Proc. Natl. Acad. Sci. USAVol. 90, pp. 10529-10533, November 1993Genetics

Specific UV-induced mutation spectrum in the p53 gene ofskin tumors from DNA-repair-deficient xerodermapigmentosum patients

(skin cancer/p53 tumor suppressor gene)

NICOLAS DUMAZ, CHRISTIANE DROUGARD, ALAIN SARASIN, AND LEELA DAYA_GROSJEAN*Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, B.P. no. 8, 94801 Villejuif, France

Communicated by Richard B. Setlow, July 2, 1993 (received for review March 1, 1993)

ABSTRACT The UV component of sunlight is the majorcarcinogen involved in the etiology of skin cancers. We havestudied the rare, hereditary syndrome xeroderma pigmento-sum (XP), which is characterized by a very high incidence ofcutaneous tumors on exposed skin at an early age, probably dueto a deficiency in excision repair of UV-induced lesions. It isinteresting to determine the UV mutation spectrum in XP skintumors in order to correlate the absence of repair of specificDNA lesions and the initiation of skin tumors. The p53 gene isfrequently mutated in human cancers and represents a goodtarget for studying mutation spectra since there are >100potential sites for phenotypic mutations. Using reverse tran-scription-PCR and single-strand conformation polymorphismto analyze >40 XP skin tumors (mainly basal and squamouscell carcinomas), we have found that 40% (17 out of 43)contained at least one point mutation on the p53 gene. AU themutations were located at dipyrimidine sites, essentially at CCsequences, which are hot spots for UV-induced DNA lesions.Sixty-one percent of these mutations were tandem CC -k TTmutations considered to be unique to UV-induced lesions; thesemutations are not observed in internal human tumors. AU themutations, except two, must be due to translesion synthesis ofunrepaired dipyrimidine lesions left on the nontranscribedstrand. These results show the existence of preferential repairof UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand inhuman tissues.

One of the major consequences of UV radiation, a potentDNA-damaging agent, is the induction of skin cancers. Infact, the incidence of cutaneous tumors induced by repeatedexposure to sunlight is constantly increasing, making themthe most common type ofhuman cancer. The extremely highfrequency of malignancies found in exposed skin of individ-uals with the rare genetic disorder xeroderma pigmentosum(XP) suggests that defective repair of UV-induced DNAdamage plays a major role in skin carcinogenesis (1). It hasbeen well characterized that the somatic cells from XPpatients are hypersensitive to killing and mutation inductionby UV radiation due to a defect in excision repair of UV-induced pyrimidine dimers and/or (6-4) photoproducts (2).Therefore, it is clear that the efficient and accurate removalofDNA lesions by cellular repair processes is critical in orderto avoid lethality, mutation, and cellular transformation,which are all important events leading to tumor formation.

Carcinogenesis is a complex multistage process, and epi-demiological and molecular studies have shown that theinduction of cancer may require the accumulation of severalindependent mutations. In animal skin carcinogenesis, theactivation of oncogenes has been well established in the

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complex processes that involve initiation, promotion, andprogression (3). Several investigators (4-6) have shown thatthe ras family of oncogenes is activated in skin tumors due tomutations occurring at pyrimidine-rich sequences known tobe targets for UV-induced DNA damage. In a comparativestudy carried out in our laboratory, we have demonstrated a>2-fold higher mutation frequency of ras genes in XP skintumors in contrast to the same type of tumors from normalindividuals (7). Furthermore, the repair-deficient XP skintumors also showed high levels (70%o) of Ha-ras gene ampli-fication and rearrangement. These data on mutations ofdominantly acting oncogenes have therefore well corrobo-rated the role of unrepaired UV lesions as an initiating eventin skin carcinogenesis.Recent research on tumor suppressor genes has clearly

shown the importance of their inactivation as a major step inthe development of human cancers (8). Among them, themutation of the p53 gene has turned out to be the mostcommon genetic alteration found in human malignancies (9).The basic function of the p53 protein is not fully understoodyet, but it is clear that it slows down the cell cycle progressionat the G1/S boundary and probably also plays a crucial rolein feedback control, whereby DNA damage arrests cells atthe restriction point (10, 11). It was, therefore, particularlyinteresting to see whether skin tumors from repair-deficientXP patients also contained inactivated p53, which may playa cooperative role with oncogenes in cutaneous carcinogen-esis. Since the p53 gene can be mutated on >100 sites (12),it is a particularly good target for determining the in vivomutation spectrum of a given type of cancer. We wanted touse this property to analyze skin cancers induced by solar UVin XP patients. Indeed, UV light produces distinctive muta-tions in DNA, essentially C -- T transitions at dipyrimidinesites where CC -+ TT double mutations appear as a veritablesignature of UV-induced lesions. These mutations are be-lieved to be due to a translesion synthesis of either cyclo-butane dimers or (6-4) photoproducts (13, 14). Furthermore,the involvement of cyclobutane dimers has already beendemonstrated in skin tumor formation in fish and opossum(15, 16). We were interested in determining if p53 may be atarget that particularly reflects this UV mutagenic specificity.We report here a study of XP skin tumors where mutationsin the p53 tumor suppressor gene are seen to be directlycaused by unrepaired UV-induced lesions. Furthermore, thedata presented here are, to our knowledge, the first directindication of the occurrence of preferential repair in a humantissue-namely, in the skin of XP patients.

Abbreviations: XP, xeroderma pigmentosum; SSCP, single-strandconformation polymorphism; UDS, unscheduled DNA synthesis;XPA, XPC, and XPD, XP complementation groups A, C, and D,respectively; XPV, XP variant.*To whom reprint requests should be addressed.

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MATERIALS AND METHODSTissue Samples. Skin tumors from XP patients were ob-

tained at the time of surgical resection; the samples werefrozen immediately in liquid nitrogen and stored at -70°C.When possible, normal skin tissue was also sampled fromunexposed skin, and primary skin fibroblast cultures wereestablished in F10 medium (GIBCO/BRL) supplementedwith 15% (vol/vol) fetal calf serum and 1% antibiotics andantifungicides.The majority of the biopsy samples were obtained from

Institut Gustave Roussy (Villejuif, France), H6pital Saint-Louis (Paris), and Hopital H. Mondor (Creteil, France); somewere from Algeria (Centre Hospitalier Universitaire of Al-ger) or Tunisia (H6pital C. Nicolle, Tunis and University ofSousse).The biological characteristics of the samples used in this

study are presented in Table 1.Unscheduled DNA Synthesis (UDS). When available, XP

skin fibroblast cell cultures were examined for DNA repairactivities as described (7).

Nucleic Acid Preparations from Tumors and Cultured Cells.Cell culture pellets or large (>200 mg) frozen tumor sampleswere ground to a fine powder, and RNA and DNA wereextracted after lysis in guanidine thiocyanate as described (7).When tumor samples were very small, only DNA wasextracted.

Reverse Transcription-PCR. Five micrograms of totalRNA from XP tumors was denatured at 90°C for 2 min andquick-chilled on ice; reverse transcription was carried out in25 ul of PCR buffer (10 mM TrisHCl, pH 8.3/1.5 mMMgCl2/50 mM KCl/0.01% gelatin) containing 400 ng ofrandom hexamer, each dNTP at 200 ,.M, 180 units ofRNAsin(Promega), and 100 units of Moloney murine leukemia virusreverse transcriptase (GIBCO/BRL). After a 30-min incuba-tion at 42°C, the reaction was stopped by heating at 90°C for2 min and was quick-chilled. The first strand cDNA was PCRamplified by adjusting the reaction volume to 50 ,ul with PCRbuffer, each dNTP at 100 AM, 10 pmol each of upstream anddownstream primer, 2.5 ,ul of formamide, and 1-2 units ofTaq DNA polymerase (Perkin-Elmer/Cetus). Amplificationwas carried out for 30 cycles of denaturation at 92°C for 20sec, annealing at 62°C for 20 sec, and elongation at 72°C for90 sec. The two pairs of primers used are as follows: set 1,5'-TTCCACGACGGTGACACGCTTC-3' and 5'-CTCAG-GCGGCTCATAGGGCACC-3'; set 2, 5'-ACTTTTCGA-CATAGTGTGGTGGTGCCCTAT-3' and 5'-GGGGGTGG-GAGGCTGTCAGTGGGG-3'.

Cloning of PCR Products. PCR products were purified onCentricon 30 (Amicon) and cloned using either the TA Cloningsystem (Invitrogen) or the SureClone ligation kit (Pharmacia).Table 1. List of samples studied from XP patients and number ofbiopsy samples containing a mutation in the p53 gene

Number of samples

Sample Analyzed Mutated

Diploid fibroblast line 5 0Nonexposed skin biopsy 10 0Sun-exposed skin biopsy 6 0Preepithelioma lesion 3 0Benign lesion 2 0Nevus 1 1Histologically undefined tumor 4 1Metastasic melanoma cell line 1 0Malignant melanoma 2 0Basal cell carcinoma 11 3 (27%)Squamous cell carcinoma 23 11 (48%)Sarcoma 1 1

Total tumors 43 17 (40%o)

Plasmid vectors containing amplified DNA inserts were puri-fied, and several independent clones were sequenced.

Single-Strand Conformation Polymorphism (SSCP) Analy-sis. The coding region of the p53 gene was amplified by usingfour sets of primers (17), covering exons 4, 5-6, 7, and 8-9.Genomic DNA (200-500 ,g) was incubated in a total volumeof 25 ,ul containing each dNTP at 100 ,tM, 10 pmol of eachprimer, and 1 unit of Tfl DNA polymerase (Epicentre Tech-nologies) or Taq DNA polymerase (Perkin-Elmer/Cetus), 5,uCi (1 Ci = 37 GBq) of [a-32P]dCTP (DuPont/NEN) bufferedin 50 mM Tris HCl, pH 9.0/20 mM NH4SO4/1.5 mM MgC92.The reaction mixture was heated at 94°C for 3 min, andamplification was carried out for 35 cycles of 94°C for 1 min,66°C or 55°C for 90 sec, and 72°C for 90 sec. The reaction wasstopped after a 5-min extension at 72°C by a 10-fold dilutioninto 0.1% SDS/10 mM EDTA; 4 ,ul of this solution was thenmixed with 7 ,ul of 95% (vol/vol) formamide, 20 mM EDTA,0.05% bromophenol blue, and 0.05% xylene cyanol. Thesamples were denatured 5 min at 100°C, quick-chilled on icefor 3 min, and loaded immediately onto 0.5 x MDE (BioprobeSystems) gels containing 0.6x TBE buffer (lx TBE contains89 mM Tris, 80 mM boric acid, and 2 mM EDTA). Electro-phoresis was carried out at 8 W of constant power for 12-24hr at room temperature. SSCP bands were analyzed byautoradiography, and tumorDNAs presenting mobility shiftswere reamplified for verification. The shifted bands wereeluted from the gel, amplified, purified, and sequenced di-rectly or cloned.

Direct Sequencing of PCR Products. Fifty femtomoles ofamplified DNA was sequenced by using a double-strandedDNA cycle sequencing system (GIBCO/BRL).

RESULTSOur XP patients originated mainly from North Africa, whereintermarriage has resulted in many more cases of this rarerecessive syndrome than in the European population. Mostpatients presented multiple skin tumors in early childhood,and UDS studies of their normal fibroblasts showed them tobe highly repair deficient (5-10% UDS levels) compared tonormal individuals. The UDS levels, clinical features, andproportional representation of the different XP groups inNorth Africa allow us to assign most ofthem to the classic XPcomplementation group C (XPC; 50%o), and a few to XPA(25%). Some XP patients (25%) developed skin tumors atmuch later ages (20 years or more), exhibited nearly normalUDS levels, and were identified as XP variants by Lehmannet al. (18).

In this study we have analyzed over 40 XP tumors, whichare listed in Table 1. Control analyses were carried out onnormal or XP diploid fibroblast cultures, biopsy samples ofunexposed and exposed skin, and two benign lesions (Table 1).

Initially, to assay for p53 alterations, RNA from tumorswas used to produce p53 first-strand cDNA by reversetranscription-PCR followed by sequence analysis. Seven XPtumors have been studied this way, and 4 (A.Z., B.B.1, I.B.,and S.T.) had at least one point mutation in the p53 gene(57%). For the remaining tumors, genomic DNA was exam-ined by the SSCP technique (Fig. 1). All DNAs presentingmobility shifts were sequenced to define the mutations re-sponsible for their altered behavior. SSCP analysis showed13 XP tumors had point mutations out of 36 tumors tested(36%). This percentage of mutations is lower than that foundusing the reverse transcription-PCR approach because theSSCP technique cannot resolve all mutations. Nevertheless,out of a total of 43 tumors analyzed, 40% (17 out of 43)presented a mutated p53 gene, whereas diploid XP fibroblastsfrom the same patients contained only wild-type p53 se-quences. Furthermore, wild-type p53 sequences were alwayspresent with mutated sequences in XP tumors, due to eitherthe presence of normal tissue in our tumor biopsy samples or

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EXONS 5 and 6

WT m.4. i.

_lQEXONS 8 and 9WT M.M. H.B.

EXON 7

WT B.B.I

FIG. 1. SSCP analysis of some amplified exons from various XPskin tumors. The presence of shifted bands (arrowheads) comparedto wild-type (WT) sample indicates the presence of at least onemutation on the DNA sequence; initials correspond to patients fromTable 2.

because only one allele carried a mutation. Among the celllines tested, we have not detected p53 alterations in normalhuman or XP fibroblasts or in the XP metastasic melanomacell line. The two malignant melanoma biopsy samples as-sayed only contained wild-type p53. Among 23 squamous cellcarcinomas analyzed, 48% contained a mutated p53 as did 3out of 11 (27%) basal cell carcinomas.

All the p53 mutations detected in the XP tumors are shownin Table 2, and all are found to have occurred at dipyrimidinesites. This indicates that mutations are targeted mainly atpyrimidine dimers or pyrimidine-pyrimidone (6-4) photo-products. Indeed, 83% (19 out of 23) of mutations are foundat CC sites while only 17% (4 out of 23) are at TT or CT sites(Fig. 2 and Table 2). Fourteen out of the 19 mutations at CCsites are tandem double base CC -* TT transitions, and the

remaining are single-base transitions. The four mutations atTT or CT sites consist of a T C transition, a T -- A

transversion, and two T -- G transversions. Five tumor

samples (A.M., B.B.1, H.B., I.B., and M.J.1; Table 2) carrytwo distinct mutations in the p53 gene, which are on differentalleles, as verified by cloning and sequencing.Assuming that the mutation can be linked to dipyrimidine

dimers, all the point mutations (except two, at least one beingfrom a XP variant) occurred opposite unrepaired lesionslocated on the nontranscribed DNA strand of the p53 gene.All mutations resulted in the amino acid changes detailed inTable 2, and all double mutations CC -* TT always resulted

in a single amino acid change.

DISCUSSIONIt is clear from a large number of studies that there is a highlevel of molecular abnormalities in the p53 gene in a widerange oftumor types (8, 9, 12, 21). Sequencing ofthe p53 genein mammals, amphibians, birds, and fish has revealed fivehighly conserved domains ofwhich four are within exons 5-8(20). Greater than 90% of the base substitution mutations ofthe p53 gene found in human tumors encompass theseevolutionary conserved regions (Fig. 2) and create mutationhot spots (12, 21). The p53 mutations that we have identifiedin the XP cutaneous tumors all have, with the exception ofone at codon 113, fallen within these regions (Fig. 2). All areat pyrimidine doublets, and five tumors containing twodistinct mutations present different mutations on each allele.Eighty-three percent of mutations are C -* T transitions,

which are characteristic of UV-induced mutations in modelsystems. Sixty-one percent of all mutations are tandem CC -*

TT double base substitutions, which have also been previ-ously reported to be specific for UV as seen by studies usingphage, bacteria, and simian virus 40 or shuttle vectors (25-27)and endogenous genes in cultured cells (28, 29). Indeed,UV-irradiated shuttle vectors replicated in normal humancells show 65% C -- T transitions compared to 90% in XP

cells. However, the incidence of double mutations is low invectors replicated in XP cells with a variation between the

Table 2. Characteristics of XP skin tumors in which mutations have been found in the p53 gene

Repair Tumor p53 genePatient deficiency, % Histology Location Mutated codon Mutation Amino acid substitutionA.K.2 90 SCC Forehead 113 cTtc - cGtc Phe ValA.L. SCC Eye 179 acCa - acTa His - TyrA.M. SCC Forehead 278t tCct tTct Pro Ser

278t tCCt tTTt Pro - PheA.ZJ Nevus Arm 135 tTgc tAgc Cys SerB.B.1 90 S Temple 247-248t aCC*g - aTTg Asn-Arg Asn-Trp

281-282t aCCg - aTTg Asp-Arg Asp-TrpB.G. 95§ SCC Forehead 179 acCa - acTa His - TyrH.B. BCC Forehead 152t cCCg cTTg Pro Leu

278t tCCt - tTTt Pro - PheI.B. 95 BCC Cheek 178-179t aCCa aTTa His-His His-Tyr

281-282t aCCg - aTTg Asn-Arg - Asn-TrpJ.A. 95§ SCC Head 152 cCCg - cTTg Pro - LeuM.J.1t BCC Cheek 135t tTgc -tCgc Cys -Arg

135-136t gCCa gTTa Cys-Gln Cys-StopM.M. 95 SCC Scalp 281-282 aCCg - aTTg Asp-Arg Asp-TrpM.N.J SCC Lip 249 aGgc aAgc Arg - LysN.H. 90 Lip 178-179 aCCa - aTTa His-His His-TyrR.N. SCC Cheek 247-248 aAcC*g - aCcTg Asn-Arg - Thr-TrpS.K.1$ SCC Nose 281-282 aCCg aTTg Asp-Arg Asp-TrpS.Ka. SCC Head 159 gCCa - gTTa Ala ValS.T. 95 SCC Cornea 247-248 aCC*g aTTg Asn-Arg - Asn-TrpThe patients are indicated by their initials. Their deficiency in UDS as compared to controls is given. BCC, basal cell carcinoma; SCC,

squamous cell carcinoma; S, sarcoma. Mutations are indicated in capital letters on the coding strand, written 5' to 3'.*Cytosine known to be methylated in vivo (19).tMutations on different alleles.tXP variant.§Patients B.G. and J.A. have been characterized as XPC and XPA, respectively.

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different groups (26, 27, 30). Yagi et al. (30) have shown thatUV-irradiated shuttle vectors replicated in XPC cells con-tained 20% tandem mutations compared to less than 10% inXPA, XPD, or XP variant (XPV) fibroblasts. This tendencyis reflected by our results, because the majority of our XPtumor samples may be from XPC patients, and we find a veryhigh level of tandem mutations. Spontaneous mutations invivo can result in C -* T substitutions (about 44% in acollection of 1000 mutations), but double-base mutations arerarely produced by mutagens other than UV. In the vastnumber (>1000) of internal malignancies analyzed for p53alterations, only 13 double mutations have been found; theonly CC -) TT tandem mutation, at codon 250 in a non-small-cell lung carcinoma, is probably due to a GG -+ AAmutation, because of polycyclic hydrocarbon reactivity withguanosine nucleotides (31). This frequency is significantlylower (P < 10-7 using the Fisher exact test) than that foundwith XP tumors (61%). Finally, tandem CC -+ TT mutationshave also been found to be induced by oxidative damage (32).This is particularly relevant in our XP tumors, because wehave previously shown that XP cells are deficient in catalaseactivity, an important enzyme in antioxidant defense (33).The predominance of CC -- TT double transitions is thus

clearly a sunlight-induced event. Among five recent studies(17, 19, 22-24) on p53 mutations in skin tumors, only Brash andcoworkers (19, 22) revealed seven tandem CC -- TT substi-tutions in 29 positive tumors for p53 mutations out of 51 skincarcinomas. In the report by Moles et aL (23), 30% of the 38epithelial skin cancers presented p53 alterations, ofwhich 80%owere point mutations and 20o were deletions. In the tworemaining studies, sample numbers were smaller; Pierceall etal. (17) have found 20% ofthe mutations in the p53 gene havinganalyzed-only 10 squamous cell carcinomas and Rady et al.(24) found 50%o p53 mutations in their study covering 14 basalcell carcinomas (Fig. 2). As in our case, most of the p53mutations were located at pyrimidine doublets, but no tandemUV-specific CC -* TT substitutions were seen in these latterstudies. Ifwe pool the data on non-XP skin tumors, only 13%of the p53 mutations (7 out of 55) are tandem CC -) TTmutations. This is significantly lower (P < 10-2 using theFisher exact test) than the 61% we find in XP tumors. All XPtandem mutations give rise to a single amino acid change sinceeither the first C -* T transition is silent (third codon base) orthe two mutations are in the same codon (Fig. 2 and Table 2).Hence, there is no bias in our study since the mutated proteins

113 135 136 152 159 178 1 79

TTC TGC-CAA CCG GCC CAC-CAT

G A T-T TT TT (T)-TC

resulting from double mutations are identical to those foundfor C - T hot spots found in internal tumors (e.g., codons 248and 282). Therefore the tandem mutations must really be dueto dimer lesions and not to some kind of artifact in the p53protein structure. To ensure that the double mutations werenot due to PCR errors, we specifically used two differentthermostable polymerases with the same results.We have only seen l9o ofthe XP p53 mutations at thymine

doublets, although UV-induced DNA lesions are usuallyhigher at TT sites compared to other pyrimidine doublets(34). This correlates very well with model studies and withthe hypothetical "A rule" whereby the DNA polymerase isthought to incorporate preferentially an adenine opposite apyrimidine-pyrimidine lesion (35). Interestingly, two out offour XPV tumors contained the only T -- A and T -. Cmutations in this study, fitting in well with the fact that thistype of mutation is more frequent (3-fold) in UV-irradiatedshuttle vectors replicated in XPV cells compared to normalhuman cells. It has been suggested that XPV may be partiallydeficient in the activity that specifically incorporates adenineresidues at UV-induced TT lesions (36).

Another, significant difference between XP and non-XPp53 mutations in skin tumors lies in the location ofthe lesions(i.e., transcribed versus nontranscribed DNA strands). In-deed, all XP p53 mutations we have revealed in this study,with the exception oftwo, could be attributed to the presenceof lesions on the nontranscribed strand of the gene. It is wellestablished that normal human cells show preferential repairwith rapid and complete repair of the transcribed strandtogether with a slower repair of the nontranscribed strand.Also normal individuals are usually exposed to much higherdoses ofUV than XP patients, who avoid sunlight due to theirphotophobia. Hence, the large number of lesions encoun-tered in normal cells may result in slower repair levelscompared to replication, leaving mutations on both strands.Therefore, no strand bias is seen in normal human cells interms of mutagenesis, whereas in the hamster DHFR gene(37) or the mouse p53 gene (38), strand bias, as well aspreferential repair, is observed. This is of particular interestin our XP study, because it has been well established that inXPC there is preferential repair of the transcribed strand ofactively expressed genes (39, 40). XPA does not showpreferential repair, and the very low level of repair that doesexist results in mutations mostly located on the transcribedstrand (29) even though patient J.A. (Table 2), a confirmed247 248 249 2)78 281 282AAC-CGG AGG CCT GAC-CGG

C (T)-T A T(T) T-T

79 282

248 278TI135 52 v TV

3 1 36 VI 59 t 247V249 tV

A AAA AA AA A A A AAAA7 47 56 A '4A78 A 234 M A AAA294 3AA

51 ,05 5 777 A 244A249 273 A280 3 0 74252 A 245 A258 277A28'

248 7282

i96 A 28286AA248

FIG. 2. Mutation spectrum of the p53 gene in skin tumors from XP and non-XP patients. o, conserved domains (20); ED, location of p53mutation hot spots in internal tumors (12, 21); A, p53 mutations in skin tumors from non-XP patients (17, 19, 22-24); v, p53 mutations reportedin this study on XP skin tumors. The numbers correspond to the mutated codons.

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XPA among our samples, had a tandem mutation on thenontranscribed strand of the p53 gene. As the majority of thealterations we have observed in the p53 gene must be due tounrepaired lesions remaining on the nontranscribed strand ofthe DNA while the transcribed strand is repaired, this clearlyindicates that the majority of these unclassified patientsprobably do belong to the XPC group. Besides at least one ofthe two mutations on the transcribed strand is from a XPVtumor where no strand bias is expected.To our knowledge, our finding in XP tumors is the first

demonstration of preferential repair in a human tissue and ischaracteristic of XP patients since skin tumors from non-XPindividuals have their mutagenic lesions more or less equallydistributed between the two strands (43% on the transcribedstrand and 57% on the nontranscribed strand; refs. 17, 19, and22-24). This correlates with the number of YC or CY (Y =pyrimidine) sites (25 on the transcribed and 31 sites on thenontranscribed strand), where C -- T transitions of the p53gene have already been detected in human tumors.As shown in the Fig. 2, of the p53 mutations in cutaneous

tumors from normal individuals and XP patients, the majorityfall in the p53 mutation hot spots, which are extremelyimportant functional domains of the tumor suppressor gene(20). Codon 248 seems to be a hot spot for cutaneous p53mutations in general, and in XP there are two more hot spots(codons 178-179 and 281-282), both at CC sites, which maybe due to local DNA structure rendering these regions moreprone to mutation in XP cells.

In one tumor (R.N.; Table 2), two mutations separated bya normal nucleotide could have arisen either from adjacentUV-induced lesions (one on each strand) or from a CC lesionon the nontranscribed strand that resulted in a C -. Tsubstitution followed by a second mutation one base awaydue to a misinsertion by the DNA polymerase. This "semi-targeted" mutagenesis is exactly what we found using aUV-irradiated shuttle vector (41), and it may occur by thelocal distorsion produced by the bypass of the UV lesion,which then produces an untargeted mutation one base afterthe UV lesion.Our previous study (7) showing higher ras oncogene mod-

ifications in the repair-deficient XP skin tumors compared tothose from normal individuals has already established therole of unrepaired UV lesions is skin carcinogenesis. Thepresent results show an unquestionable relationship betweenUV-specific lesions and p53 mutations, demonstrating theimportance of unrepaired UV DNA damage in cells for theloss of essential growth control genes. Up to now, apart fromskin tumors, carcinogen specificity in p53 has only beendescribed for aflatoxin lesions in some hepatocarcinomas andsuggested for polycylic hydrocarbons in lung malignancies(8). These observations confirm the hypothesis that theinactivation of tumor suppressor genes during tumorigenesismay represent an interesting model for looking at specificmutation spectra. However, mutated p53 is not always foundin the skin tumors from both normal individuals or XPpatients, and it will be interesting to see what other genes arealso targeted by UV in skin carcinogenesis.

Note Added in Proof. A recent report by Sato et al. (42) of p53mutations in five XP skin tumors agrees on the whole with our data.

We are grateful to all physicians who send us tumor samples. Wethank Dr. J. Armier, Mrs. A. Margot, and A. Benoit for technicalassistance and Drs. A. Gentil and P. May for a critical reading of themanuscript. We thank Dr. T. Soussi for the cDNA amplificationprimers and the data base of p53 mutations. This work was supportedby grants from the Association pour la Recherche sur le Cancer(Villejuif, France), The Ministere de la Recherche et de l'Espace

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