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Xeroderma Pigmentosum Variant Heterozygotes Show Reduced Levels of Recovery of Replicative DNA Synthesis in the Presence of Caffeine after Ultraviolet Irradiation Toshiki Itoh,Stuart Linn,* Ryoichi Kamide,Hiroyuki Tokushige,§ Nobutada Katori,§ Yoshiaki Hosaka,§ and Masaru Yamaizumi² *Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California, U.S.A.; ²Department of Cell Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan; Department of Dermatology, Jikei University School of Medicine, Japan; §Department of Plastic and Reconstructive Surgery, Showa University School of Medicine, Japan Patients with xeroderma pigmentosum variant show clinical photosensitivity, skin neoplasias induced by ultraviolet light, and defective postreplication repair, but normal nucleotide excision repair. We recently reported an alternative, simple method for the diagnosis of xeroderma pigmentosum variant that measures by autoradiography three cellular markers for DNA repair after ultraviolet irradiation: un- scheduled DNA synthesis, recovery of RNA syn- thesis, and recovery of replicative DNA synthesis. Among hereditary photosensitive disorders, includ- ing other xeroderma pigmentosum groups, Cockayne syndrome, and a newly established ultra- violet-sensitive syndrome, only xeroderma pigmen- tosum variant cells exhibited normal unscheduled DNA synthesis, normal recovery of RNA synthesis, but reduced recovery of replicative DNA synthesis (51 6 6% the rate relative to normal controls). This reduction of recovery of replicative DNA synthesis was enhanced in the presence of a nontoxic level of caffeine to 36 6 5%. In this study we assess the cellular markers in two independent families that included two photosensitive patients that were identified as xeroderma pigmentosum variant. Cells from heterozygotic parents showed normal levels of unscheduled DNA synthesis, recovery of RNA syn- thesis, and recovery of replicative DNA synthesis, but reduced rates of recovery of replicative DNA synthesis in the presence of 1 mM caffeine (53 6 8% relative to the normal control). Furthermore, with a colony-forming assay, the cells showed normal sur- vival by ultraviolet without caffeine, but slightly reduced survival by ultraviolet with 1 mM caffeine present. In one family, we confirmed inheritance of two heterozygous mis-sense mutations. One muta- tion is an AG transition at nucleotide 1840 that generates a K535E mis-sense mutation. Another mutation is an AC transversion at nucleotide 2003 that generates a K589 mis-sense mutation. Each of these mutations were absent in 52 unrelated Japanese individuals. These results suggest that xeroderma pigmentosum variant heterozygotes can be identified by their sensitivity to ultraviolet irradiation in the presence of nontoxic levels of caffeine. Key words: DNA polymerase eta/DNA replication/mutations. J Invest Dermatol 115:981–985, 2000 X eroderma pigmentosum (XP), an autosomal reces- sive disease characterized by photo-induced deterioration of the skin and eyes, often leads to early onset of malignant skin neoplasias (Kraemer and Slor, 1985; Cleaver and Kraemer, 1995). There are seven complementation groups, A through G (XPA–XPG), that are deficient in nucleotide excision repair (NER). A separate group, XP variant (XPV), shows normal NER but an exaggerated delay in recovery of replicative DNA synthesis (RDS) after ultraviolet (UV) irradiation (Lehmann et al, 1975, 1977; Rude and Friedberg, 1977; Cleaver and Kraemer, 1995). Recently, the XPV cDNA was cloned and its product identified as DNA polymerase eta, an enzyme that specifically inserts to deoxyadeno- sine triphosphate residues opposite a cyclobutane thymine dimer (Masutani et al, 1999; Johnson et al, 1999). It is difficult to diagnose patients with XPV at an early age because most XPV patients do not develop clinical manifestations and skin neoplasias until a later age. It is particularly difficult to identify heterozygous parents of XPV patients, as no unique cellular characteristics of XPV heterozygotes have been elucidated. We recently reported a simple method for the diagnosis of XPV that utilizes the measurement of three cellular markers of DNA repair by autoradiography: unscheduled DNA synthesis (UDS), recovery of RNA synthesis (RRS), and RDS after UV irradiation (Itoh et al, 1996a). The method provides a systematic basis for the diagnosis of photosensitive disorders, including XP, Cockayne syndrome, or UV-sensitive syndrome (Itoh et al, 1994, 1995, 1996b). In this study, we used this method to characterize cells of two XPV Manuscript received June 17, 2000; revised August 15, 2000; accepted for publication August 22, 2000. Reprint requests to: Dr. Toshiki Itoh, Department of Molecular and Cell Biology, Division of Biochemistry and Cell Biology, University of California, Berkeley, 229 Stanley Hall, Berkeley, CA, 94720-3206. Email: [email protected] Abbreviations: XP, xeroderma pigmentosum; XPV, xeroderma pig- mentosum variant; UDS, unscheduled DNA synthesis; RRS, recovery of RNA synthesis; RDS, recovery of replicative DNA synthesis; NER, nucleotide excision repair. 0022-202X/00/$15.00 · Copyright # 2000 by The Society for Investigative Dermatology, Inc. 981

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Xeroderma Pigmentosum Variant Heterozygotes ShowReduced Levels of Recovery of Replicative DNA Synthesis inthe Presence of Caffeine after Ultraviolet Irradiation

Toshiki Itoh,*² Stuart Linn,* Ryoichi Kamide,³ Hiroyuki Tokushige,§ Nobutada Katori,§ Yoshiaki Hosaka,§and Masaru Yamaizumi²*Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California, U.S.A.;

²Department of Cell Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, Japan; ³Department of Dermatology, Jikei

University School of Medicine, Japan; §Department of Plastic and Reconstructive Surgery, Showa University School of Medicine, Japan

Patients with xeroderma pigmentosum variant showclinical photosensitivity, skin neoplasias induced byultraviolet light, and defective postreplication repair,but normal nucleotide excision repair. We recentlyreported an alternative, simple method for thediagnosis of xeroderma pigmentosum variant thatmeasures by autoradiography three cellular markersfor DNA repair after ultraviolet irradiation: un-scheduled DNA synthesis, recovery of RNA syn-thesis, and recovery of replicative DNA synthesis.Among hereditary photosensitive disorders, includ-ing other xeroderma pigmentosum groups,Cockayne syndrome, and a newly established ultra-violet-sensitive syndrome, only xeroderma pigmen-tosum variant cells exhibited normal unscheduledDNA synthesis, normal recovery of RNA synthesis,but reduced recovery of replicative DNA synthesis(51 6 6% the rate relative to normal controls). Thisreduction of recovery of replicative DNA synthesiswas enhanced in the presence of a nontoxic level ofcaffeine to 36 6 5%. In this study we assess thecellular markers in two independent families thatincluded two photosensitive patients that were

identi®ed as xeroderma pigmentosum variant. Cellsfrom heterozygotic parents showed normal levels ofunscheduled DNA synthesis, recovery of RNA syn-thesis, and recovery of replicative DNA synthesis,but reduced rates of recovery of replicative DNAsynthesis in the presence of 1 mM caffeine (53 6 8%relative to the normal control). Furthermore, with acolony-forming assay, the cells showed normal sur-vival by ultraviolet without caffeine, but slightlyreduced survival by ultraviolet with 1 mM caffeinepresent. In one family, we con®rmed inheritance oftwo heterozygous mis-sense mutations. One muta-tion is an A®G transition at nucleotide 1840 thatgenerates a K535E mis-sense mutation. Anothermutation is an A®C transversion at nucleotide 2003that generates a K589 mis-sense mutation. Each ofthese mutations were absent in 52 unrelated Japaneseindividuals. These results suggest that xerodermapigmentosum variant heterozygotes can be identi®edby their sensitivity to ultraviolet irradiation in thepresence of nontoxic levels of caffeine. Key words:DNA polymerase eta/DNA replication/mutations. J InvestDermatol 115:981±985, 2000

Xeroderma pigmentosum (XP), an autosomal reces-sive disease characterized by photo-induceddeterioration of the skin and eyes, often leads toearly onset of malignant skin neoplasias (Kraemerand Slor, 1985; Cleaver and Kraemer, 1995). There

are seven complementation groups, A through G (XPA±XPG),that are de®cient in nucleotide excision repair (NER). A separategroup, XP variant (XPV), shows normal NER but an exaggerateddelay in recovery of replicative DNA synthesis (RDS) after

ultraviolet (UV) irradiation (Lehmann et al, 1975, 1977; Rudeand Friedberg, 1977; Cleaver and Kraemer, 1995). Recently, theXPV cDNA was cloned and its product identi®ed as DNApolymerase eta, an enzyme that speci®cally inserts to deoxyadeno-sine triphosphate residues opposite a cyclobutane thymine dimer(Masutani et al, 1999; Johnson et al, 1999).

It is dif®cult to diagnose patients with XPV at an early agebecause most XPV patients do not develop clinical manifestationsand skin neoplasias until a later age. It is particularly dif®cult toidentify heterozygous parents of XPV patients, as no unique cellularcharacteristics of XPV heterozygotes have been elucidated. Werecently reported a simple method for the diagnosis of XPV thatutilizes the measurement of three cellular markers of DNA repairby autoradiography: unscheduled DNA synthesis (UDS), recoveryof RNA synthesis (RRS), and RDS after UV irradiation (Itoh et al,1996a). The method provides a systematic basis for the diagnosis ofphotosensitive disorders, including XP, Cockayne syndrome, orUV-sensitive syndrome (Itoh et al, 1994, 1995, 1996b). In thisstudy, we used this method to characterize cells of two XPV

Manuscript received June 17, 2000; revised August 15, 2000; acceptedfor publication August 22, 2000.

Reprint requests to: Dr. Toshiki Itoh, Department of Molecular andCell Biology, Division of Biochemistry and Cell Biology, University ofCalifornia, Berkeley, 229 Stanley Hall, Berkeley, CA, 94720-3206.Email: [email protected]

Abbreviations: XP, xeroderma pigmentosum; XPV, xeroderma pig-mentosum variant; UDS, unscheduled DNA synthesis; RRS, recovery ofRNA synthesis; RDS, recovery of replicative DNA synthesis; NER,nucleotide excision repair.

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc.

981

patients and their heterozygous parents. This application hasallowed us to identify unique characteristics of the XPVheterozygotes.

MATERIALS AND METHODS

Cells and culture conditions The cell strains used in this study areshown in Table I. Ops4 and Ops36 cells are primary ®broblast strainsdesignated XPV, whereas Ops17, Ops18, and Ops39 cells are primary®broblast strains designated heterozygotes of XPV. A pedigree of the twofamilies giving rise to these strains is shown in Fig 1. Mori, Turu, and Sonocells are primary normal ®broblast strains established and characterizedpreviously (Itoh et al, 1996a, 2000). Fifty-two ®broblast strains, eachderived from unrelated Japanese individuals, including 18 normal and 34photosensitive patients, were used to test whether the Ops4 mutations werea polymorphism. All cells were cultured in a humidi®ed incubator at 37°Cin 5% CO2 in Dulbecco's modi®ed Eagle's Minimum Essential Medium(ICN, Costa Mesa, CA) supplemented with 15% (vol/vol) fetal bovineserum (HyClone Laboratories, Logan, UT), 100 units penicillin G per ml,and 100 mg streptomycin per ml.

Measurement of UDS, RRS, and RDS after UV irradiation UDSwas measured as described (Itoh et al, 1994) with some modi®cations.Brie¯y, to minimize variations between different preparations and tofacilitate comparison, 20 ml aliquots (1±2 3 104 cells) of the experimental orcontrol (Mori) cell suspensions were plated with micropipettes in two rowson the same 18 3 18 mm coverslip (Itoh et al, 1994) and incubated for 7±10 d until they were in stationary phase. They were then washed withphosphate-buffered saline, irradiated with UV light (254 nm) at a dose rateof 1 J per m2 per s for 30 s, immediately labeled with [3H]thymidine (40 mCiper ml) for 2.5 h, and ®xed. Coverslips were mounted on glass slides,dipped in Kodak NTB-3 emulsion, and exposed for 24 h or 48 h at 4°C.Grains above 20 nuclei were then counted under a microscope.

RRS (Itoh et al, 1994) and RDS (Itoh et al, 1996a) were measured asfollows: 1±2 3 104 cells were plated on a coverslip as described above andincubated for 1±2 d to reach log phase. They were then washed withphosphate-buffered saline and irradiated with UV light (254 nm) at a doserate of 1 J per m2 per s for 15 s. Cells were next incubated for 23 h for RRSor 6 h with or without 1 mM caffeine for RDS and labeled with[3H]uridine (40 mCi per ml) or [3H]thymidine (15 mCi per ml), respectively,for 1 h. Autoradiography was performed as described above.

UV survival assay UV survival of cells was measured as describedelsewhere (Itoh et al, 1994, 2000). Brie¯y, appropriate numbers of cellswere inoculated on to 60 mm dishes and left to attach for 10 h.Subsequently, cells were rinsed with phosphate-buffered saline andexposed to UV light at a ¯uence rate of 0.7±1.0 J per m2 per s for thetimes indicated. Then, after incubation for 10±21 d, the dishes were washedwith phosphate-buffered saline, and the colonies ®xed with 80% (vol/vol)methanol, stained with Giemsa, and counted. Three dishes were utilized foreach dose.

Sequence analysis Reverse transcription±polymerase chain reaction(reverse transcription±PCR) and DNA sequencing were performedessentially as described (Itoh et al, 1999) with PCR primers described

previously (Masutani et al, 1999). Brie¯y, PCR was performed betweenforward primer S0 (GATCCCTTCTCGGTTTTCC, base pairs 57±76)and reverse primer AS25 (TCCATGCCTGTGAAGAGATG, base pairs2531±2550), nested PCR was performed between forward primer S1(ACTGGACCTCCTAGAAAG, base pairs 110±129) and reverse primerAS24 (ATCCTACAGGCAAGCCTGAG, base pairs 2387±2416),between forward primer S1 and reverse primer AS9 (TGCCAGGACC-TTATTGTGTGT, base pairs 885±905), and between forward primer S8(TCCACAATAAGGTCCTGGC, base pairs 887±906) and reverse primerAS24. PCR fragments were subcloned into pGEM-T Easy Vector(Promega, Madison, WI). Sequencing was performed utilizing T7 andSP6 DNA polymerases and forward primer S1, S5 (AGCCAGTGTTGA-AGTGATGG, base pairs 519±538), S8, S13 (AGCTGGTTGTGAGCA-TTCG, base pairs 1331±1349), or S17 (CCATGAGCAATTCACCA-TCC, base pairs 1760±1779). To exclude the possibility that either or bothof the Ops4 mutations are a polymorphism in the Japanese population,RNA was extracted from diploid ®broblast strains from 52 unrelatedJapanese individuals as described (Itoh et al, 1999), and reversetranscription±PCR was performed using primers S0 and AS25; nestedPCR was then performed using primers S17 and AS24. The PCR productswere puri®ed with the QIAquick PCR puri®cation kit (Qiagen, Valencia,CA) and direct sequencing was performed utilizing primer S17.

RESULTS

Case reports Ops4, an 8 y old boy and the second of threesiblings (Fig 1), was ®rst recognized to be photosensitive at the ageof 2±3 y. The parents were not consanguineous. He burned easilyafter long periods of exposure to the sun. At present, he showsslight tanning, and a number of small pigmented freckles around thelower eyelids and on the cheeks (Table I). Ops36, a 67 y old manand the ®rst of four siblings (Fig 1), showed photosensitivity at anearly age. He suffered from squamous cell carcinomas on his nose atthe ages of 46 and 54, and these tumors were resected. Recently, hesuffered from nine metastatic malignant melanomas on the bodyand basal cell carcinoma on the face at age 67. After resection oftumors, he underwent the following treatment regimen:peplomycin, nimustihe hydrochloride (ACNU), vincristine, andnatural interferon-b (Feron, Toray Industries, Tokyo, Japan). Theparents of Ops36 were consanguineous (Fig 1b).

Application of an alternative method for the diagnosis ofXPV We applied an alternative method (Itoh et al, 1996a) forthe diagnosis of XP complementation group to cells from thetwo photosensitive patients (Table I). In this method ®broblaststrains are plated on four coverslips in parallel with normal cells,and then RDS with or without caffeine present, UDS, andRRS are measured. XPV cells speci®cally exhibit normal UDS,normal RRS, but a reduction of the rate of RDS that isenhanced in the presence of 1 mM caffeine (Itoh et al, 1996a,2000); both the Ops4 and Ops36 cells showed normal UDS

Table I. Patient pro®les and cell strains used in this studya

PatientsCell

ClinicalStrain Genotype Sex Age photosensitivity Remarks

Mori Normal Male 21 ± Itoh et al (1996a, 2000)Turu Normal Male 22 ± Itoh et al (1996a, 2000)Sono Normal Male 48 ± Itoh et al (1996a, 2000)Ops4 XPVb Male 8 + freckles, slight pigmentationOps17 XPV Heteroc Male 44 ± consanguinity (±)Ops18 XPV Heteroc Female 38 ± consanguinity (±)Ops36 XPVb Male 67 + squamous cell carinomas, malignant melanomas,

basal cell carcinomas; consanguinity (±)Ops39 XPV Heteroc Female 87 ± consanguinity (+)

aSee Fig 1 for the patient pedigrees.bCells were classi®ed as homozygotes xeroderma pigmentosum variant in this study.cCells were classi®ed as heterozygotes of xeroderma pigmentosum varient in this study.

982 ITOH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(Table II), normal RRS (Table III), but reduced RDS, whichwas enhanced in the presence of 1 mM caffeine (Table IV).These properties and the patients' phenotypes are consistentwith a classi®cation as XPV.

Cellular DNA repair markers in heterozygotes ofXPV When the cellular DNA repair markers of parents of theXPV patients, Ops17, Ops18, and Ops39, were examined, UDSand RRS levels were normal (Tables II and III). RDS levels ofthese cells were also normal, but were reduced in the presence of1 mM caffeine (Table IV). As the aphenotypic parents of the XPVpatients are presumed to be heterozygous for XPV because of theautosomal recessive inheritance of XP, these results indicate thatheterozygotes of XPV show normal levels of UDS, RRS, andRDS, but reduced levels of RDS in the presence of 1 mM caffeine.

UV survival in the presence and absence of caffeine TheXPV cells Ops4 and Ops36 were abnormally sensitive to killing byUV irradiation only in the presence of 1 mM caffeine (Fig 2a, b).These results are consistent with the established characteristics ofmost XPV cells (Arlett et al, 1975; Lehmann et al, 1975; Cleaverand Kraemer, 1995; Itoh et al, 1996a, 2000) and also con®rm thatOps4 and Ops36 cells are XPV. The XPV heterozygote Ops17,Ops18, and Ops39 were similarly examined (Fig 2c±e). These cellswere not sensitive to UV without caffeine, but were slightly moresensitive than normal cells in the presence of 1 mM caffeine.

Novel mutations present in the XPV (DNA polymerase eta)cDNA By sequence analysis, Ops4 cells were determined to havecompound heterozygous mutations (TableV). An A®G transitionat nucleotide 1840 generates a K535E mis-sense mutation in oneallele, whereas a A®C transversion at nucleotide 2003 generates a

Table II. Levels of unscheduled DNA synthesis observed incells of two XPV families

UDSa Range ofFamily Cell strains (grains/nucleus 6 SEM) relative rateb (%)

I Ops4/Mori 13.6 6 0.9/13.5 6 1.2 86±118Ops17/Mori 13.4 6 0.6/14.6 6 1.1 82±104Ops18/Mori 12.3 6 1.0/12.6 6 1.1 82±116

II Ops36/Mori 11.6 6 0.4/12.6 6 0.6 85±100Ops39/Mori 14.8 6 0.4/14.4 6 1.0 94±113

aThe exposure time was 24 h. Data are means 6 SEM of 50 determinations.bRange of relative rate is the range of UDS values of cell strains tested

compared with those of Mori (normal control) cells. The ranges of variation oftwo other normal cells (Turu and Sono) versus Mori cells was 87±113% (Itoh et al,2000).

Table III. Levels of recovery of RNA synthesis observed incells of two XPV families

RRSa Range ofFamily Cell strains (grains/nucleus 6 SEM) relative rateb (%)

I Ops4/Mori 19.9 6 1.2/19.9 6 1.2 89±113Ops17/Mori 18.5 6 0.8/19.4 6 0.8 88±104Ops18/Mori 21.7 6 0.7/17.1 6 0.6 119±136

II Ops36/Mori 21.0 6 1.1/24.2 6 1.4 77±97Ops39/Mori 20.4 6 1.5/17.2 6 0.8 105±133

aThe exposure time was 24 h. Data are means 6 SEM of 50 determinations.bRange of relative rate is the range of RRS values of cell strains tested

compared to those of Mori (normal control) cells. The range of variation of RRSvalues for two other normal cells (Turu and Sono) versus Mori cells was 111±115%(Itoh and Yamaizumi, unpublished data).

Table IV. Levels of recovery of replicative DNA synthesis observed in cells of two XPV families

RDSa Range of relative rateb (%)

Family Cell strains With caffeine Without caffeine With caffeine Without caffeine

I Ops4/Mori 21.0 6 1.3/60.3 6 6.8 18.0 6 1.3/44.2 6 5.8 29±42 33±50Ops17/Mori 23.2 6 1.2/56.1 6 5.7 52.6 6 2.8/54.3 6 3.6 35±56 86±109Ops18/Mori 35.6 6 2.4/71.8 6 6.0 52.2 6 3.0/51.8 6 5.0 43±58 87±118

II Ops35/Mori 26.0 6 1.8/52.8 6 5.3 32.9 6 2.8/50.3 6 4.2 42±59 55±77Ops39/Mori 36.0 6 3.6/53.0 6 4.2 46.7 6 3.8/49.7 6 5.0 57±80 78±113

aThe exposure time was 20±24 h (with caffeine) or 10±13 h (without caffeine), respectively. Data are means 6 SEM of 50 determinations.bRange of relative rate is the range of RDS values of cell strains tested compared to those of Mori (normal control) cells. Normal variation between RDS values for two

other normal cells (Turu and Sono) versus Mori cells was 91±115% or 90±111% in the absence or presence of caffeine, respectively (Itoh and Yamaizumi, unpublisheddata).

Figure 1. Pedigrees of two XPV families. Filled symbols representaffected individuals, half-®lled symbols are individuals heterozygotes for XPvariant, and open symbols are individuals who were unavailable for analysis.Parents of Ops36 are consanguineous. Ops4, Ops36, and the youngestbrother of Ops36 showed clinical photosensitivity (*). The father of Ops36died of gastric cancer at the age of 58, and the youngest brother of Ops36had photosensitivity and skin cancer, strongly suggesting xerodermapigmentosum.

VOL. 115, NO. 6 DECEMBER 2000 HETEROZYGOTES OF XP VARIANT 983

K589T mis-sense mutation in the other allele. Ops17 cells haveonly the A®C transversion at nucleotide 2003 in one allele(TableV), whereas Ops18 cells have only the A®G transition atnucleotide 1840 in one allele (TableV). These basic Lys residuesare highly conserved between human and mouse (Yamada et al,2000). On the other hand, Glu or Thr are acidic or hydroxylresidues, respectively, and therefore these mutations would likelyaffect the conformation and functionality of the DNA polymerase.Furthermore, we screened for these mutations in 52 unrelatedJapanese individuals (104 chromosomes) by direct sequencing andthey were absent. Therefore, these changes are not a polymorphismin the Japanese population. On the basis of these results weconclude that Ops17 and Ops18 are the obligate heterozygousparents of XPV Ops4. Similar sequence analyses of Ops36 andOps39 cells are in progress.

DISCUSSION

XPV cells showed reduced RDS levels without caffeine after UVirradiation (Itoh et al, 1996a, 2000). RDS levels relative to normalin all XPV cells examined to date are 51 6 6% without caffeine and36 6 5% with caffeine (Itoh et al, 1996a, 2000; this study), whereasXPV heterozygotes showed reduced RDS levels in the presence ofcaffeine at 53 6 8%. A similar intermediate effect was seen inheterozygotes of Cockayne syndrome, a rare autosomal recessive

disorder defective DNA repair: cells from parents of patients withCockayne syndrome were intermediately sensitive to UV and thedegree of sensitivity was between that of normal and Cockaynesyndrome cells (Lehmann, 1987).

XPV and XPV heterozygous cells appear to have normal UVsensitivity in the absence of caffeine. Ops36 (XPV) and Ops39(XPV heterozygote) cells, however, showed impaired UV survivalin the absence of caffeine. This sensitivity might be a generalproperty of XPV and XPV heterozygous cells or it may have beencaused by these particular individuals' histories. Ops36 sufferedfrom multiple skin neoplasias (squamous cell carcinomas, basal cellcarcinoma, and malignant melanomas) and underwent chemother-apy as described above. DNA damages induced by the chemother-apy regimen could have led to the reduced UV survival of cellscultured from Ops36. Ops39 was 87 y old when her skin biopsywas performed and as a consequence these cells rapidly senescebecause of her age and possibly also because of an accumulation ofDNA damages caused by sun exposure during her long life.

It is dif®cult to diagnose patients with mild photosensitivity (Itohet al, 2000). The observation that the heterozygotes of XPVshowed abnormal RDS is an important ®nding as many XPVpatients have been reported in Japan and it is therefore necessary fordiagnosis to distinguish among XPV, heterozygotes of XPV, andXPE (Itoh et al, 1999, 2000) from normal cells. For example,GM01389 cells were formerly assigned to XPV, but recentlyreclassi®ed as XPE by Otrin et al (1998) and the mutationscon®rmed in the DDB2 gene (Nichols et al, 2000).

Although the XPV gene product has recently been identi®ed asDNA polymerase eta (Johnson et al, 1999; Masutani et al, 1999),little is known about the relation of the mutant XPV proteins to thecaffeine effect. The molecular mechanisms of inhibition of repairprocesses by caffeine were studied by Selby and Sancar (1990).They concluded that caffeine inhibits DNA repair by two differentmechanisms: (i) interference with speci®c binding of the DNArepair enzymes to damaged DNA, and (ii) promotion ofnonspeci®c binding of DNA repair enzymes to DNA. DNApolymerase eta inserts to deoxyadenosine triphosphate residuesspeci®cally opposite a cyclobutane thymine dimer (error-freebypass) (Johnson et al, 1999; Masutani et al, 1999) and as XPVhomozygotes and heterozygotes might be compromised in thisregard, other DNA polymerases might compensate to allow suchbypass. Were caffeine to interfere with these DNA polymerases asdescribed above, XPV homozygous and heterozygous cells mightthen be abnormally sensitive to caffeine. We are currently studyingthe effect(s) of caffeine on a number of human DNA polymerases inthis regard.

Figure 2. Survival to UV irradiation in thepresence and absence of caffeine. The cellsutilized were: (a) Ops4, (b) Ops36, (c) Ops17, (d)Ops18, and (e) Ops39. After UV irradiation, cellswere incubated for 10±21 d, ®xed with 80%methanol, and stained with Giemsa. Caffeine wasadded to a ®nal concentration of 1 mM just afterUV irradiation. Open circles and solid lines aremeasurements in the absence of 1 mM caffeine,and closed circles and dotted lines in the presenceof 1 mM caffeine. The data of Mori are a normalcontrol and are overwritten in each ®gure (no cir-cles). Points for these lines are omitted for clarity.Each point represents an average of two or threeindependent experiments (one experiment is anaverage of two dishes for each point). The errorbar indicates SEM.

TableV. Mutation analysis of DNA polymerase eta cDNAin Ops4, Ops17, and Ops18 cells

DNA polymerase eta mutation Aminoacid

Cell strains Nucleotide Mutation change Occurrencea

Ops4 1840 A®G K535E 8/101840 none ± 2/102003 A®C K589T 2/102003 none ± 8/10

Ops17 1840 none ± 26/262003 A®C K589T 2/262003 none ± 24/26

Ops18 1840 A®G K535E 2/61840 none ± 4/62003 none ± 6/6

aRatio of number of clones with mutations to the number of clones sequenced.Note: Each of these mutations are absent in 50 unrelated Japanese individuals.

984 ITOH ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

We thank Mrs Yuka Itoh and Dr. Toshiro Kageshita (Kumamoto University of

Medicine) for help with the preparation of the manuscript. We are also grateful to

Dr. Francesca Zollezi, Dr. Anne Nichols and Dr. Hitomi Asahara (Berkeley) for

helpful discussions. This work was supported partly by Grants-in-Aid from the

Ministry of Education, Science, Sports, and Culture of Japan (11770468 to T.I.),

the Kao Foundation for Arts and Sciences (to T.I.), the Nakatomi Foundation (to

T.I.) and USPHS grant P30ES08196 (to S.L.).

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