Defective Global Genome Repair in XPC Mice Is Associated with Skin Cancer Susceptibility But Not with Sensitivity to UVB Induced Erythema and Edema Rob J.W. Berg, Hendrik J.T. Ruven,* Arthur T. Sands,† Frank R. de Gruijl, and Leon H.F. Mullenders* Department of Dermatology, University Hospital Utrecht, Utrecht, The Netherlands; *Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Leiden, The Netherlands; †Lexicon Genetics, The Woodlands, Texas, U.S.A. It is generally presumed that xeroderma pigmentosum (XP) patients are extremely sensitive to developing UV erythema, and that they have a more than 1000-fold increased skin cancer risk. Recently established mouse models for XP can be employed to investigate the mechanism of these increased susceptibilities. In line with human data, both XPA and XPC knockout mice have been shown to have an increased susceptibility to UVB induced squamous cell carcinomas. In XPA knockouts, nucleotide excision repair of UV induced DNA photolesions is completely defective (i.e., both global genome repair and transcription coupled repair are defective). We determined the strand specific removal of cyclobutane pyrimidine dimers and pyrimidine [6–4] pyrimidone photoproducts from the p53 gene in cells from XPC knockout mice and wild-type littermates. Analogous to human XPC cells, embryonic fibroblasts from XPC knockout mice are only capable of performing transcription coupled repair of DNA photolesions. We show that these XPC knockout mice, in striking contrast to XPA knockout mice, do not have a lower minimal erythema/edema dose than their wild-type littermates. Hence, defective global genome repair appears to lead to skin cancer susceptibility, but does not influence the sensitivity to acute effects of UVB radiation, such as erythema and edema. The latter phenomena thus relate to the capacity to perform transcription coupled repair, which suggests that blockage of RNA synthesis is a key event in the development of UV erythema and edema. Key words: Cockayne syndrome/cyclobutane pyrimidine dimers/ pyrimidine [6–4] pyrimidone photoproducts/transcription coupled repair. J Invest Dermatol 110:405–409, 1998 S stimulate the synthesis of prostaglandins that activate the inflammatory mechanism (Naylor, 1997). The pathway from DNA damage to sunburn may also include different mediators such as histamine (Hruza and Pentland, 1993), interleukin-1 (Luger and Schwarz, 1995), interleukin-8, and tumor necrosis factor α (Strickland et al, 1997). The vascular response to UVB radiation reaches a maximum between 8 and 24 h after exposure of human skin (Van der Leun, 1965; Andersen et al, 1991), and between 24 and 48 h after exposure of mouse skin (Trevithick et al, 1992). The difference in the time course of the response in human and murine skin is probably due to dominance of either erythema or edema (Cole et al, 1983). Owing to anatomical differences between man and mouse in the cutaneous vascular density, the threshold response to UVB radiation of human skin is erythema, whereas the threshold response to UVB radiation of mouse skin is edema. The action spectrum for mouse edema at 48 h is very similar to the action spectrum for human erythema at 24 h, which strongly suggests that the initial steps are the same (Cole et al, 1983). The predominant types of UVB induced DNA damage are cyclobutane pyrimidine dimers (CPD) and pyrimidine [6–4] pyrimidone photoproducts (6–4PP), which are removed by the nucleotide excision repair (NER) system. This complex system (Ma et al, 1995; Wood, 1996) involves the concerted action of multiple proteins to eliminate lesions from the genome (‘‘global genome repair’’). A specialized subpathway of NER selectively removes lesions from the transcribed strand of active genes (Bohr et al, 1985; Mellon et al, 1987), and is called transcription coupled repair (TCR). The function of TCR is to alleviate blockage of RNA synthesis caused by the presence of DNA damage, as only cell lines expressing TCR are capable of restoring UV inhibited transcription (Ma et al, 1995). unburn is a UV induced inflammatory reaction that is characterized by cutaneous vasodilatation (erythema), and that can be followed by an increase in vascular permeability with exudation of fluid (edema) in the affected skin. The pathway leading to sunburn is most commonly thought to be activated by UV induced DNA damage. The evidence that supports a linkage between DNA damage and sunburn is that genetic diseases with defects in the removal of UV photoproducts, such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), are accompanied by increased sensitivity to sunburn (Cleaver and Kraemer, 1989; Bootsma, 1993). In addition, it has been shown in the opossum Monodelphis domestica that immediate removal of UV photoproducts by photoreactivation leads to a suppression of the erythemal response (Ley, 1985). Furthermore, the wavelengths that are the most effective in producing UV photolesions (namely, the UVB region) are also the most effective in producing erythema (Hacham et al, 1991) or edema (Johnson, 1978). The mechanism that connects DNA damage to the inflammatory reaction is unclear. UVB induced DNA damage may Manuscript received September 19, 1997; revised December 23, 1997; accepted for publication January 14, 1998. Reprint requests to: Dr. Rob J.W. Berg, Department of Dermatology, University Hospital Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands. Abbreviations: CPD, cyclobutane pyrimidine dimer(s); CS, Cockayne syndrome; NER, nucleotide excision repair; 6–4PP, pyrimidine [6–4] pyrimidone photoproduct(s); TCR, transcription coupled repair; XP, xeroderma pigmentosum. · 0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc. 405 406 BERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Mutations in genes that participate in NER cause XP, which is clinically characterized by photosensitivity and a high incidence of solar radiation induced skin tumors. The extremely high susceptibility of XP patients to sunburn suggests that UV induced erythema and edema are causally related to persistence of UV photoproducts in the DNA as a consequence of defective NER. Experimental studies on this relationship are difficult to carry out as XP is a rare disease, and it consists of at least seven NER defective subgroups (complementation groups XPA through XPG), each caused by mutations in a different NER gene, and a NER proficient variant group (Bootsma, 1993; Ma et al, 1995). Within the same complementation group different XP patients can vary quantitatively in their responses, as different mutations in the same gene can lead to different levels of residual enzyme activity. In addition, experimental in vivo studies on erythema in XP patients can be considered questionable for obvious ethical reasons. Recently, mouse models for XP have been established that are very well suited to studying the relationship between deficiencies in NER and UV induced erythema/edema and the ultimate tumors. Knockout mice for the XPA gene (De Vries et al, 1995; Nakane et al, 1995) as well as for the XPC gene (Sands et al, 1995; Cheo et al, 1997) have been described. Dysfunctional XPA genes lead to a complete NER deficiency (i.e., both in global genome repair and in TCR), and dysfunctional XPC genes lead to a deficiency in global genome repair only (Venema et al, 1990; Evans et al, 1993). Both XPA and XPC knockout mice have been shown to have an increased susceptibility to UVB induced squamous cell carcinomas of the skin (De Vries et al, 1995; Nakane et al, 1995; Sands et al, 1995). Here we first characterize the NER deficiency in the XPC mice under study, and then show that these XPC mice are only capable of removing UV photoproducts from the transcribed strand of the transcriptionally active p53 gene, i.e., to perform TCR. For an accurate determination of the sensitivity of mouse skin to UV induced erythema or edema it is necessary to cross the knockout genotype into a strain of hairless mice, as it is very difficult to distinguish a minimal effect on the skin of haired mice that have been shaven. Determination of the MED in hairless XPA mice has revealed that these mice have an MED that is about 10 times lower than that of their wild-type littermates (Berg et al, 1997). Here we report that XPC mice, in striking contrast to XPA mice, do not have a lower MED than their wild-type littermates. These results indicate that persistence of photoproducts in transcriptionally active DNA triggers the pathway that leads to erythema and edema in the affected skin, whereas persistence of photoproducts in nontranscribed DNA does not activate this pathway, but clearly contributes to skin carcinogenesis. Determination of acute UVB sensitivity For all three genotypes (XPC–/–, XPC1/–, and XPC1/1), two male and two female hairless mice were exposed on the dorsal skin to UVB radiation from a filtered (using a Schott-WG305 filter) Hanovia Kromayer lamp (dose rate 5 135 J per m2 per s; 280–400 nm). This is a hand-held lamp that allows short exposures to limited skin areas by placing the circular port (about 2.5 cm2) of the source in close contact with the skin (Sontag et al, 1994). For all genotypes male and female mice were exposed to 4, 8, 16, and 24 s of UV radiation from the Kromayer lamp. All exposures were given in duplicate on separate mice. The mice were checked separately by three experienced, but noninformed, observers for erythema and/or edema at 6, 20, 28, 46, 55, 69, and 127 h after the exposures. MATERIALS AND METHODS RESULTS In the mice used in this experiment, the XPC gene was inactivated by replacing a fragment of about 4.5 kilobases (spanning exons 3–6) with a replacement vector followed by homologous recombination in embryonic stem cells (Sands et al, 1995). This replacement eliminates a coding portion of the mouse gene corresponding to bases 159 to 1546 of the human XPC cDNA (Legerski and Peterson, 1992). Embryonic fibroblasts from the XPC knockout mice appeared to have a decreased survival upon UV irradiation, and the XPC knockout mice appeared to have an increased susceptibility to UV induced squamous cell carcinomas of the skin (Sands et al, 1995). XPC mice only have TCR of CPD and 6–4PP Lack of XPA leads to a complete NER deficiency, i.e., both in global genome repair and in TCR (Evans et al, 1993; Ma et al, 1995). To establish the NER phenotype of the XPC mice used in this experiment, we have measured strand specific removal of CPD and 6–4PP in the p53 gene. As shown in Fig 1(A), in both XPC and wild-type mice CPD are efficiently removed from the transcribed strand (µ90% removal in 24 h), but not from the nontranscribed strand (,5% removal in 24 h). Figure 1(B) shows that 6–4PP are efficiently removed from the transcribed strand in both XPC and wild-type cells (.70% removal in 24 h), and also from the nontranscribed strand in wild-type cells (µ60% removal in 24 h); however, hardly any removal of 6–4PP from the nontranscribed strand in XPC cells was found (µ10% removal in 24 h). This NER phenotype is in agreement with that determined for another XPC knockout mouse, in which XPC has been inactivated by a smaller replacement vector (Cheo et al, 1997). Nucleotide excision repair in XPC mice Mouse embryonic fibroblasts were isolated from day 13.5 embryos of XPC–/– mice and XPC1/1 littermates by standard procedures. For analysis of repair of UV induced photolesions, mouse embryonic fibroblast cells were grown to confluence, rinsed with phosphate buffered saline buffer, and irradiated with 10 J per m2 or 30 J per m2 UVC radiation (predominantly 254 nm) for determination of CPD and 6–4PP, respectively. Subsequently, cells were lyzed or incubated in conditioned medium for various periods of time. Strand specific analysis of CPD and 6–4PP in the p53 gene was performed as described previously (Ruven et al, 1994a; Van Hoffen et al, 1995). Briefly, purified genomic DNA was restricted with EcoR1 and incubated with the CPD specific enzyme T4 endonuclease V. For determination of 6–4PP, the restricted DNA was digested with Escherichia coli Uvr ABC excinuclease, and prior to digestion the CPD were removed from the DNA by in vitro photoreactivation. Therefore, the DNA was mixed with photolyase derived from Anacystis nidulans, and exposed to 425 nm light for 1 h at room temperature. The digested DNA samples were then subjected Figure 1. XPC mice are proficient in TCR of (A) CPD and (B) 6–4PP. UV induced CPD and 6–4PP were measured as described in Materials and Methods in the transcribed and the nontranscribed strand of the p53 gene in primary mouse embryonic fibroblasts from XPC–/– mice and their wild-type littermates. ., XPC–/–, nontranscribed strand; m, XPC1/1, nontranscribed strand; j, XPC –/–, transcribed strand; d, XPC1/1, transcribed strand. to alkaline gel electrophoresis, transferred to membranes, and hybridized with strand specific probes recognizing a 16 kb fragment of the p53 gene. Breeding of hairless XPC mice In order to compare the MED for the XPC knockout mice with that of their wild-type littermates, the XPC deficient phenotype was crossed into a hairless mouse strain. Therefore, XPC knockout mice in a 129/ola-C57Bl/6 background were crossed with albino hairless mice (HRA/SKH). The offspring was backcrossed with HRA/SKH, and this offspring was selected for being hairless, for being albino (we selected for animals with red eyes), and for being heterozygous for the XPC gene. Hairless albino XPC1/– animals were then intercrossed to generate XPC–/– animals with heterozygous and wild-type littermates as controls. XPC–/–, XPC1/–, and XPC1/1 animals were identified by Southern blot analysis of genomic DNA isolated from the tail tips, as described earlier (Sands et al, 1995). MED not reduced in XPC mice, but strongly reduced in XPA mice All hairless mice of the three different genotypes (XPC–/–, XPC1/–, and XPC1/1) showed a clear UV inflammatory response at 46 h after 16 or 24 s of exposure to the Kromayer lamp. In general, in the male animals the predominant effect was swelling of the exposed skin, whereas in the female animals besides swelling a slight redness was also seen. No macroscopically visible effect was detected on any VOL. 110, NO. 4 APRIL 1998 MED NOT REDUCED IN XPC MICE Table I. XPC mice do not have a higher sensitivity to UVB induced erythema/edema than their heterozygote and wildtype littermates XPC–/– 4 sa 8s 16 s 24 s XPC1/– 4s 8s 16 s 24 s XPC1/1 4s 8s 16 s 24 s 6 hb 20 h 28 h 46 h 55 h 69 h 127 h –c – – – – – – – – – – – – – 1 1 – – 1 1 – – 1 1 – – 1 1 – – – – – – – – – – – 6 – – 1 1 – – 1 1 – – 1 1 – – 6 1 – – – – – – – – – – 6 6 – – 1 1 – – 1 1 – – 1 1 – – 6 6 aTimes are exposure times to the Kromayer lamp (dose rate 5 135 J per m2 per s; 280–400 nm). bTime after the exposure. c–, no macroscopically visible response; 1, development of clear edema/erythema; 6, variable response between separate animals, or variable judgement of independent noninformed observers. Table II. In contrast to XPC mice, XPA mice have a highly increased sensitivity to UVB induced erythema/edemaa 1 sb 2s 8s 16 s XPA–/–c –d XPA1/– XPA1/1 XPC–/– XPC1/– XPC1/1 nd nd nd nd nd 1 nd nd nd nd nd nd – – – – – nd 1 1 1 1 1 aAll mice were an F2 intercross of the parental strains 129/ola-C57Bl6 and HRA/SKH. are exposure times to the Kromayer lamp (dose rate 5 135 J per m2 per s; 280–400 nm). cXPA data from Berg et al (1997). d–, no macroscopically visible response at any time point; 1, development of edema/ erythema; nd, not done. bTimes of the mice after 4 or 8 s of exposure (see Table I). Hence, the MED of both the XPC–/– mice and the heterozygote and wild-type littermates is reached between 8 and 16 s of exposure, which corresponds to a UV dose between 1080 and 2160 J per m2. In an earlier experiment we determined the MED for the Kromayer lamp in XPA knockout mice, which were also breeded as an F2 intercross of the parental strains 129/olaC57Bl6 and HRA/SKH (Berg et al, 1997). In that study the XPA1/– and the XPA1/1 animals showed the same response as the mice in this study, i.e., no effect at 8 s and a clear effect at 16 s; however, the XPA–/– animals had their threshold between 1 and 2 s of exposure (see Table II for comparison). Hence, mice lacking XPA have an MED that is about a factor 10 lower than that of their wild-type littermates, whereas mice lacking XPC have an MED that is not lower than that of their wild-type littermates. DISCUSSION UV induced inflammation of the skin (erythema and edema) has been well documented clinically and histologically; however, the mechanism that leads from the absorption of UV radiation to the clinical response remains poorly defined. Persistence of UV photoproducts in the DNA is likely to be an important step: mice with a complete deficiency in NER (XPA knockouts; Miyauchi-Hashimoto et al, 1996; Berg et al, 1997) and mice with a deficiency in only TCR (CSB knockouts; Van der Horst et al, 1997) have an increased sensitivity to UV induced inflammation of the skin. In this paper we show that mice with a deficiency solely in global genome repair do not have an increased 407 sensitivity to UV induced inflammation, i.e., no lower MED than their wild-type littermates. Hence, the level of sensitivity to UVB induced erythema and edema appears to be determined by persistence of photoproducts in transcriptionally active DNA, but not in transcriptionally inactive DNA. Consistent with human XPC (Van Hoffen et al, 1995), cells from XPC mice appear deficient in the removal of 6–4PP from the nontranscribed strand of the p53 gene, but proficient in the removal of 6–4PP from the transcribed strand of this gene. Also in agreement with human XPC (Venema et al, 1990; Van Hoffen et al, 1995), cells from XPC mice appear proficient in the removal of CPD from transcriptionally active DNA. In contrast with humans, mice are very inefficient in the removal of CPD from transcriptionally inactive DNA (Ruven et al, 1993, 1994b). This latter difference between the two species does not appear to lead to qualitative differences between the human XPC and the murine XPC phenotype: fibroblasts from human XPC (Cleaver and Kraemer, 1989) as well as from mouse XPC (Sands et al, 1995; Cheo et al, 1997) show an increase in sensitivity to killing by UVC radiation that is of the same order of magnitude. Both XPC patients (Kraemer et al, 1987; Kondo et al, 1992) and XPC mice (Sands et al, 1995; Cheo et al, 1997) have an increased susceptibility to UV induced nonmelanoma skin cancer, and both are prone to pathologic eye abnormalities, but do not show obvious neurologic abnormalities. Although the XPC knockout mice used in this study do not have a lowered UVB induced MED, they do have an increased susceptibility to UVB induced epidermal hyperplasia and to UVB induced squamous cell carcinomas (Sands et al, 1995). An increased susceptibility to UVB induced epidermal hyperplasia and to UVB induced squamous cell carcinomas has also been reported for CSB knockout mice (defective in TCR only; Van der Horst et al, 1997) and for XPA knockout mice (defective in both global genome repair and TCR, De Vries et al, 1995; Nakane et al, 1995). In apparent contrast to erythema and edema, susceptibility to epidermal hyperplasia and to skin cancer is determined by accumulation of photoproducts in both transcriptionally active DNA and transcriptionally inactive DNA. For reasons already mentioned in the introduction, adequate and systematic studies on the sensitivity to UV induced erythema in XP patients are cumbersome: the small number of patients, variations in response and genetic background, ethically questionable, etc. There are, however, reports on the erythemal response of XP patients. An old report by Rottier (1954) describes two XP patients that did not have an extremely low MED, but these XP patients could at that time not be assigned to any of the complementation groups. In a report by Andrews et al (1978), three XP patients were mentioned that did not suffer from acute sun sensitivity (no experimental determination of MED was carried out). In later studies XP patients from separate complementation groups were described. XPA patients are clearly skin cancer prone and have a low MED (Ichihashi and Fujiwara, 1981; Kondo et al, 1992). The majority of XPD and XPE patients appear to have a low MED and are mildly skin cancer prone, i.e., most of them get skin cancer but at a much later age than XPA patients (Mamada et al, 1988; Ichihashi et al, 1988; Kondo et al, 1988; Ishii et al, 1991). Most XPF patients have an MED within the range of healthy humans, and the ones that get skin cancers also get them at a relatively late age (Kondo et al, 1989). Nine XPC patients had early skin cancers and showed MED within the range of healthy humans (Kondo et al, 1992). In this paper we confirm this finding under well controlled circumstances (i.e., omitting genetic variability between normal as well as XPC genotypes, differences in history of exposure, pigmentation, age, etc.) using a mouse model for XPC. If defective TCR leads to an enhanced susceptibility to erythema, one would expect patients with disrupted XP or CS genes that are involved in TCR to have a lowered MED. The CSB gene codes for a protein that is specifically involved in TCR (Ma et al, 1995), and, indeed, CSB patients (Norris et al, 1991) and mice (Van der Horst et al, 1997) have been shown to have a lowered MED. The products of XPA, XPD, and XPF appear to be involved in TCR (Ma et al, 1995; Wood, 1996), whereas TCR does not require the XPC gene product (Mu and Sancar, 1997). The role of the XPE gene product in TCR has not been established yet, but in view of the general 408 BERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY impairment of NER in XPE cells the defect is likely to affect TCR as well (Ma et al, 1995). In general, XPA, XPD, and XPE patients have a low MED, whereas most XPF patients have a normal MED. Although the MED in most XPF patients is within the normal range, the development of erythema in many XPF patients is abnormally delayed, peaking at 48 h or even 72 h after exposure (Kondo et al, 1989). This phenomenon of delayed erythemal reaction has also been found in XPA patients (Ichihashi and Fujiwara, 1981), in XPD and XPE patients (Kondo et al, 1989; Kondo et al, 1992), and in CSB patients (Norris et al, 1991), whereas the tested XPC patients showed a peak at 24 h after exposure (Kondo et al, 1992). Although there are reports of XPC patients that have ‘‘sun sensitive’’ skin (e.g., Cleaver et al, 1981), this condition may well have resulted from accumulated effects of a series of successive (suberythemal) exposures, and not from an acute sunburn. We have not been able to find a report of a proven XPC patient with an objectively assessed deviant MED. In summary, the available data suggest that disruption of TCR leads to an abnormal erythemal reaction (lowered treshold and/or delayed development), whereas specific disruption of global genome repair (as in XPC) does not affect the erythemal response. Interestingly, it has recently been shown that cells from XPA and CSB patients, but not from XPC patients, exhibit UV induced apoptosis at a considerable lower dose than wild-type cells (Ljungman and Zhang, 1996). This observation parallels our present finding on erythemal UV sensitivity in XPA, CSB, and XPC mice. The induction of apoptosis correlated with the inhibition of RNA synthesis, and, based on these findings, Ljungman and Zhang propose that blockage of RNA polymerase at sites of UV photoproducts is the initial step in the pathway leading to UV induced apoptosis. They, and others (Yamaizumi and Sugano, 1994), have also shown that nuclear accumulation of the p53 tumor-suppressor gene product occurs at lower UV exposure levels in XPA and CSB, but not in XPC, when compared with normal cells. This suggests that after blockage of RNA polymerase, nuclear p53 accumulation is an intermediate step towards the execution of apoptosis. For UV induced inflammation the first two steps might be the same as for UV induced apoptosis (i.e., persistence of photoproducts in transcriptionally active DNA, and blockage of RNA polymerase). It is very unlikely, however, that nuclear p53 accumulation is also an important step towards erythema and edema. First, it has been shown that indomethacin inhibits the development of UVB induced erythema in human skin without reducing nuclear p53 accumulation (Healy et al, 1994). Second, our own experiments with hairless p53 knockout mice did not reveal any influence of absence of p53 on sensitivity to UVB induced erythema or edema (data not shown), whereas the experiments of Ziegler and Brash have revealed that being nullizygous or heterozygous for p53 leads to a marked reduction in UV induced apoptosis in mouse skin (Ziegler et al, 1994). Sensitivity to sunburn is generally considered to be an indicator of skin cancer risk (Naylor, 1997). This study shows that skin cancer susceptibility is not always paralleled by sensitivity to UV induced inflammation, which implies that sensitivity to sunburn may not always be an adequate clinical marker of an individual’s skin cancer risk. This study also indicates that NER capacity (in particular, the capacity for global genome repair) may be a better predictor for skin cancer risk than sensitivity to sunburn. A comparative tumor induction study in XPC knockout mice versus CSB knockout mice will further unveil the relative contributions of the NER subpathways global genome repair and TCR to skin cancer susceptibility. We thank H.G. Rebel, C.M.J. Seelen, and H. van Weelden for experimental help. This work was supported by grants from the Environment Program of the European Community (EV5V-CT91-0030 and ENV4-CT96–0172), and the Dutch Cancer Society (UU 97–1531). 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