., 2000van Oosten M. Rebel H. Friedberg E.C. et al.Differential role of transcription-coupled repair in UVB-induced G2 arrest and apoptosis in mouse epidermis.Proc Natl Acad Sci USA. 2000; 97: 11268-11273Crossref PubMed Scopus (70) Google Scholar;Wijnhoven et al., 2000Wijnhoven S.W. Kool H.J. Mullenders L.H. et al.Age-dependent spontaneous mutagenesis in xpc mice defective in nucleotide excision repair.Oncogene. 2000; 19: 5034-5037Crossref PubMed Scopus (50) Google Scholar). This pathway differs from apoptosis induced by agents such as ionizing radiation, for which DNA double-strand breaks are a signal (Nelson and Kastan, 1994Nelson W.G. Kastan M.B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.Mol Cell Biol. 1994; 14: 1815-1823Crossref PubMed Scopus (848) Google Scholar). In contrast, a number of in vitro studies indicate the involvement of non-nuclear UV-induced signals such as ligand-independent activation of membrane receptors by UVC or UVB (Devary et al., 1993Devary Y. Rosette C. DiDonato J.A. Karin M. NF-κB activation by ultraviolet light not dependent on a nuclear signal.Science. 1993; 261: 1442-1445Crossref PubMed Scopus (567) Google Scholar;Sachsenmaier et al., 1994Sachsenmaier C. Radler-Pohl A. Zinck R. Nordheim A. Herrlich P. Rahmsdorf H.J. 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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: 7974-7979https://doi.org/10.1073/pnas.96.14.7974Crossref PubMed Scopus (174) Google Scholar). In vivo, on the other hand (see Discussion), apoptosis can be reduced by blocking receptors or ligands, as if a ligand–receptor interaction transmits an apoptosis signal that originates elsewhere. Yet, introducing ligand does not itself substitute for UVB. We therefore sought to determine in keratinocytes in vivo: (i) the fraction of the signal for UVB-induced apoptosis that requires DNA photoproducts; (ii) the extent to which this signal regulates two key molecular components of the UV apoptosis pathway, Trp53 and Mdm2; and (iii) whether this DNA photoproduct signal arises from the small proportion of genes that are actively transcribed. We focused on haired mice, because hairless mice exhibit abnormal follicle development, including cysts, and have a thickened epidermis (Sundberg and Sundberg, 1994Sundberg J.P. The hairless (hr) and rhino (hrrh) mutations, chromosome 14.in: Sundberg J.P. Handbook of Mouse Mutations with Skin and Hair Abnormalities. CRC Press, Boca Raton1994: 291-312Google Scholar). Moreover, the use of haired mice allows us to compare our results directly with previous studies in which Trp53 knockout mice in a similar genetic background were essential for UV-induced apoptosis (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 (1302) Google Scholar). Mice defective in nucleotide excision repair of a genomic region critical to signaling UVB apoptosis will accumulate unrepaired photoproducts in those regions. Such mice are predicted to generate sunburn cells at lower UVB doses than wild-type cells. In contrast, an apoptosis pathway initiated by membrane events would be unaffected by a DNA repair deficit. Csb−/− mutant mice carry homo zygously inactivated homologs of the human CSB gene, mimicking a protein-truncation mutation found in a human Cockayne syndrome patient. The mice are defective only in transcription-coupled excision repair, leading to a defect in repairing the transcribed strand (but not the coding strand) of the subset of genes being actively transcribed in any particular differentiated cell type (van der Horst et al., 1997van der Horst G.T. van Steeg H. Berg R.J. et al.Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition.Cell. 1997; 89: 425-435Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). After UVB irradiation, these mice accumulate cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6–4) photoproducts in the transcribed strand of active genes. Xpc−/− mice are inactivated for the murine homolog of the human XPC gene, mutations which underlie xeroderma pigmentosum complementation group C. These mice are defective in global excision repair (Cheo et al., 1997Cheo D.L. Ruven H.J. Meira L.B. et al.Characterization of defective nucleotide excision repair in XPC mutant mice.Mutat Res. 1997; 374: 1-9Crossref PubMed Scopus (100) Google Scholar) and thus accumulate UVB photoproducts in the majority of the DNA, including nontranscribed genes, the nontranscribed strand of active genes, and extragenic regions. Xpa−/− mice, inactivated for the murine homolog of the human XPA gene, are defective in one of the initial incision steps of DNA excision repair and are defective in both transcription-coupled and global excision repair pathways. After UVB irradiation, they accumulate photoproducts throughout the genome (de Vries et al., 1995de Vries A. van Oostrom C.T. Hofhuis F.M. et al.Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.Nature. 1995; 377: 169-173Crossref PubMed Scopus (353) Google Scholar). If the dose–response of apoptosis due to DNA or membrane signals is not linear, the relative importance of these signals will depend on the particular dose chosen. For this reason, photobiologists quantitate and compare the underlying molecular causes (x-axis of a dose–response) rather than the resulting biologic response (y-axis). Causes cannot be compared at a single dose. Dose–responses for modified (e.g., knockout) organisms and unmodified organisms are compared to determine the “dose modification factor”—the amount by which the dose must be increased in, for example, the wild-type mouse to give the same level of apoptosis seen in the knockout (Harm, 1976Harm H. Repair of UV-irradiated biological systems: photoreactivation.in: Wang S.Y. Photochemistry and Photobiology of Nucleic Acids. Vol. II. Academic Press, New York1976: 219-263Google Scholar). The “modifiable sector” (i.e., the fraction of the dose modifiable by the knockout) is then calculated as 1–1/(dose modification factor). For excision repair knockouts and apoptosis, this number (the “excision repairable sector”) represents the fraction of the UVB dose (and thus the fraction of DNA photoproducts) that leads to apoptosis and is modifiable by excision repair of the type affected by the knockout. Because the same biologic effect is compared (at different doses) in the knockout and wild type, the same amount of relevant DNA damage is present in each. Therefore, the excision repairable sector figure applies to both organisms. Comparing equal levels of apoptosis at different UVB doses, rather than asking whether repair-defective knockouts induce more apoptosis at the doses used for wild type, has a second advantage in addition to correcting for nonlinear dose–responses. The latter approach would bias the results toward a DNA-mediated apoptosis mechanism by effectively flooding the repair-defective keratinocyte with unrepaired DNA photoproducts. We therefore use the dose-modification factor to calculate the fraction of UVB-induced apoptosis that is due to excision-repairable DNA photoproducts in particular genomic regions. Mice inactivated in the Xpa, Xpc, or Csb genes were as described (de Vries et al., 1995de Vries A. van Oostrom C.T. Hofhuis F.M. et al.Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.Nature. 1995; 377: 169-173Crossref PubMed Scopus (353) Google Scholar;Cheo et al., 1997Cheo D.L. Ruven H.J. Meira L.B. et al.Characterization of defective nucleotide excision repair in XPC mutant mice.Mutat Res. 1997; 374: 1-9Crossref PubMed Scopus (100) Google Scholar;van der Horst et al., 1997van der Horst G.T. van Steeg H. Berg R.J. et al.Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition.Cell. 1997; 89: 425-435Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The genotype of progeny were determined in polymerase chain reaction amplifications specific for the inactivated alleles (neomycin cassette) and for the wild-type allele. Animals were used for experiments at age 6–9 wk, with individual animals excluded if there was extensive hair regrowth 40 h after shaving, indicative of being in the hair cycle. Groups of wild-type and homozygous-mutant littermates were shaved on the back under Ketamine–Rompun–Atropine anesthesia (AUV, Inc., Cuyk, the Netherlands) approximately 24 h before the experiment. The next morning, between 9 and 10 a.m., mice in both groups were irradiated from broadband Philips FS40 sunlamps (250–400 nm). The UVB output of the lamp was measured prior to each session by a UVX meter (UV Products, Upland, CA). During irradiation, the animals were allowed to move freely but were prevented from standing upright by a stainless steel wire mesh with 4 cm × 1 cm openings at a height of 3 cm. Twenty-four hours later, the animals were killed by cervical dislocation. Skin areas (1 cm × 2 cm) were taken from the mid-dorsal region and fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. The experimental protocol was reviewed and approved by the RIVM Animal Committee. Five micrometer thick sections were cut and every third section stained with hematoxylin–eosin. Sunburn cells were identified under a light microscope at × 150 magnification based on their characteristic morphology: pyknotic, darkly basophilic nuclei, eosinophilic cytoplasm, and intercellular gap (halo) formation (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 (1302) Google Scholar). In contrast to previous work (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 (1302) Google Scholar;Brash et al., 1996Brash D.E. Ziegler A. Jonason A. Simon J.A. Kunala S. Leffell D.J. Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.J Invest Dermatol Symp Proc. 1996; 1: 136-142PubMed Google Scholar), scoring in this study also included cells with eosinophilic cytoplasm but no nuclei, which time-course studies indicated are late-stage apoptotic cells (D.E.B, unpublished observations). At a fixed time-point, here 24 h, this inclusion has the effect of increasing the number of sunburn cells scored at the higher doses and results in a curvilinear dose–response. Sunburn cells were counted on six nonadjacent sections and the length of each epidermal section was measured. The apoptosis frequency was expressed as number of sunburn cells per centimeter of epidermis. The apoptosis frequency was averaged for each mouse and these averages were used in computing the mean and SEM. Twenty-four hours after UVB irradiation, skin was isolated from three to four mice per genotype and from their wild-type littermates. Skin samples were divided in half to create duplicate samples, embedded in paraffin, and immunostained with CM5 antibody for Tpr53 protein (Midgley et al., 1995Midgley C.A. Owens B. Briscoe C.V. Thomas D.B. Lane D.P. Hall P.A. Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo.J Cell Sci. 1995; 108: 1843-1848Crossref PubMed Google Scholar;van Kranen et al., 1995van Kranen H.J. de Gruijl F.R. de Vries A. et al.Frequent p53 alterations but low incidence of ras mutations in UV-B-induced skin tumors of hairless mice.Carcinogenesis. 1995; 16: 1141-1147Crossref PubMed Scopus (76) Google Scholar). Xpc−/− mice were examined in an experiment performed separately from Xpa−/− and Csb−/−, with each experiment containing internal wild-type controls. Cells were categorized according to the intensity of immunopositivity as minimal (light brown), medium (brown), and strong (dark brown). Two 2.5 mm sections were scored for each mouse, with two to four mice per point, and the level of Trp53 induction was expressed as the number of strongly immunopositive basal cells per centimeter of epidermis. The Trp53 induction frequency was averaged for each mouse and these averages were used in computing the mean and SEM. Paraffin-embedded sections adjacent to those analyzed for Trp53 were immunostained for Mdm-2 using similar methods, including antigen retrieval. SMP14 mouse monoclonal antibody for Mdm-2 protein (Santa Cruz Biotech, Santa Cruz, CA) was diluted 1:50 in MoM diluent (Vector, Burlingame, CA), which allows mouse monoclonal antibodies to be used on mouse tissues. Immuno positivity was scored in four to six sections per mouse, with two to four mice per point, and the level of Mdm-2 induction wa
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