trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane...

Trichothiodystrophy Fibroblasts Are Deficient in the Repairof Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and(6–4)Photoproducts

Yoko Nishiwaki,�w Nobuhiko Kobayashi,w Kyoko Imoto,�w Taka-aki Iwamoto,�w Aya Yamamoto,� SachikoKatsumi,w Toshihiko Shirai,w Shigeki Sugiura,z Yu Nakamura,y Alain Sarasin,yy Sachiko Miyagawa,w andToshio Mori��Radioisotope Research Center, wDepartment of Dermatology, zMedical Genetics Research Center, and yDepartment of Psychiatry, Nara Medical University,Kashihara, Nara, Japan; yyLaboratory of Genetic Instability and Cancer, Gustave Roussy Institute, Villejuif, France

A photosensitive form of trichothiodystrophy (TTD) results from mutations in the same XPD gene as the DNA-

repair-deficient genetic disorder xeroderma pigmentosum group D (XP-D). Nevertheless, unlike XP, no increase in

skin cancers appears in patients with TTD. Although the ability to repair ultraviolet (UV)-induced DNA damage has

been examined to explain their cancer-free phenotype, the information accumulated to date is contradictory. In this

study, we determined the repair kinetics of cyclobutane pyrimidine dimers (CPD) and (6–4)photoproducts (6–4PP)

in three TTD cell strains using an enzyme-linked immunosorbent assay. We found that all three TTD cell strains are

deficient in the repair of CPD and of 6–4PP. UV sensitivity correlated well with the severity of repair defects.

Moreover, accumulation of repair proteins (XPB and proliferating cell nuclear antigen) at localized DNA damage

sites, detected using micropore UV irradiation combined with fluorescent antibody labeling, reflected their DNA

repair activity. Importantly, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA

damage sites and the TFIIH expression. Our results suggest that there is no major difference in the repair defect

between TTD and XP-D and that the cancer-free phenotype in TTD is unrelated to a DNA repair defect.

Key words: CPD/6–4PP/TTD/xeroderma pigmentosum/XPB.J Invest Dermatol 122:526 –532, 2004

DNA damage in cells exposed to ultraviolet (UV) radiationplays significant roles in cell-cycle arrest, activation of DNArepair, cell killing, mutation, and neoplastic transformation(Setlow, 1978; Suzuki et al, 1981; Maher et al, 1982; Otoshiet al, 2000; Zhou and Elledge, 2000). Two major types ofDNA lesions produced by UVB (280–315 nm) and by UVC(200–280 nm) are cyclobutane pyrimidine dimers (CPD) and(6–4)photoproducts (6–4PP) (Clingen et al, 1995). 6–4PP areformed at a rate 15% to 33% that of CPD (Mitchell, 1988;Clingen et al, 1995; Eveno et al, 1995). Although 6–4PP areremoved from the global genome at a much higher speedthan are CPD, both types of DNA lesions are repaired bynucleotide excision repair (NER) in normal human cells(Mitchell et al, 1985). NER is initiated by recognition of DNAdamage, which can occur either by the binding of XPC/hHR23B to damaged DNA (Sugasawa et al, 1998) or by thestalling of RNA polymerase at a DNA lesion (Hanawalt et al,1994). The DNA duplex around the lesion is subsequentlyopened by the concerted action of RPA, XPA, and TFIIH

(Evans et al, 1997; Mu et al, 1997). This allows incisions ofthe damaged DNA strand on both sides of the lesion by thestructure-specific endonucleases XPG (O’Donovan et al,1994) and ERCC1/XPF (de Sijbers et al, 1996), which arefollowed by excision of the lesion-containing oligonucleo-tide (Huang et al, 1992). The gap in the duplex is filled in bya proliferating cell nuclear antigen (PCNA)-dependent DNApolymerase (Nichols and Sancar, 1992; Shivji et al, 1992)and is then sealed by a DNA ligase which regenerates theintact DNA structure (de Boer and Hoeijmakers, 2000).Defects in NER are associated with an autosomal recessivedisease termed xeroderma pigmentosum (XP), which isprimarily characterized by extreme UV sensitivity and anincreased incidence of sunlight-induced skin cancers(Kraemer et al, 1987; van Steeg and Kraemer, 1999). Thereare seven different genetic complementation groups of XP(XP-A to XP-G) and a variant form. Another defective NER-related disease is trichothiodystrophy (TTD), which ischaracterized in patients by brittle hair with reduced sulfurcontent, impaired mental and physical development, apeculiar face, ichthyosis, and in approximately half of thepatients, cutaneous photosensitivity but no skin cancer(Bergmann and Egly, 2001; Itin et al, 2001). Photosensitivepatients with TTD belong to three genetic complementationgroups (TTD-A, TTD/XP-B, and TTD/XP-D), cells from thelatter 2 groups of patients falling into the XP-B and XP-Dgroups, respectively (most of those belonging to the

Abbreviations: CPD, cyclobutane pyrimidine dimers; DPBS, Dul-becco’s phosphate-buffered saline; ELISA, enzyme-linked immu-nosorbent assay; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NER, nucleotideexcision repair; 6–4PP, (6–4)photoproducts; PCNA, proliferatingcell nuclear antigen; TTD, trichothiodystrophy; XP, xerodermapigmentosum.

Copyright r 2004 by The Society for Investigative Dermatology, Inc.

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TTD/XP-D group). XPB and XPD are ATP-dependenthelicases with opposite polarities and are subunits of TFIIH,which is not only an NER factor but is also a transcriptionfactor. Thus, it has been assumed that the function of TFIIH,both in DNA repair and in transcription, is to unwind thedouble-stranded DNA helix. To understand how mutationsin the same NER gene may produce different skin cancersusceptibilities in patients with XP or TTD, a better under-standing of the defects in NER is essential. Nevertheless,reports on the ability to repair the two major types of UV-induced DNA damage in TTD cells have been limited andeven controversial. One early study showed that some TTD/XP-D cells had a marked reduction in the repair of 6–4PP incellular DNA (Broughton et al, 1990). In contrast, anothermore recent study (which used a more sensitive assay)indicated that in photosensitive TTD cell lines, the defectiverepair was mainly confined to CPD (Eveno et al, 1995), aconclusion generally accepted today. Nevertheless, thatlater study used exponentially growing TTD cells to measurerepair, which can result in the dilution of DNA damagethrough replication, in addition to the removal of DNAdamage by NER, during the post-UV incubation period.Because those data were not corrected for the replication-related decrease in DNA damage, it probably led to theoverestimation of the DNA repair level of the cells. Thus, itremains possible that TTD cells are deficient in the repairnot only of CPD but also of 6–4PP. In this study, we carefullydetermined the repair kinetics of CPD and 6–4PP in threetypical TTD/XP-D cell strains using a sensitive enzyme-linked immunosorbent assay (ELISA) probed with DNA-lesion-specific monoclonal antibodies. Care was taken tocorrect for replication-related reductions in photolesions.Our results demonstrate that all three TTD cell strains areindeed deficient in the repair of both CPD and 6–4PP andare not confined to defective repair of one of thesephotoproducts.

Results

TTD cells are deficient in the repair of UV-induced CPDand 6–4PP from genomic DNA To determine the repairkinetics of CPD and 6–4PP in normal and in TTD cells, wemeasured these photolesions in genomic DNA at differenttimes after UV radiation using an ELISA (Fig 1). Normalhuman MSU-1 cells repaired 90% of the initial 6–4PP within3 h after UV irradiation, whereas they removed only 40% ofthe initial CPD within 8 h and only 60% at 24 h. In contrast,all TTD cell strains examined were significantly deficient inthe ability to repair CPD and 6–4PP, although to variousdegrees. In TTD9VI cells, the extent of removal of DNAdamage did not exceed 15% for 6–4PP and 5% for CPDwithin 24 h, demonstrating their severe repair deficiency,which was comparable to that of XP-A cells (Mitchell et al,1985). TTD1VI cells were more competent in this regard andrepaired 50% of 6–4PP within 8 h and removed 10% of CPDwithin 24 h. Moreover, TTD2VI cells repaired 80% of 6–4PPwithin 8 h and removed 20% of CPD within 24 h. Theseresults indicate that different TTD cell strains have variabledefects in the repair of CPD and 6–4PP from genomic

DNA and that CPD repair is more severely compromisedthan 6–4PP repair.

UV sensitivity of TTD cell strains correlates with theirseverity of DNA repair deficiency To verify the repair dataobtained by ELISA, we examined the UV sensitivities ofnormal and of TTD cells using an MTS assay (Fig 2). All TTDcell strains showed a higher sensitivity to UV than normalcells. TTD9VI cells were the most sensitive, followed byTTD1VI cells and by TTD2VI cells, in that order. That resultindicates that the UV sensitivity of different TTD cell strains

Figure1TTD cells are deficient in the repair of UV-induced CPD and 6–4PPin genomic DNA. Cells were irradiated with UV (10 J/m2) andincubated to allow for repair. The percentage of the initial number ofphotoproducts was determined at various times after UV irradiationusing an ELISA with monoclonal antibodies specific for each type oflesion. Each point shows the mean ( � SD) of four independentexperiments.

Figure2TTD cells are more sensitive to UV than normal cells. On the fourthday after UV irradiation, cell viability was determined using the MTSassay. Percentage of viability is expressed relative to unirradiated cells.Each point shows the mean ( � SD) of three independent experiments.

IMPAIRED REPAIR OF UV DNA DAMAGE IN TTD CELLS 527122 : 2 FEBRUARY 2004

correlates well with the severity of their DNA repairdeficiency.

Fluorescence images of localized CPD at various repairtimes reflect the DNA repair kinetics To confirm the repairdata reported above, we induced CPD in localized areas ofindividual cell nuclei by micropore UV irradiation andexamined the repair of those lesions at various repair timesusing immunofluorescence with an anti-CPD antibody(Fig 3). No DNA damage was observed in unirradiatedcells, but UV irradiation immediately produced severalfluorescent CPD foci per nucleus in normal and in TTDcells. In normal cells, the CPD fluorescence graduallyweakened over time after UV exposure, confirming theefficient repair determined by ELISA. In contrast, the brightfluorescence persisted in the nuclei over the entire repairperiod of 24 h in TTD9VI cells, confirming the severity oftheir repair deficiency. TTD1VI cells showed an intermediaterepair pattern between normal cells and TTD9VI cells. Theseresults indicate that the fluorescent images of localized CPDat various repair times after UV exposure reflect well therepair kinetics obtained by ELISA, both in normal cells andin TTD cells. Similar results were also obtained in the caseof 6–4PP (data not shown).

The level of NER factors at DNA damage sites at earlyrepair times correlates with the repair capacity of eachcell strain Our original technique of micropore UV irradia-tion combined with fluorescent antibody labeling enables usto visualize not only the localized DNA damage but also theaccumulation of repair proteins at the DNA damage sites.We first examined how levels of the detergent-insolubleform of PCNA vary at damage sites during DNA repair.PCNA is a processivity factor for DNA polymerase and isessential both for NER and for DNA replication (Celis andMadsen, 1986). It is accepted that PCNA participates in arepair synthesis step of NER by forming a trimeric ringaround duplex DNA near a gap site in UV-irradiated humancells (Aboussekhra and Wood, 1995). This form of PCNA isthus resistant to extraction with nonionic detergents,although PCNA is largely in a soluble form in non-S-phasecells in the absence of UV (Toschi and Bravo, 1988). In Fig 4,detergent-insoluble PCNA was visualized under the sameconditions as used in Fig 3. PCNA fluorescence was notobserved without UV exposure or immediately after UVirradiation either in normal or in TTD cells. Nevertheless,several bright fluorescent foci per nucleus can be seen 0.5 hafter irradiation in normal cells. The fluorescence of thosePCNA foci gradually weakened at 3 h and at 9 h andbecame undetectable at 24 h after UV exposure, correlating

Figure3Fluorescent images of localized CPD atvarious repair times reflect the DNA repairkinetics obtained by ELISA. Cells were cul-tured in 35-mm glass-bottom dishes for 48 h.Immediately after micropore UV irradiation (100J/m2) or at various times after UV irradiation,cells were treated with detergent solution andwere then fixed. CPD (green) were thenvisualized with TDM-2 antibody. Nuclear DNA(red) was counterstained with propidium iodide.A filter with 3-mm pores was used.

Figure4The localization of PCNA at DNA damagesites at early repair times reflects the repaircapacity in TTD cell strains. Cells weretreated according to the procedure describedin Figure 3. Detergent-insoluble PCNA (green)was then visualized with a PCNA antibody.Nuclear DNA (red) was counterstained withpropidium iodide. A filter with 3-mm pores wasused.

528 NISHIWAKI ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

well with the activity of DNA repair (Figs 1,3). In contrast, noor little PCNA fluorescence was observed at 0.5 or at 3 hafter UV, respectively, in TTD9VI cells, which is consistentwith their severe defect in DNA repair ability. Importantly,however, we detected several fluorescent PCNA foci at 9and at 24 h after UV irradiation, a pattern that has also beenobserved in XP-A cells (Katsumi et al, 2001). TTD1VI cellsshowed an intermediate response pattern between normalcells and TTD9VI cells. The PCNA fluorescence was veryweak at 0.5 h, peaked at 9 h, and decreased at 24 h afterUV irradiation.

Next, we wanted to examine how mutations in the XPDgene affect the recruitment of XPD protein to sites of DNAdamage in TTD cells, but no antibody highly specific forXPD is available. Therefore, cells were doubly stained forXPB and for CPD 0.5 h after UV irradiation, because XPBand XPD are subunits of TFIIH and have similar functions inNER. In normal cells, one or two XPB foci per nucleus withbright fluorescence were observed in addition to weakstaining throughout the nucleus (Fig 5). The XPB focioverlapped with the corresponding CPD foci, indicatingthat XPB is quickly recruited to the sites of DNA damage.Similar results were obtained in TTD2VI cells even thoughthe level of XPB at damage sites was lower compared withnormal cells. In contrast, no XPB foci at the DNA damagesites were observed in TTD9VI cells. In TTD1VI cells, almostno significant accumulation of XPB occurred at the DNAdamage sites with the exception that a few cells showedfoci with very weak staining. The results of Figs 4 and 5indicate that the localization of NER factors at DNA damagesites at early repair times correlates well with the repaircapacity of each cell strain.

The degree of reduction in the level of XPB in TTD cellstrains does not correlate with their severity of DNArepair deficiency To further understand the differences inXPB accumulation at DNA damage sites among TTD cellstrains, the level of XPB was analyzed by western blots of

cell lysates. In parallel, we measured the content of actin asa loading control. Figure 6 shows that the level of XPB issignificantly lower in TTD cells than in normal cells. The XPBcontent in TTD1VI cells, TTD2VI cells, and TTD9VI cells is67, 52, and 56% of that found in control cells, respectively.This result indicates that the degree of reduction of XPB inTTD cell strains does not correlate with their severity of DNArepair defects.

Discussion

Accurate information about DNA repair in photosensitiveTTD cells is essential to explain why they do not result inincreased skin cancer susceptibility. Nevertheless, availabledata have been limited and even controversial to date.Thus, we determined the repair kinetics of CPD and of6–4PP in three typical TTD/XP-D cell strains using asensitive ELISA with monoclonal antibodies specific foreach type of UV-induced DNA damage. In these experi-ments, we corrected for replication-related reductions inDNA damage during the repair period using radioisotope-labeled cells. This simple approach proved to work wellbecause NER-unrelated decreases in photolesions 24 hafter UV irradiation of XP12ROSV (XP-A) cells was exten-sively reduced by this correction (data not shown). Thiscarefully controlled study led to the following three findingsabout DNA repair in TTD cells (Fig 1). The first and mostimportant finding is that all TTD cell strains examined aredeficient in the repair of both CPD and 6–4PP, and repair isnot confined to just one of those photoproducts. This is notconsistent with some published results that indicate that thedefective repair in some TTD cell strains are confined to6–4PP (Broughton et al, 1990) or to CPD (Eveno et al, 1995).The apparent contradiction between their results and oursmay stem from the different sensitivities of the protocolsused or different treatments for the replication-relateddecreases in DNA damage. XP-D cells are known to bedeficient in the repair of both CPD and 6–4PP (Mitchell et al,1985; Hiramoto et al, 1989). Thus, these results suggest thatthere is no major difference in the repair defect betweenTTD/XP-D and XP-D cells. This supports the hypothesis that

Figure 5The level of XPB at localized CPD sites at early repair timesreflects the repair capacity in TTD cell strains. Cells were doublystained for XPB (green) using a XPB antibody and for CPD (red) withTDM-2 antibody 0.5 h after micropore UV irradiation. A filter with 5-mmpores was used.

Figure6The level of XPB is significantly lower in TTD cells than in normalcells. Western blot analyses of XPB and actin proteins were performedon cell lysates of normal or TTD cell strains. Protein levels werequantitated by a luminescent image analyzer. The amount of XPB wasnormalized to the actin content.


the cancer-free phenotype of patients with TTD is unrelatedto its repair defect. It may result from a transcription defect,which prevents the skin abnormalities from being expressed(Berneburg et al, 2000).

The second important finding of this study is that theremoval of 6–4PP is less severely inhibited than that of CPDin TTD cells. It is known that 6–4PP form bigger distortionsin double-stranded DNA and show higher affinities for repairfactors of NER compared with CPD (Buterin et al, 2002).Thus, 6–4PP are removed more efficiently than CPD fromthe global genome in normal human cells. Our resultssuggest that this preferential repair of 6–4PP over CPD isfurther increased when the cellular repair activity is loweredby mutation(s) in a DNA repair gene. This hypothesis issupported by a study on repair-deficient XP-E cells (Hwanget al, 1999).

The third important finding of this study is that the level ofthe residual DNA repair capacity varies substantially amongTTD cell strains. TTD9VI cells are the most defective in therepair of photoproducts, followed by TTD1VI cells and byTTD2VI cells, in that order. The UV sensitivity correlatedperfectly with the severity of the DNA repair deficiency inthese cell strains, supporting this result (Fig 2). Theimmunofluorescent staining of localized CPD at variousrepair times also confirmed the repair kinetics in normalcells, in TTD1VI cells and in TTD9VI cells (Fig 3). A similarapproach was used to examine the spatial interactionbetween NER factors and DNA damage (Figs 4, 5). Brightfluorescent foci of PCNA appeared at DNA damage sites at0.5 h after UV exposure in normal cells, whereas little or noPCNA fluorescence was observed in TTD1VI cells and inTTD9VI cells. Importantly, the fluorescence of XPB at DNAdamage sites at 0.5 h after UV exposure was strong inMSU-1 cells, weak in TTD2VI cells, very weak or negligiblein TTD1VI cells, and negligible in TTD9VI cells. These resultsdemonstrate that the localization of NER factors at DNAdamage sites at early repair times correlates well with therepair capacity of each cell strain. It was recently reportedthat the content of TFIIH subunits in photosensitive TTDfibroblasts is markedly lower than in normal fibroblasts(Botta et al, 2002). In particular, in TTD patients carryingdifferent mutated XPD alleles, the reduction in their levels isthe most striking in the case of the N-terminal R112Hsubstitution, like TTD9VI cells. Our western blot analysisrevealed that the level of XPB, a subunit of TFIIH, in thethree TTD cell strains is indeed lower than in normal cells(Fig 6). Nevertheless, the degree of reduction in XPB isrelatively similar among the TTD cell strains (approximately60% of the control), suggesting that the reduction in TFIIH isnot related solely to their severity of repair deficiency. TTDcells with the same mutation as TTD9VI cells are reported toexpress reduced levels of XPD protein similar to the otherTFIIH subunits, including XPB and p62 (Botta et al, 2002).Considering that XPB does not accumulate at DNA damagesites at 0.5 h after UV exposure in TTD9VI cells, it is mostlikely that the R112H substitution in XPD interferes with therecruitment of the TFIIH complex to the sites of DNAdamage in addition to reducing its cellular level whichresults in the marked severe decrease in DNA repairdeficiency. It has been suggested that XPC/hHR23B, aDNA damage sensor, plays a crucial role in the recruitment

of TFIIH to sites of DNA damage through its specific affinityfor XPB and/or p62 (Yokoi et al, 2000; Volker et al, 2001).The R112H substitution in XPD might therefore induceconformational changes in the TFIIH complex, which wouldlead to interference with the proper interactions betweenXPC and XPB or between XPC and p62. Mutations ofTTD1VI cells and TTD2VI cells are located in the C-terminalregions of XPD (R722W and R592P, respectively) (Takayamaet al, 1996). Considering that reduced levels of XPB proteinaccumulate at DNA damage sites in TTD2VI cells, theR592P substitution in XPD may exclusively affect theexpression of TFIIH subunits, resulting in the mild repairdeficiency. The severe repair deficiency of TTD1VI cells mayrelate to the marked reduction in XPD that affects therecruitment of the TFIIH complex to the DNA damage sites,because they are functionally hemizygous (Bergmann andEgly, 2001).

PCNA foci appeared at UV damage sites as late as 9 and24 h after UV irradiation in TTD9VI cells, even though thosecells could only minimally repair CPD and 6–4PP within24 h. We have observed similar results in XP-A cells, whichsuggests that TTD9VI cells have a marked defect in DNArepair that is comparable to XP-A cells (Katsumi et al, 2001).Interestingly, TTD1VI cells, which had a less severe DNArepair phenotype, also expressed delayed PCNA, in addi-tion to the early one related to the residual repair ability. Thefollowing explanations may underlie the delayed expressionof PCNA at DNA damage sites in repair-deficient cells. Firstit might be due to DNA repair that occurs normally inTTD9VI cells. Defects in NER do not affect the globalgenome repair of oxidative DNA damage or single-strandbreaks, which are minor types of DNA lesions caused by UV(Cadet et al, 1992). Second, it might relate to DNA frag-mentation resulting from apoptosis. The UV irradiation level(100 J/m2) used in this study produces a high density of DNAdamage at localized nuclear areas and might causeapoptotic DNA breaks, which are possibly associated withPCNA-dependent repair. Third, it might reflect blocked or de-layed DNA replication. When partially irradiated cells enterthe S phase, DNA replication in damaged nuclear areas maybe slowed down more than in undamaged areas because ofthe block by DNA damage. Finally, it might be attributable toan incomplete or stalled NER of CPD or 6–4PP. If the lack ofone functional NER factor interrupts the normal NERprocess and keeps it stalled for more than several hours,other NER factors might be recruited slowly to the sites ofDNA damage. Experiments to examine these hypothesesare under way and the results will be published elsewhere.

In summary, we found that all three TTD cell strainsexamined are deficient in the global genome DNA repair ofCPD and 6–4PP and not confined to one of thesephotoproducts. This conclusion is supported by the resultsof UV sensitivity and of the immunofluorescent staining ofDNA damage and NER factors. The repair defects in TTDcells may relate not only to the reduction in the level of theTFIIH complex but also to the inability to recruit the TFIIHcomplex to the sites of DNA damage. Thus, these resultssuggest that there is no major difference in the repair defectbetween TTD/XP-D and XP-D cells and that the cancer-freephenotype in TTD cells is therefore unrelated to its repairdefect.


Materials and Methods

Cells Normal human fibroblasts (MSU-1) derived from newbornforeskins were provided by J.E. Trosko (Michigan State University)(Mori et al, 1989). Three kinds of fibroblasts (TTD1VI, TTD2VI,TTD9VI) were obtained from skin biopsies of patients with TTD(complementation group TTD/XP-D) (Sarasin et al, 1992; Evenoet al, 1995). The passage numbers of these cells were between 13and 19. Cells were cultured in Dulbecco’s modified Eagle’s medium(Nissui, Seiyaku, Tokyo) supplemented with 10% fetal bovineserum (Dainippon Pharmaceutical, Osaka, Japan) and antibiotics.The patients we studied gave their written consent for skin biopsiesand our work with TTD and XP patients has been accepted by ourlocal ethics committee and conducted according to the Declara-tion of Helsinki principles.

UV irradiation Micropore (localized) UV irradiation was performedessentially as described previously (Katsumi et al, 2001). Briefly,cells were cultured in 35-mm glass-bottom dishes (MatTek,Ashland, MA) for 2 days. After being washed with Dulbecco’sphosphate-buffered saline (DPBS), cells were carefully coveredwith a polycarbonate isopore membrane filter (pore size, 3 or 5 mm;Millipore, Bedford, MA). The filter-masked cells were then UV-irradiated with five low-pressure mercury lamps (GL-10, Toshiba,Tokyo, Japan; predominantly 254-nm UV) at a dose rate of 1.67 Jper m2 per s at a distance of 1.6 m. For whole-cell (uniform)irradiation, cells without a filter were irradiated with a low-pressuremercury lamp at a dose rate of 0.42 J per m2 per s at the samedistance. Dose rates were monitored using a UV radiometer (UVR-1, Topcon, Tokyo, Japan). Localized UV irradiation at 100 J per m2

causes similar levels of DNA damage to that of uniform irradiationat 5 J per m2 (Imoto et al, 2002). In this study, we used UVC but notUVB, because similar amounts of CPD and 6–4PP and similarlevels of cell killing are induced in human cells irradiated with UVCand with 30 times more UVB (sunlamp) (data not shown).

Measurement of UV sensitivity UV sensitivity was evaluatedusing a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison,WI) (Imoto et al, 2002). Cells (3 � 103) were cultured in each well of48-well culture plates for 24 h. After being washed with DPBS, cellswere irradiated with UV and were further cultured for 4 d. Viabilitywas assessed by the ability of cells to convert MTS into formazan,which was quantitated spectrophotometrically. Percentage ofviability was expressed relative to unirradiated cells.

Quantitation of induction and repair of UV-induced DNAdamage by ELISA Exponentially growing cells were labeled with1.85 kBq per mL [2-14C]thymidine (Amersham, Piscataway, NJ;2.18 GBq per mmol) in 175-cm2 flasks for 3 d. After being washedwith DPBS, labeled cells (42 � 106) were plated in each 10-cmFalcon dish and cultured for 48 h to reach subconfluent monolayerin a radioisotope-free medium. After being washed with DPBS,cells were irradiated with UV and incubated to allow repair.Immediately after, or at various times after, UV irradiation, cellswere harvested by a cell scraper from the dishes, and cell pelletswere stored at �801C until processing. Genomic DNA was thenpurified using a QIAamp blood kit (Qiagen, Hilden, Germany). DNAconcentrations were calculated from the absorbance at 260 nm,and 14C-radioactivity of DNA samples was also measured using aliquid scintillation counter (LSC-5100, Aloka, Tokyo). CPD and6–4PP were quantitated by an ELISA using TDM-2 and 64M--2monoclonal antibodies (Mori et al, 1991; Kobayashi et al, 2001).Those antibodies specifically bind to CPD or 6–4PP, respectively, inall dipyrimidine sequences (TT, TC, CT, and TT) in denatured DNA.Details of this method have been described previously (Nakagawaet al, 1998; Imoto et al, 2002). In brief, 96-well polyvinylchlorideflat-bottom microtiter plates, precoated with 0.003% protaminesulfate, were coated with heat-denatured sample DNA (1.6 Bq perwell for CPD, 32 Bq per well for 6–4PP). In this study, equalamounts of radioactive DNA, instead of equal amounts of DNA

calculated from the absorbance at 260 nm, were coated in thewells to correct for decreases in photolesions per DNA by possibleDNA replication during the post-UV incubation period. Quantitiesof 1.6 and 32 Bq of DNA corresponded to 15 and 300 ng of DNA,respectively, of cells without post-UV incubation. The binding ofmonoclonal antibodies to photolesions in immobilized DNA in wells(in quadruplicate) was detected with biotinylated F(ab0)2 fragmentof goat anti-mouse IgG (Zymed, South San Francisco, CA) andthen with streptavidin-peroxidase. The absorbance of coloredproducts derived from o-phenylenediamine was measured at 492nm by Titertek Multiskan (Labsystems, Helsinki, Finland). Forexamining repair kinetics, the percentage of the initial number ofphotolesions was calculated at various times after UV irradiationusing damage induction standard curves.

In situ visualization of CPD, PCNA, and XPB after microporeUV irradiation Immediately after, or at various times after,micropore UV irradiation (100 J per m2), cells were treated withice-cold detergent solution (0.5% Triton-X 100, 0.2 mg/mLethylenediaminetetraacetic acid, and 1% bovine serum albuminin PBS) for 15 min and were then fixed with methanol:acetone (1:1)for 10 min at –201C. After denaturation of DNA using 2 M HCl (30min), CPD was visualized by the immunologic method using TDM-2 monoclonal antibody as described before (Nakagawa et al, 1998;Katsumi et al, 2001). Detergent-insoluble PCNA was visualized withthe same method except we used a PCNA monoclonal antibody(Clone 24, Transduction Laboratory, Lexington, KY) and omittedthe denaturation of DNA (Katsumi et al, 2001). Nuclear DNA wascounterstained with propidium iodide. For double staining of CPDand XPB, cells were allowed to repair for 30 min after micropore UVirradiation, fixed with 1.6% formaldehyde for 10 min at roomtemperature, and then permeabilized with 2% Triton-X 100 at 41Cfor 5 min. Cells were sequentially labeled with an XPB rabbitantibody (SC-293, Santa Cruz Biotechnology, Santa Cruz, CA) andAlexa Fluor 488 goat anti-rabbit IgG conjugate (Molecular Probes,Eugene, OR). Antigen–antibody complexes were fixed with 1.6%formaldehyde for 10 min, and DNA was denatured. Cells were thensequentially labeled with TDM-2 and Alexa Fluor 594 goat anti-mouse IgG conjugate. Fluorescence microscopy was performedusing a Leica DMIRB. Fluorescent images of CPD and XPB weremerged using Adobe Photoshop software.

Western blot analysis Samples of subconfluent monolayer cellsgrown in 10-cm dishes were resuspended in lysis buffer (100 mMNaCl, 10 mM Tris-HCl, pH 7.5, 0.5% Triton X-100, completeprotease inhibitor cocktail (Roche, Mannheim, Germany)) andsonicated three times for 30 s on ice, and cellular debris wasremoved by centrifugation. Different samples of lysates wereseparated on 7.5% polyacrylamide-sodium dodecyl sulfate gelsand transferred onto Immobilon-p transfer membranes (Millipore).The membranes were incubated for 2 h at room temperature inblocking buffer (PBS containing 5% skim milk) and were thenhybridized for 1 h with primary antibodies against XPB or actin(Santa Cruz) diluted in PBS. The membranes were then washedfour times for 10 min with PBS/Tween 0.1% and incubated for 1 hwith goat anti-rabbit IgG conjugated with horseradish peroxidase(Zymed). Proteins were visualized and quantitated using enhancedchemiluminescence (Perkin-Elmer, Boston, MA) and a luminescentimage analyzer (LAS-1000, Fujifilm, Tokyo). The amount of XPBwas normalized to the actin content.

We thank Dr James E. Trosko (Michigan State University) for the normalhuman fibroblasts. This work was supported by Grants-in-Aids forScientific Research from The Ministry of Education, Culture, Sports,Science and Technology of Japan, and from The Japanese Dermato-logical Association.

DOI: 10.1046/j.0022-202X.2004.22226.x

Manuscript received June 13, 2003; revised September 3, 2003;accepted for publication October 5, 2003


Address correspondence to: Toshio Mori, PhD, Radioisotope ResearchCenter, Nara Medical University, Kashihara, Nara 634-8521, Japan.Email: [email protected]

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trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane...

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