UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome
2000; Springer Nature; Volume: 19; Issue: 5 Linguagem: Inglês
10.1093/emboj/19.5.1157
ISSN1460-2075
AutoresMark Berneburg, Jillian E. Lowe, Tiziana Nardò, Sofia J. Araújo, Maria Fousteri, Michael H.L. Green, Jean Krutmann, Richard D. Wood, Miria Stefanini, Alan R. Lehmann,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle1 March 2000free access UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome Mark Berneburg Mark Berneburg MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Clinical and Experimental Photodermatology, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany Search for more papers by this author Jillian E. Lowe Jillian E. Lowe School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Brighton, BN2 4GJ UK Search for more papers by this author Tiziana Nardo Tiziana Nardo Istituto di Genetica Biochimica ed Evolutionistica CNR, Via Abbiategrasso 207, Pavia, Italy Search for more papers by this author Sofia Araújo Sofia Araújo Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Maria I. Fousteri Maria I. Fousteri MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Search for more papers by this author Michael H.L. Green Michael H.L. Green School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Brighton, BN2 4GJ UK Search for more papers by this author Jean Krutmann Jean Krutmann Clinical and Experimental Photodermatology, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany Search for more papers by this author Richard D. Wood Richard D. Wood Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Miria Stefanini Miria Stefanini Istituto di Genetica Biochimica ed Evolutionistica CNR, Via Abbiategrasso 207, Pavia, Italy Search for more papers by this author Alan R. Lehmann Corresponding Author Alan R. Lehmann MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Search for more papers by this author Mark Berneburg Mark Berneburg MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Clinical and Experimental Photodermatology, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany Search for more papers by this author Jillian E. Lowe Jillian E. Lowe School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Brighton, BN2 4GJ UK Search for more papers by this author Tiziana Nardo Tiziana Nardo Istituto di Genetica Biochimica ed Evolutionistica CNR, Via Abbiategrasso 207, Pavia, Italy Search for more papers by this author Sofia Araújo Sofia Araújo Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Maria I. Fousteri Maria I. Fousteri MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Search for more papers by this author Michael H.L. Green Michael H.L. Green School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Brighton, BN2 4GJ UK Search for more papers by this author Jean Krutmann Jean Krutmann Clinical and Experimental Photodermatology, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany Search for more papers by this author Richard D. Wood Richard D. Wood Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Miria Stefanini Miria Stefanini Istituto di Genetica Biochimica ed Evolutionistica CNR, Via Abbiategrasso 207, Pavia, Italy Search for more papers by this author Alan R. Lehmann Corresponding Author Alan R. Lehmann MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK Search for more papers by this author Author Information Mark Berneburg1,2, Jillian E. Lowe3, Tiziana Nardo4, Sofia Araújo5, Maria I. Fousteri1, Michael H.L. Green3, Jean Krutmann2, Richard D. Wood5, Miria Stefanini4 and Alan R. Lehmann 1 1MRC Cell Mutation Unit, Sussex University, Falmer, Brighton, BN1 9RR UK 2Clinical and Experimental Photodermatology, Heinrich-Heine-Universität, Moorenstrasse 5, 40225 Düsseldorf, Germany 3School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Brighton, BN2 4GJ UK 4Istituto di Genetica Biochimica ed Evolutionistica CNR, Via Abbiategrasso 207, Pavia, Italy 5Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:1157-1166https://doi.org/10.1093/emboj/19.5.1157 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells. Introduction The process of nucleotide excision repair (NER) removes many types of DNA damage from the genome. It is a highly conserved mechanism (Taylor and Lehmann, 1998) that involves >30 proteins acting in a tightly regulated manner. Damage in DNA is recognized either by the XPC–HR23B protein complex in so-called global genome repair, or by the transcribing RNA polymerase II in transcription-coupled repair, together with the XPA protein. After these recognition steps the two helicases XPB and XPD open up the DNA around the lesion allowing incisions to be introduced on both sides of the damage by the endonucleases XPF-ERCC1 and XPG. This is followed by removal of the damage-containing oligonucleotide and filling of the resulting gap by DNA polymerase δ or ϵ and a DNA ligase (for recent reviews, see Wood 1996; de Laat et al., 1999). To ensure the removal of damaged sites without disruption to the rest of the genome it is of paramount importance that the correct order of events is maintained. Defects in NER can lead to three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). These diseases show distinct clinical phenotypes. The hallmarks of XP are sun-sensitivity, pigmentation changes and an ∼2000-fold increased incidence of skin cancer (Kraemer et al., 1987). TTD is characterized by sulfur-deficient brittle hair together with growth and mental retardation (Itin and Pittelkow, 1990). CS also exhibits growth and mental retardation and a characteristic bird-like face as well as neurodegeneration and retinal degeneration (Nance and Berry, 1992). Neither TTD nor CS is cancer-prone. Seven genes, XPA–G are associated with NER defects in XP. Whereas the features of TTD have not been found in combination with XP, mutations in three of the XP genes, XPB, XPD and XPG can give rise to combined symptoms of XP and CS (XP/CS). The XPB and XPD proteins are subunits of TFIIH, a transcription factor with dual functions in both basal transcription and NER (Hoeijmakers et al., 1996). The two known XP–D/CS patients, XP8BR (Broughton et al., 1995) and XP-CS-2 (Lafforet and Dupuy, 1978), are the subject of the current study. These patients both had extremely severe clinical features. Both are compound heterozygotes. The mutations in the XPD gene in XP8BR are G675R in one allele and deletion of nucleotide 2083 in the second allele, the latter causing a frameshift at codon 669 and a truncated 708 amino acid protein, which is likely to be completely non-functional (Broughton et al., 1995). In XP-CS-2, only one allele is expressed and contains the mutation G602D (Takayama et al., 1995). Cellular studies showed that despite a level of unscheduled DNA synthesis (UDS) of ∼30% of normal, similar to or greater than that in other XP-D cells, both XP-D/CS cell strains were considerably more sensitive to killing by UV irradiation than XP-D cells. In fact they are the most sensitive fibroblasts that we have analysed (Broughton et al., 1995). We have constructed a series of mutations analogous to those found in various cell strains from the XP-D complementation group in the homologous rad15 gene of Schizosaccharomyces pombe. The mutation corresponding to that found in the XP-D/CS cell strain XP8BR was much the most sensitive of all the mutations tested (Berneburg et al., 2000). The repair of different photoproducts was found to be indistinguishable in XP-D and XP-D/CS cells (van Hoffen et al., 1999). Elimination of cyclobutane pyrimidine dimers (CPD) was undetectable and removal of 6–4 photoproducts was inefficient in both. Nevertheless, the XP-D/CS cells failed to recover normal rates of RNA synthesis even after very low doses of UVC irradiation (2 J/m2), whereas RNA synthesis did recover after this dose in XP-D cells (van Hoffen et al., 1999). To gain further insight into the underlying defect in XP/CS cells we have investigated the proficiency of XP-D/CS cells to introduce incisions into their DNA during NER. Our results show that XP-D/CS cells are able to detect DNA damage and appear to initiate incisions; however, these incisions are aberrant and distant from the sites of the damage. Results Extreme sensitivity of XP-D/CS cells to inhibition of gene expression by UV Details of the cell lines used in this paper are shown in Table I. Our previous work showed that XP-D/CS fibroblasts were extremely sensitive to killing by UV irradiation (Broughton et al., 1995). This was correlated with a reduced ability to restore overall RNA-synthesis levels to normal even after very low UV doses (van Hoffen et al., 1999). We have now measured the response to UV of a specific gene, which codes for intercellular adhesion molecule 1 (ICAM-1), whose expression in human fibroblasts can be induced by treatment with γ-interferon. We have shown previously that this upregulation is suppressed by irradiation with UVB (Krutmann et al., 1994). The reduction in expression of ICAM-1 by UVB treatment of XP-D cells is significantly greater than in normal cells (Ahrens et al., 1997). Based on these observations, we investigated post-UVB ICAM-1 expression in the two known XP-D/CS cell lines (Figure 1). Strikingly, both XP-D/CS cell lines revealed an extreme inhibition of the transcription of this immunologically relevant molecule. ICAM-1 expression was inhibited only above doses of 100 J/m2 in normal cells and was reduced to 50% in the XP-D cell line at 100 J/m2. In contrast, doses of UVB >50 J/m2 resulted in >80% inhibition of ICAM-1 transcription in both XP–D/CS lines. This inhibition is greater than that observed in any other XP cell line that we have studied. This end-point therefore correlates with the extreme sensitivity to killing by UV irradiation (Broughton et al., 1995), and the failure of RNA synthesis to recover even after very low doses of UV irradiation (van Hoffen et al., 1999). Figure 1.Inhibition of ICAM-1 expression by UVB. Normal, XP-D (XP16BR) or XP-D/CS fibroblasts were exposed to different doses of UVB irradiation and then treated with interferon-γ. Four hours later the levels of ICAM-1 mRNA were measured by semi-quantitative RT–PCR. Error bars represent the SEMs of three experiments. Download figure Download PowerPoint Table 1. Cell lines used in this study Cell type Phenotype Cell lines Mutation in XPDa Primary fibroblasts normal 1BR, 48BR, C3PV XP-D XP1BR R683W/R683W XP16 BR R683W/R616P XP3NE R683W/R683W XP-D/CS XP8BR G675R/Fs669 XP-CS-2 G602D XP-G/CS XP20BE Transformed fibroblasts normal 1BR pre-crisisb XP-D/CS XP8BR pre-crisisb G675R/Fs669 Lymphoblastoid XP-A GM2345 (XP2OS) XP-B GM2252 (XP11BE) XP-D XP7BE R683W/Del exon 3 XP-D/CS XP8BR G675R/Fs669 XP-G XPG83 (XP125LO) a The amino acid changes resulting from mutations in the two alleles of the XPD gene are shown. Fs, frameshift; Del, deletion. Only one allele is expressed in XP-CS-2. Data from Taylor et al. (1997) (XP1BR, XP16BR, XP3NE), Broughton et al. (1995) (XP8BR), Takayama et al. (1995) (XP-CS-2) and our unpublished results (XP7BE). b Pre-crisis cells were derived from primary fibroblasts following transfection with pSV3neo (Mayne et al., 1986). The SV40 T-antigen expressed from this plasmid transforms the cells morphologically and extends their life-span, but the lines are not immortal. XP-D/CS cells accumulate nicks after UV irradiation Previous measurements in XP-D/CS cells showed that, as in many other XP-D cell strains, the level of UV-induced UDS was ∼30% of that in normal cells, when measured either by autoradiography or liquid scintillation counting (Broughton et al., 1995), and that photoproduct removal was similarly deficient in both XP-D and XP-D/CS cells (van Hoffen et al., 1999). These studies did not therefore reveal any obvious differences in NER that could account for the much greater sensitivity of the XP-D/CS cells. In the following experiments we investigated another aspect of NER, the ability of XP-D/CS cells to incise DNA following UV exposure. To measure incision in XP-D/CS cells we employed the single cell gel electrophoresis (‘comet’) assay. The incision steps of NER are normally rate limiting and incised intermediates are not readily observable following UV irradiation. If, however, hydroxyurea (HU) and 1-β-D-arabinofuranosylcytosine (ara-C) are included in the post-irradiation medium, the subsequent repair steps of NER are inhibited, and incised intermediates accumulate (Squires et al., 1982) and can be measured using this assay (Green et al., 1992). We measured incision in normal, XP-D and XP-D/CS cells irradiated with either UVB (Figure 2A) or UVC (Figure 2B). As expected, the normal cell line 1BR generated increasing comet lengths, representing the accumulation of incised intermediates, with increasing UVB or UVC doses. As shown previously (Berneburg et al., 2000), in XP-D cells (XP1BR, XP16BR) comet formation was barely detectable with increasing UV dose, the incision efficiency being at least 10-fold lower than in normal cells (Figure 2A). These data are consistent with the very low rate of photoproduct removal in these cells. In striking contrast, however, both XP-D/CS cell lines (XP8BR and XP-CS-2) appeared to generate incision intermediates efficiently with a dose–response for both UVB and UVC only slightly lower than that of normal cells. Figure 2.Accumulation of incised intermediates after UV irradiation of normal and XP-D/CS cells. Fibroblasts were irradiated with different doses of UVB (A) or UVC (B) and incubated for 1 h in the presence of HU and ara-C. Incised intermediates were measured using the comet assay. The average increase in length of the comet tails is plotted as a function of UV dose. Download figure Download PowerPoint To see if this phenomenon was observed in XP/CS cells from other complementation groups, we also examined the response of the XP-G/CS cell line XP20BE (Moriwaki et al., 1996). Results in Figure 2A show that incision intermediates were undetectable in this cell line. The generation of incision intermediates appeared therefore to be specific for XP-D/CS cells, and was most unexpected in view of the striking sensitivity of these cells to UV irradiation. We therefore considered the possibility that the XP-D/CS cells might be able to incise the damaged DNA, but not complete the subsequent steps in the NER process. If this were the case, then incision intermediates might accumulate even in the absence of the repair synthesis inhibitors, ara-C and HU. As anticipated, in normal 1BR cells, incision intermediates accumulated only in the presence of the inhibitors (●) but not in their absence (○) (Figure 3A). In the absence of ara-C and HU, the removal and repair synthesis steps occur too quickly in normal cells for incision intermediates to accumulate. As anticipated, no intermediates were detected in the XP-D cell line XP16BR, either in the presence (▾) or absence (▿) of the inhibitors. Strikingly, however, the XP-D/CS cell line XP8BR accumulated incisions even in the absence of the inhibitors (□). A time-course showed that incisions could be observed as early as 30 min after irradiation and reached a maximum after 3–4 h (Figure 3B). No incisions were detected in normal cells even after 12 h incubation (data not shown). We noted that the accumulation of breaks in XP8BR fibroblasts in the presence of the inhibitors was independent of their proliferation status, whereas incisions in the absence of the inhibitors could only be detected in non-proliferating cells. Figure 3.Accumulation of incised intermediates in the absence of repair synthesis inhibitors after UV irradiation. Non-proliferating cells were (A) irradiated with different doses of UVB and incubated for 1 h, or (B) irradiated with 30 J/m2 UVB and incubated for different times prior to analysis of the DNA using the comet assay. Download figure Download PowerPoint These data, showing the accumulation of DNA breaks in UV-irradiated XP8BR cells, led us to test the idea that XP-D/CS cells are able to initiate the NER process by incising the DNA, but might be unable to complete the process. XP-D/CS cell extracts are completely defective in in vitro repair activities Various in vitro assays have been developed to measure the different steps in the NER process using cell-free extracts. In particular these assays are able to detect uncoupled incisions on a damaged template (Moggs et al., 1996; Evans et al., 1997). Incision on either the 3′ or 5′ side of the damage without incision on the other side has been reported for several cell lines (Evans et al., 1997). The high rates of incision without the actual removal of existing damage observed in XP-D/CS cells might be explained by uncoupled 3′ or 5′ incision. To test this idea, we carried out in vitro experiments in a reconstituted NER repair system. Whole-cell extracts derived from XP8BR lymphoblastoid cell lines were investigated for their ability to carry out dual or uncoupled incision as well as repair synthesis using a plasmid substrate containing a single cisplatin adduct (Moggs et al., 1996; Evans et al., 1997). Dual incisions result in the release of an ∼30 nucleotide fragment, which can be radioactively labelled and analysed on a denaturing polyacrylamide gel as described previously (Moggs et al., 1996). Figure 4A shows that HeLa cell extracts have efficient excision activity, whereas extracts from XP7BE (XP-D) or XP8BR (XP-D/CS) had no detectable activity. In both cases, activity could be restored by addition to the extracts of either purified TFIIH (which contains the XPD protein subunit) or extract of NER-defective 43-3B cells (defective in the NER protein ERCC1). Figure 4.Absence of NER in cell-free extracts of XP-D/CS cells. (A) Cell-free extracts of HeLa, XP7BE or XP8BR cells were incubated with plasmid DNA containing a single cisplatin lesion. Excised fragments were directly end-labelled with [32P]dCTP and separated on polyacrylamide gels. Different amounts of cell extract protein were used as indicated, and in some samples they were supplemented either with TFIIH or with 30 μg of protein extract from the NER-deficient Chinese hamster ovary cell line 43-3B. (B) To analyse incision activity of cell extracts, the amounts of extract protein indicated were incubated with substrate and a DNA fragment containing the lesion was isolated by restriction digestion and 3′-end-labelled. M, size marker. (C) Repair synthesis. Substrate was incubated with extracts and triphosphates including radioactive dCTP to label repair patches. The DNA was then digested with BstNI and the products separated on polyacrylamide gels. Download figure Download PowerPoint The experiment shown in Figure 4B was designed to detect single or dual incisions in the DNA. The plasmid containing the single cisplatin lesion was cleaved with AvaII and end-labelled 140 nucleotides from the 3′ side of the site of the damage (Constantinou et al., 1999). It was then incubated with cell extracts and purified proteins as indicated. Incisions 3′ to the lesion will generate labelled fragments of ∼130 bases, whereas uncoupled incisions 5′ to the lesion will generate a 155 base fragment. HeLa cells efficiently incised the DNA 3′ to the adduct (Figure 4B, lanes 1 and 2), whereas extracts from XP-A (lane 3), XP-G (lane 11), XP-D (lanes 8 and 9) and XP-D/CS cells (lanes 5 and 6) were almost completely defective in incision ability. Addition of XPA, XPG or TFIIH proteins to the appropriate extracts restored 3′ incision activity (Figure 4B, lanes 4, 12, 10 and 7). Finally, in the experiments of Figure 4C, the ability of the extracts to carry out repair synthesis following excision of the cisplatin adduct was measured. Following incubation with the extracts in the presence of [32P]dNTP to label the repair patches, the DNA was extracted and digested with BstNI. Most of the repaired DNA is located in a 33 bp fragment, which contains the site of the lesion, and to a lesser extent in surrounding fragments of 38, 57, 68 and 127 bp. Repair activity was seen in HeLa cells in Figure 4C, lane 1. In contrast, activity was very low or absent in XP-A, XP-B, XP-D and XP8BR extracts (Figure 4C, lanes 2, 4, 6, 7, 9 and 10), but could be restored by addition of complementing activity (lanes 3, 5, 8 and 11). These results obtained with cell-free extracts therefore contrasted with our in vivo analysis and provided no evidence for uncoupled NER incision activity. XP8BR cells accumulate breaks in genomic DNA in the presence of ectopic damage The in vitro studies did not provide any evidence for the induction of breaks in the proximity of the damage. We therefore considered the possibility that the breaks generated in the cellular DNA of XP8BR following UV irradiation (Figure 3) were not targeted to the sites of the damage. To test this hypothesis, we introduced irradiated plasmid DNA into the cells and used the comet assay to look for the ability of this damaged DNA to induce breaks in undamaged genomic DNA. We used two independent approaches, transfection and microinjection, to introduce the damaged DNA into the cells. In each case we introduced unirradiated or irradiated plasmid into both normal and XP8BR cells. Transfection. Cells were cotransfected with the plasmid pcDNA3.1, either unirradiated or irradiated with a UVB dose of 15 000 J/m2 (photoproduct yield approximately equivalent to that produced by 500 J/m2 UVC) together with a second (unirradiated) plasmid, pHook-1. The latter expresses the gene phOx sFv, which encodes a single-chain antibody directed against phOx that is displayed on the cell surface. Cells expressing this antibody can be rapidly separated by binding to phOx hapten linked to magnetic beads. The molar ratio of pcDNA3.1 to pHook-1 was 4:1, and it was anticipated that many of the cells that had taken up the pHook-1 plasmid would also have incorporated the pcDNA3.1. With this procedure we were able to separate the transfected cells from the 99% of the population that remained untransfected. The transfected cells were then analysed using the comet assay. After cotransfection, the cells were incubated for 24 h to allow for the expression of the phOx antibody. The expressing cells were separated, embedded in agarose, and comets analysed as described above. Cells were considered to have comets when the comet length exceeded 20 μm. Less than 10% of normal cells had comets when transfected with either unirradiated or irradiated plasmid (Figure 5A, first column). Similarly, when XP8BR cells were transfected with unirradiated plasmid, 20 μm. (B) Microinjection. Normal, XP-D (XP3NE) or XP8BR cells in a marked area on a dish were microinjected with UV-irradiated pcDNA3.1 and incubated for 6 h, before harvesting and analysis using the comet assay. Details as in (A). (C) The data are summarized as a percentage of cells with comet tails >20 μm under different conditions. Download figure Download PowerPoint Microinjection. In order to provide further independent evidence for this conclusion, we microinjected irradiated and unirradiated plasmid molecules into the nuclei of normal, XP3NE (XP–D) and XP8BR fibroblasts. This procedure also had the advantage that we were able to use primary fibroblasts, whereas for the transfection experiments we were obliged to use transformed cells to obtain a workable transfection frequency. We injected ∼200–400 cells within a small area of the Petri dish containing a coverslip. After injection, the cells were incubated for 6 h, and the population of injected cells was harvested and subsequently analysed by the comet assay. The results were very similar to those generated with the transfection procedure. After a 6 h incubation following injection, very few or no comets were seen in normal (Figure 5B, left) or XP–D cells (Figure 5B, middle) or in XP8BR cells injected with unirradiated plasmid. In striking contrast, microinjection of irradiated plasmid into XP8BR cells led to comet formation in ∼45% of the injected cells (Figure 5B, right). The length of the comet tails ranged from 20 to 60 μm. Results of the microinjection experiments are summarized in Figure 5C (right). These results indicate that in XP-D/CS cells, sensing of the damage is uncoupled from the incisions measured by the comet assay, which take place away from the site of the DNA damage. For this to take place the damage must transmit a signal to the machinery that generates the nick in the genomic DNA. We considered the possibility that this signal transduction might be mediated by p53. The transfection experiments described above, however, were carried out with fibroblasts transformed with SV40 T-antigen. Such cell lines are in general functionally p53−, because the T-antigen sequesters the p53 protein. In order to confirm that this was indeed the case, we measured levels of both p53 and its downstream effector p21 in irradiated and unirradiated cells by immunoblotting. When primary fibroblasts were used, both p53 and p21 levels were increased following irradiation. In the T-antigen-transformed cells, the constitutive levels of p53 were at least 10–fold higher than those in untransformed cells, but there was little increase on irradiation. In contrast, p21 levels were low in the transformed cells and showed little change on irradiation (results not shown). From these data we infer that the signal transduction is unlikely to be mediated by p53, since it occurs in cells in which the p53 appears to be functionally inactive. Incision observed by comet assay is independent from apoptosis We considered the possibility that the DNA damage-induced incisions that we observed in XP8BR cells could be carried out by apoptotic pathways. To investigate this hypothesis, we measured apoptosis in normal, XPD and XP-D/CS primary fibroblasts as a function of time after irradiation. Apoptosis was measured by an end-labelling method and by cleavage of the PARP protein by caspase 3. Using the former procedure, apoptotic signals were detected 48 and 72 h after irradiation with 100 J/m2 in 15 and 35% of the XP-D cells (XP16BR), respectively, and in 45 and 66% of the XP-D/CS cells (Figure 6A). No apoptosis was observed in either of the normal cell strains investigated. Similar results were obtained with the PARP cleavage assay (Figure 6B). No apoptotic signals were observed at any of the earlier times, at which comet formation could be readily detected. These data show that although XP-D and XP-D/CS cells do undergo apoptosis after relatively high doses of UV irradiation, the incisions observed using the comet assay are unlikely to be generated by apoptotic pathways. Figure 6.Apoptosis in normal and XP8BR cells after UVB irradiation. Cells were irradiated with 100 J/m2 UVB and at various times after irradiation ap
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